Professional Documents
Culture Documents
Edited by
EDITORIAL
This issue of Methods in Cell Science is devoted molecular techniques, the genetics and molecular
entirely to methods, or new research tools, developed biology of the Gram-positive cocci lagged behind
in and for studies of members of the bacterial family, that of the Enterobacteriaceae for many years. This
Streptococcaceae. The methods included were seems somewhat ironic, since the origins of bacterial
specifically designed for analysis of different species genetics and molecular biology can be traced to two
belonging to what was once a single genus, seminal papers that describe the results of studies on
Streptococcus, but which now includes three genera S. pneumoniae. The first of these papers, of course,
of Streptococcaceae: the original genus, Strep- represented the beginning of bacterial genetics, i.e.,
tococcus, in which are retained the ~-hemolytic the transformation experiements of Griffith, in 1928
species, such as S. pyogenes and S. agalactiae, the [5]. The second, and a major boost to both bacterial
oral streptococci, e.g., S. mutans and S. parasanguis, genetics and molecular biology, was the identifica-
and S. pneumoniae; Enterococcus, formerly repre- tion of DNA as Griffith's transforming principle,
sented among the Lancefield Group D streptococci, by Avery, MacLeod, and McCarty, in 1944 [1].
and including such species as E. faecalis and E. However, very little work in streptococcal genetics
faecium; and Lactococcus, formerly referred to as the was published until 30 years later, when Jacob and
Group N, or dairy, streptococci, most notably L. Hobbs reported the first definitive demonstration of
lactis. Many investigators conducting research on conjugation between streptococcal (enterococcal)
the so-called 'pathogenic streptococci' seldom strains [6]. Despite the pioneering work presented
interact with those who study dairy organisms, such in those three papers on members of the
as the lactococci, and many of the former also fail Streptococcaceae, four years after the appearance of
to recognize the pathogenic potential of the entero- the latter paper, the ever widening gap between the
cocci. Yet, it has become clear in recent years that streptococci and other bacterial groups, relative to
procedures developed specifically for members of advances in genetics, molecular biology, and by that
one genus are very often applicable to investigations time recombinant DNA technology, as well, was
of all three genera. Thus, it is hoped that those pointed out in the inaugural address at the VIlth
working with anyone streptococcal genus will find International Symposium on Streptococci and
valuable information in articles written by investi- Streptococcal Diseases, by Sir Robert Williams,
gators involved with species classified in one of the President of the Symposium [8]. He noted that
other two genera. although there were a large number of papers to be
Certainly, many of the studies that have been presented on 'the fine details of the structure and
conducted on the Streptococcaceae were initiated behaviour of the antigens of Streptococcus pyogenes,
because of the diseases they cause, or to enhance but in comparison with most other areas of bacteri-
their utility from an industrial perspective. However, ology, rather little on genetics and antibiotic suscep-
the results of many of these investigations have tibility'. In fact, of the 156 presentations at that
demonstrated a complexity among some members of meeting, only seven were concerned with genetic
the family that would seem to warrant an interest in transfer mechanisms, mostly on the conjugative
them in their own right, apart from, or in addition transfer of antibiotic resistance plasmids. Sir Robert
to, any biomedical or industrial considerations. For jokingly referred to a stage play in London called 'No
example, many of the streptococci and pneumococci sex please, we're British ... ', and to the fact that
have as many as 30 multifunctional surface proteins. for most symposia on streptococci prior to the present
Some of these prokaryotic organisms are capable one, one might give credence to the comparable
of glycosylating proteins in a manner similar to assertion, 'no sex please, we're streptococci'.
eukaryotes, a concept that just a few years ago would However, that symposium, with its seven papers on
have been thought to be heresy. Clearly, there is genetic transfer presented in a single poorly attended
much to be learned from these complex organisms. session, served as a new beginning for streptococcal
Although many of the papers appearing in this genetics, a field which continues to grow to this day.
issue describe highly sophisticated genetic and The enthusiastic and optimistic speakers at that
VIll
session met at dinner in the evening to discuss the tocols for gene transfer between bacterial strains via
possibility of an international conference on strepto- transformation (Gaustad and Morrison), conjugation
coccal genetics, sometime in the future. Three years (Mills et aI.), electroporation (Hirt et aI.), and by
later, in November of 1981, the first ASM mobilization or electroporation (LeBlanc et aI.). The
International Conference on Streptococcal was held papers in section 3 describe methods for gene isola-
in Sarasota, FL, and was attended by 140 scientists tion (Xu et aI., and Nizet et al.), gene mapping (Lee
from 14 countries, who had come to listen to 33 oral et aI.), and for the study of gene expression
presentations, and read and discuss 44 posters (Djordejvic and Klaenhammer, Shiroza and
devoted exclusively to streptococcal genetics [7]. Of Kuramitsu, and Grey et aI.). Section 4 concerns
the many highlights of that meeting, two stand out studies on the regulation of gene expression at the
in particular, the keynote address by Maclyn transcriptional (Froeliger and Fives-Taylor, and
McCarty, titled 'Streptococci and the Birth of Peruzzi et aI.) and translational (Quivey et aI.) levels,
Molecular Genetics' , and the session titled or by cells in an adherent versus planktonic state
'Development and Use of Recombinant DNA (Burne and Chen). The fifth section includes methods
Technology'. There have now been a total of four for the study of parasite-host interactions in vitro
International Streptococcal Genetics meetings since (Winram et aI.) and in vivo (Munro), or by analyses
then, the latest in 1999 in Vichy, France, and atten- of bacterial cell surface proteins (McNab and
dance has exceeded 300 scientists from more than 20 Jenkinson). Section 6 contains papers that provide
different countries. Many new genetic and molec- tools for epidemiologic analysis, including species-
ular tools have been introduced at these meetings, specific isolation media (Spatafora and Moore), and
and presented in publications stemming from them methods of species identification (Paster et aI.), and
[2-4]. However, the streptococci are often difficult strain differentiation (Patterson and Kelly). Finally,
to work with, and there are often minute, yet section 7 contains a single paper on the in vitro
extremely critical details that are omitted from the production of antibodies (Stephenson et aI.).
proceedings of scientific conferences, primarily Most of the papers in this issue deal with micro-
because of space constraints. In the articles appearing organisms that are pathogenic for humans. Such a
in this issue of Methods in Cell Science devoted to host:parasite relationship involves a complex array
the Streptococcaceae, the experimental methods have of highly specific molecular and cellular interactions.
been written in such a manner as to permit someone A great deal of progress has been made in the
unfamiliar with the field to repeat the experiments identification of the causes of epidemic diseases such
presented without the need of additional sources. as tuberculosis and plague. However, many of the
Two additional occurrences were noted in the diseases associated with organisms like the strepto-
inaugural address of the VIIth International cocci and enterococci, that can coexist with their host
Symposium on Streptococci and Streptococcal indefinitely, and in some instances may even be
Diseases. These included the resuscitation of S. considered normal members of the human flora, are
mutans as a cause of dental caries, and an upsurge more intractable. Koch's postulates cannot be ful-
of interest in Lancefield group B streptococci and in filled for the most part. The real challenge is to
group B infections. Recognition of these two species understand the mechanisms of the disease processes
as the causes of important diseases has resulted in a of these bacteria that have evolved together with their
large number of studies on their virulence over the human hosts, resulting in the elaboration of well
past 20 years, and consequently, in the development developed and subtle strategies for coexistence. Some
of a significant amount of the new methodology, properties of these bacteria, such as the ability to
much of which will be applicable to other strepto- usurp host functions, or to invade host cells and
coccal species. In fact, strains of S. mutans, or one tissues, may facilitate peaceful coexistence with the
or more other oral streptococcal species, and the host, or contribute to pathogenesis. Other properties,
group B streptococci, were employed in the devel- such as the ability to secrete toxins and other factors,
opment of more than half of the methods to be and to cause tissue damage through stimulation of the
described in the articles appearing in this issue. host inflammatory response, would seem to be overt
The 27 papers appearing in this issue are grouped virulence traits. Clearly, whether coexistence or
into seven sections. The first includes methods of pathogenesis prevails must depend on changes in the
mutagenesis, which is accomplished by the use of regulation of many of these traits. How, then, does
transposons (Cvitkovitch et aI.), suicide vectors (Yim one decipher the mechanisms and conditions associ-
and Rubens), or a combination of both (Qin et aI.). ated with such regulation? Perhaps the most impor-
The second section contains several different genetic tant area in need of study relates to the types of
transfer, and/or host-vector systems. Three of the signaling involved in cell-cell communication; i.e.,
papers in this section describe new integration bacteria to bacteria, bacteria to host, host to bacteria,
vectors (Leenhouts et aI., McShan et aI., and Tao), bacteria in mixed communities (biofilms), bacteria
which can be used for multiple purposes, including in their environment. Significant insights into the
mutagenesis, while the remaining four provide pro- evolution of microbial pathogenicity have been
IX
Dedication
The editors have agreed to dedicate this special issue stated: 'Although the adhesiveness of bacteria for
on methods for the study of the streptococci to the some mammalian cells was recognized as early as
memory of the late Dr. Ronald Gibbons. The 1908 and selectivity was demonstrated by Duguid
pioneering studies of Gibbons and coworkers with and Old (1980), an understanding of the role of
the oral viridans group of streptococci were among microbial adhesion in the initiation of the infectious
the first to establish the critical initial role of micro- processes obtained a major stimulus through the
bial adherence in the colonization of host mucosal studies of the attachment of oral bacteria to the
surfaces. In the late sixties and early seventies very surfaces of the mouth'. While insights into the
few papers were published that documented the molecular basis of streptococcal adhesion have
ability of microorganisms to attach to human tissues continued with the studies performed over the past
in a specific fashion. Dr. Gibbons first papers on the 25 years, much still remains to be discovered.
topic appeared in the early seventies. He clearly
showed that microorganisms must have specific
adhesins for receptors on oral tissues and that this References
specificity was responsible for their ecological niche.
His work so dramatically influenced the field that the l. Gibbons RJ (1989). Bacterial adhesion to oral tissues:
number of papers on bacterial adhesion increased A model for infectious diseases. J Dent Res 68:
dramatically (Figure 1). In 1980, Drs. Ofek and 750-760.
Beachey, well-known for their work on the adhesion 2. Ofek I, Beachey EH (1980). Bacterial adherence. Adv
Intern Med 25: 503-532.
of Gram-negative organisms, recognized the contri-
butions made by Gibbons and coworkers when they
500
450 f"
400 - r
N 350 jl
u 300 I 1\ I't
m 250
II Selective
"" r~ I:i
~,
b Adhesion
- rif
r""' ;0
"" ~
e 200 !";'
_!C1fJ~
r 150 ~
100
'<.. ".
.~:
.
,
~
0
I
-
50
o
I
""'"
. I
66 67 6869 7071 72737475 76777879 80 81 82 8384 8586
Year
Figure 1. Papers published on bacterial adhesion.
Methods in Cell Science 20: xiii-xvi (1998)
List of contributors
Abstract. In order to study virulence factors of the adenine, glutamate and argmme auxotrophy. The
opportunistic oral pathogen Streptococcus mutans we transposon has been used successfully in two strains
have used Tn917 mutagenesis to generate mutants of S. mutans, JHlOOS and NG8, where transposition
defective in specific phenotypic properties believed frequencies of 10-5 and 10-4 were observed, respec-
to be associated with virulence. This work describes tively. Rescue of inactivated genes was achieved
the procedures required to use the temperature- using a recovery vector, pTV21il2TetM, that gener-
sensitive transposon Tn917 delivery vector pTV1- ates a replicative E. coli plasmid by reciprocal
OK, along with two techniques to recover inactivated recombination with the mutant S. mutans chromo-
genes. This vector has proven useful in generating somal transposon site, and shot-gun cloning chro-
transposon mutants of a poorly transformable S. mosomal DNA of mutants into pUCI8, followed by
mutans strain. We have successfully isolated mutants selection of ampicillin and erythromycin resistant
with diminished growth at pH S.O, with defects in transformants in E. coli. The techniques described
production of a mutacin, formerly known as BLIS here can be adapted for generating mutants and
or bacteriocin-like inhibitory substance and with recovering genes from a number of other Gram-
nutritional requirements in minimal media including positive and possibly Gram-negative bacteria.
11 kbp
2. Materials
A. Equipment
1. Water bath adjustable from 28 °C-45 °C, Cat.
No. 6651-25. 7
2. Circulating cooled water bath Model D.8
Figure 1. Plasmid pTVI-OK harbors the transposon 3. Shaking incubator adjustable from 28 °C_
Tn9]7 and contains the temperature-sensitive origin of 45°C.
replication from Lactococcus lactis plasmid pWVOl
4. UV-Visible recording spectrophotometer
(repAts-pWVOl). It also carries the kanamycin resistance
gene aphA3 that is expressed in E. coli and S. mutans.
UV-160. 9
5. Bench-top centrifuge Cat. No. 67390. 7
6. GasPak Anaerobic culture jar Cat. No. 11-
814-21. 5
7. Bench-top microfuge and tubes (1.5 ml).
B. Chemicals and media
1. Tetracycline Cat. No. T3258. 1
2. Erythromycin Cat. No. E6376. 1
3. Kanamycin monosulfate Cat. No. K4378. 1
4. Ampicillin Sodium Crystalline Cat. No.
NruI
A9518. 1
EcoRI 5. Tryptone Cat. No. 0123-07-5 Lot No.
pTV21LUTetM HindIII 108093J1. \0
16.0 kb 6. Yeast extract Cat. No. 0127-17-9 Lot No.
100392JB. 10
ClaI 7. Todd- Hewitt broth Cat. No. 11736 H8292-
181202 Lot No. J5DMUS. 6
8. Bacto agar Cat No. 0140-01 Lot No.
97810JA.\O
9. Sodium Chloride Cat. No. 22351-4.1
10. ddHzO.
11. Tris crystallized free base Cat. No. BPI52-1. 5
12. EDTA Cat. No. ED255. 1
Figure 2. Recovery vector pTV21~2TetM contains repli-
cons rep-pE194 (Bacillus subtilis) and rep-pBR322, that
13. Heat inactivated horse serum Cat. No.
functions in E. coli, but not in S. mutans, where it is intro- H1138. 1
duced into strains harboring Tn9]7 insertions after being 14. Gycerol Cat. No. G5516. 1
linearized by XbaI (see Figure 3). 'Tn917 and Tn9]T are 15. 5N HCI Cat. No. AI44s1-212.5
Erm-proximal and Erm-distal ends of transposon Tn9]7, 16. EcoRI Cat. No. R6011. 3
respectively, ~erm is a deletion of the erythromycin resis- 17. HindIII Cat. No. R6041. 3
tance gene that inactivates Erm-resistance upon recombi- 18. XbaI Cat. No. R6181. 3
nation, tetM confers tetracycline resistance in the E. coli 19. NruI Cat. No. R7091. 3
replicative form and in the chromosomally integrated form 20. BglII Cat. No. R6081. 3
in S. mutans. 21. Agarose Cat. No. BP1356-100 Lot No.
965988. 5
selection of E. coli transformants for ampicillin and 22. A-HindIII biotinylated standards Cat. No.
erythromycin resistance. The mutagenesis technique 15617-012. 4
and the use of complementary rescue and recovery 23. Isoamyl alcohol Cat No. A393-4. 5
techniques described here in detail have allowed us 24. Chloroform Cat. No. C298-4. 5
to isolate a variety of mutants and recover the 25. Ethanol, Absolute 200 proof Cat. No.
relevant genetic loci. This paper will use the example 94L20QA. ll
3
26. Transfer RNA from Baker's Yeast Cat. No. (Amp) - dissolve 250 mg antibiotic in 10 ml of
109495. 12 H 2 0, filter sterilize through a 0.45 11m syringe
27. T4 DNA ligase and buffer Cat. No. M1801. 3 filter and aliquot into 1 ml volumes. Prepare LB
28. DL-threonine Cat. No.T1520.1 agar plates containing 50 Ilg/ml kanamycin by
29. Glycine Cat. No. 332-9. 1 dissolving 10 g tryptone, 5 g yeast extract and
30. Mutanolysin Cat. No. M9901.1 5 g NaCl in 1 1 of ddH 20 in a 2 1 bottle. Add
31. RNase Cat. No. R6501. 1 17 g of agar and autoclave for 20 min at 121 DC.
32. Lysozyme Cat. No. L7651 c. When the medium has cooled to 55-58 DC add
33. Sodium dodecyl sulfate (SDS) Cat. No. 2 ml of Kan stock and dispense medium into
L3771. 1 100 mm x 15 mm petri plates, cool at room
34. Ammonium acetate Cat. No. A2706. 1 temperature until solid, and store inverted in a
C. Utensils and kits sealed bag at 4 DC. Inoculate an LB agar plate
1. BRL Photogene nucleic acid detection system with a sterile loop from a glycerol or stab culture
Cat. No. 8192SA. 4 of E. coli harboring pTVI-0K. Incubate the plate
2. BRL Bionick labeling system Cat. No. for 2 days at 28 DC. Make 1 1 of LB broth medium
8247SA. 4 by dissolving 10 g tryptone, 5 g yeast extract and
3. Photogene nylon membrane Cat. No. 5 g N aCl in 1 1 of ddH 2 0 and autoclave for
8194SA. 4 20 min at 121 DC in a 2 liter screw cap flask.
4. Disposable gas generator envelopes Cat No. When cooled add 2 ml of Kan stock (25 mg/ml)
43-7140. 2 to 1 1 of LB medium. Using a sterile pipette asep-
5. 15 ml 125 x 16 mm Falcon Polystyrene, tically remove 10 ml of the medium and transfer
round-bottom tubes Cat. No. 2307. 6 to a 50 ml Falcon tube. Inoculate the 50 ml tube
6. 50 ml Falcon Blue Max 30 x 115 ml with several colonies of the 2 day old 28 DC LB
polypropylene conical tubes Cat. No. 2098. 6 agar plate containing E. coli harboring pTVI-0K.
7. 500 ml Centrifuge bottles Cat. No. B0658. 1 Incubate the culture with shaking at 200 rpm at
8. 21 Srew cap flasks Kimax Cat. No. 10-26505- 28 DC. The next day if the 15 ml culture has
1000. 5 grown, as indicated by an increase in turbidity,
9. 1 1 culture bottles Kimax Cat. No. 10-26505- use the entire 15 ml culture to inoculate the
2000. 5 remainder of the 1 1 of LB containing Kan.
10. Petri dishes 100 x 15 mm Cat. No. 08-757- Incubate the culture at 28 DC overnight with
12.5 shaking (200 rpm).
11. Micropipetors and tips. When cells have reached saturation (this may
12. Milk dilution bottles Cat. No. 03-422-13. 5 take up to 48 h) collect the cells by dividing into
13. Dialysis tubing Cat. No. D9277. 1 2 x 500 ml centrifuge bottles and centrifuging
D. Plasmids for 10 min. at 6000 xg. Plasmid DNA can be
1. pTVI-0KY recovered by the alkaline lysis method [1] and
2. pTV21i12TetM. 13 purified by polyethylene glycol precipitation [1]
3. pUC18 EcoRUBAP. 13 or CsCl-EtBr density gradient centrifugation [1].
E. Bacteria After purification, the plasmids are dissolved in
1. Escherichia coli MC 1061.13 TE buffer (10 mM Tris-HCl, 10 mM EDTA, pH
2. E. coli DH5u. 4 7.5) to a concentration of 1 Ilg/Ill.
3. Streptococcus mutans JH1005. 12 B. Transformation of S. mutans with pTVI-0K and
4. s. mutans NG8.13 generation of mutants
Todd Hewitt broth (THB) is prepared by dis-
solving 30 g of Todd Hewitt medium in 1 1 of
3. Procedures ddH 2 0 which is dispensed into milk dilution
bottles in 100 ml aliquots and autoclaved for 20
A. Preparation of pTV1-0K from an E. coli host min at 121 DC. Five ml of the medium is asepti-
Important note: All growth and maintenance of cally dispensed by pipette into a sterile 10 ml
E. coli strains harboring pTVI-0K must be per- screw-cap tube. The tube is then inoculated with
formed at 28 DC. Growth or incubation of the a single colony of S. mutans, and incubated at
cultures at a higher temperature at any stage can 37 DC overnight. One ml of heat inactivated horse
result in loss of the plasmid. serum and 5 ml of sterile THB is added to a
Prepare stock solutions of antibiotics as 10 ml screw capped tube and inoculated with
follows: erythromycin (Erm) or tetracycline (Tet) 125 III of the overnight S. mutans culture. The
- dissolve 250 mg of the antibiotic in 10 ml of optical density of the culture is monitored spec-
95% ethanol, filter sterilize through a 0.45 11m trophotometrically at 600 nm until the culture has
syringe filter, aliquot into 1 ml volumes, and store achieved an OD 600 of between 0.15 and 0.25
at -20 DC; for kanamycin (Kan) or ampicillin (usually 2-4 hours). The cells are then left at
4
room temperature for 10 minutes and 1 I1g (1 Ill) volumes of cells from each tube are plated on
of pTVI-OK is added to the tube and incubated THYE plates containing both antibiotics Erm and
at 30°C for 1 h (note temperature change). Cells Kan, and 10-2_10-6 dilutions ofthe cultures (dilute
are then diluted by the addition of 5 ml of THB with THYE broth) are plated on THYE without
supplemented with horse serum and incubation is antibiotics. One ml of sterile glycerol is then
continued for an additional hour. The cells are added to the balance of the culture tubes and they
then harvested by centrifugation in a bench-top are stored at -70°C. These pools of mutants are
centrifuge and suspended in 200 III of Todd stored at this point, prior to further analysis, to
Hewitt-yeast-extract (THYE) prepared by dis- retain optimal viability. The plates are then
solving 30 g of THB medium and 3 g of yeast incubated anaerobically at 37°C for 24-48 hand
extract in 1 1 of ddH 2 0 and autoclaving at the plates are scored for the number of colonies
121°C for 20 min. THYE Kan-Erm agar plates present. The ratio of Erm-resistant colonies (deter-
are prepared by dissolving 30 g of TH medium mined from the number of colonies on the THYE-
plus 3 g of yeast extract in 1 1 of ddH 2 0 and Erm plates) to the total number of colonies
adding 17 g of agar, autoclaving at 121°C for (determined by the number of colonies on the
20 min and, when cooled sufficiently, supple- plates devoid of antibiotic), indicates the per-
mented with 2 g of solid kanamycin and 200 III centage of the S. mutans population containing
of Erm stock solution. The agar then is poured in Tn917. The number of Erm- and Kan-resistant
25 ml aliquots into 100 mm x 15 mm petri plates colonies represents the ratio of the mutants har-
and allowed to harden. Fifty III aliquots of the boring the entire pTV1-0K plasmid, including
cells are spread onto four of the hardened plates. Tn917, in the chromosome (rep Ii con fusions).
The plates are inverted and incubated at 30°C The mutagenesis technique is deemed suc-
anaerobically. All anaerobic incubations of S. cessful if the total number of Erm-resistant
mutans are performed in a Gas Pak anaerobic mutants represented in the culture tubes is at least
culture jar containing a Gas Pak plus disposable 3000 in total. For using a genetic screen, as exem-
gas generator envelope. Colonies should appear plified here for selection of acid-sensitive
on the plate after three to five days of incubation. mutants, determine the volume of culture from
The transformants growing on the THYE plates each tube to be spread on agar plates that will
containing Kan and Erm should harbor a repli- yield 150-200 colonies per plate. If this volume
cating pTV1-0K plasmid. Several single colonies exceeds 200 III the cells should be concentrated
should be used to inoculate separate 5 ml tubes of by 5 min of centrifugation at full speed in a
THYE containing Erm and Kan. The tubes are bench-top centrifuge and resuspended in a smaller
prepared by adding 20 III Erm stock and 1.2 ml volume of the supernatant so that a suitable
of Kan stock to 100 ml of THYE and dispensing density is achieved.
5 ml volumes by pipette into 15 ml screw cap C. Screening the mutants for the desired phenotype
tubes. Once inoculated the tubes are incubated at To screen for acid-sensitive mutants, spread out
30°C for 48 hours. Between 1-5 tubes are chosen enough cells on THYE-Erm plates so that at least
as inocula for facilitating mutagenesis. Five, 10, 3000 mutants will be available for screening. The
25 and 50 III aliquots of each of the selected plates are incubated at 37°C for 2 days and
cultures (that will represent dilutions of 1: 1000, removed from the incubator. Individual colonies
1:500,1:200, and 1:100 respectively) are used to are picked using sterile toothpicks and patched
inoculate new 5 ml tubes ofTHYE (no antibiotic). in duplicate in the same orientation onto a second
The cultures are then incubated overnight in a set of plates. The first set (master plate) contains
water bath at 40-45 °C. The temperature range THYE Erm (pH 7.0), while the second set
is determined by the heat tolerance of the (pH-selective plate) contains THYE made 2x by
individual S. mutans strain. For example, S. adding 30 g of Todd Hewitt media and 3 g of
mutans strain NG8 is incubated at 41°C and S. yeast extract to 500 ml of ddH 20 in a 1 I screw-
mutans strain JH1005 is incubated at 43°C. These capped flask. The pH of this solution is adjusted
are the highest temperatures at which the to 5.0 by the addition of 5N HCl prior to auto-
organisms are able to grow well. claving. A second I 1 flask is prepared containing
The culture is incubated at the higher restric- 500 ml of ddH 2 0 and 15 g of agar. After auto-
tive temperature (40-45 0c) overnight. The cells calving for 20 min at 121°C the flasks are cooled
are then assessed for the aquisition and efficiency to aprox. 55°C and mixed immediately before
of transposition of Tn917 by virtue of their Erm pouring. Typically, the plates are placed over a
resistance. To determine the percentage of the grid to aid in orienting the patched colonies
culture containing successful transposants a direct between master and pH-selective plates.
plating of 10, 50 and 200 III of the cultures are Routinely, 100 clones are represented on each
spread on THYE plates containing Erm (200 III plate without cross-contamination between
Erm stock per liter of THYE agar). The same colonies. The plates are incubated anaerobically
5
at 37°C for 2 days and examined. Clones that do is dilute it can be concentrated by the addition of
not grow, or grow poorly at pH 5.0, are selected a half volume of 7.5 M ammonium acetate, 2.5
from the corresponding master plate and chosen volumes of ice-cold absolute ethanol, followed by
for further study. The selected clones are grown 10 min of centrifugation at 4°C, two rinses with
at 37°C overnight in 15 ml screw-cap tubes 70% ethanol (v:v), 2 h of drying at room tem-
containing 5 ml THYE Erm. One ml of sterile perature and dissolving in a smaller volume of TE
glycerol is mixed with the culture and then it is buffer.
aliquoted and stored at -70°C. The phenotype The BRL photogene kit is used to perform non-
and viability of the clone should be re-tested at radioactive Southern hybridizations. Detailed
this time for its ability to grow at pH 7, but not instructions for its use are provided with the kit.
at pH 5, by streaking a loopful of culture onto a Other non-isotopic or isotopic Southern hybridiza-
THYE Erm plate and a pH 5 THYE plate and tion techniques can also be implemented for this
confirming the negative or reduced growth at pH purpose. Chromosomal DNA (5 flg) is digested to
5 after two days of 37°C anaerobic incubation. completion using separate reactions containing the
D. Linkage of Tn917 insertion to the mutant pheno- restriction endonucleases EcoRI and XbaI accord-
type ing to the enzyme manufacturer's instructions.
Before attempting to recover the inactivated gene These enzymes are chosen since restriction
the following protocols are recommended: con- fragments generated by these enzymes harbor
firmation of a single Tn917 insertion by Southern chromosomal DNA and the erythromycin resis-
blot analysis and transference of the desired tance gene from Tn917.
phenotypic characteristic by genetic back-cross to The digested DNA and biotinylated A HindIII
a wild-type S. mutans strain. standards (BRL) are subjected to electrophoresis
To perform these procedures chromosomal on a 0.5% agarose gel and transfer to a Photogene
DNA is extracted from S. mutans as follows: nylon membrane is performed as described by the
mutanolysin stocks are prepared by dissolving manufacturer. The membrane is probed with
7,500 units of solid mutanolysin per ml of TE 0.5 flg of biotinylated pTVI-0K which is
buffer and frozen at -20°C in 1 ml volumes. prepared using the Bionick nick translation kit
RNase stocks are prepared by dissolving RNase from BRL. After completion of the procedure the
at 20 mg/ml in H20 and placing in a boiling water resultant images are examined for single bands
bath for 10 min prior to storage at -20°C. in the EcoRI lanes. A single band indicates a
Lysozyme stocks are prepared fresh immediately single chromosomal insertion of Tn917. The
before use by dissolving 15 mg of solid enzyme relative size of the fragments generated by each
in 1 ml of TE buffer. A single colony of the S. enzyme is then determined and used to confirm
mutans mutant to be characterized is used to results from the gene recovery experiments.
inoculate a 15 ml screw-capped tube containing The next step before proceeding to rescue
5 m) of THB supplemented with 20 mM DL- transposon-flanking chromosomal fragments is
threonine. After incubation at 37°C overnight, a back-cross involving transfer of the mutant
10 ml of fresh, prewarmed THB is added and the phenotype to a wild-type strain of S. mutans by
culture is allowed to grow for an additional 45 transformation using chromosomal DNA from the
min. Glycine (0.75 g) is added and incubation mutant. For this genetic back-cross experiment,
continued for 60 min. The cells are then harvested we routinely use S. mutans strain NG 8 since it
by centrifugation in a bench-top centrifuge for is highly transformable. When the parent strain
5 min at maximum speed and resuspended in 1 ml used in the construction of the original mutant
of ddH 20. Ten fll of mutanolysin, 6 fll of RNase is transformable, it is recommended that it be
and 70 fll of lysozyme stock solutions are added included to conserve the genetic background of
to the cells and 37°C incubation is continued for the original mutant. Transformation of S. mutans
one hour. with chromosomal DNA is performed as for
The cell suspension is lysed by the addition of transformation with pTVI-0K with the exception
40 fll of 20% SDS followed by gentle inversion that 5 flg of EcoRI-digested chromosomal DNA
of the tube. The lysed cell mixture is then divided is added to the cultures and incubation is
between two microfuge tubes and extracted twice continued at 37°C rather than 30 dc. After trans-
with phenol:chloroform:isoamyl alcohol (25:24:1) formation, aliquots of the cells are spread onto
and twice with chloroform:isoamyl alcohol (24:1). THYE Erm plates and incubated anaerobically for
The solution is then transferred to sterile dialysis 48 h. Erm-resistant colonies are then used to
tubing and the DNA is dialyzed against two 2 I confirm the transference of phenotype back to the
changes of TE buffer at 4 °C overnight. The DNA parent strain. In our example, the transformants
is quantitated by measuring the absorbance at are examined for their diminished growth at pH
OD26o in a spectrophotometer. The concentration 5 when compared to the parent strain.
should be between 0.2 and 0.5 flg/ml. If the DNA
6
(A)
pTV21deltaltetM
'T0917
;,;
'geneX Tn917 primer PI
Figure 3. Cloning streptococcal DNA sequences adjacent to a Tn917 insertion with the recovery vector pTV21il2TetM.
The two-step process requires, first, allelic exchange of the wild-type Tn917 with linearized pTV21il2TetM
(Tn917ilerm::pVA981 portion) (A), followed by marker rescue in E. coli (B). To clone the DNA bordering the Erm-
proximal end of the transposon insertion, chromosomal DNA from the S. mutans convertant strain is digested with
restriction enzymes that cut outside of the selectable marker TetM and the pBR322 rep, such us BglII, EcoRI, HindIII,
NruI or XbaI (e.g., EcoRI). The digests are then ligated at concentrations that promote self-ligation. Finally, ligation
products are precipitated and used to transform E. coli selecting for clones containing plasmids expressing tetracycline
resistance. Digests with restriction enzymes that do not cut within the Tn917ilerm::pVA981 element, such us BamHI,
XhoI and others as well as partial digests with the above mentioned enzymes can also be used for marker rescue (e.g.,
partial digest with EcoRI). In this case, DNA adjacent to the two ends of the transposon insertion could be cloned. Gene
designations are those in the legends to Figures 1 and 2. geneX, a Tn917-inactivated gene; PI, sequencing primer based
on homology to the Erm-proximal sequence (see Procedures). Reprinted with permission of The American Society for
Microbiology.
7
The initial requirements for using the system are pools of mutants were successfully generated at a rate
that the strain to be used is able to grow at the of only SO% of all attempts despite rigid control of
permissive temperature of 2S-30 DC and that it is all the variables involved.
sensitive to the antibiotics erythromycin and We found that the transposition frequency varied
kanamycin. This facilitates selection of successful between strains JH100S and NGS with the former
pTVI-OK transformants by virtue of growth at 30 DC strain exhibiting a lO-fold higher transposition effi-
in the presence of kanamycin and erythromycin. ciency over the latter strain. Specifically, with S.
Another requirement of the strain to be mutagenized mutans strain JH100S, in 6 independent, successful
is that it is able to grow in the restrictive tempera- experiments, we achieved transposition frequencies
ture range of 40-4S DC to allow efficient curing of of between 2.S x 10-5 and O.S x 10-4. The mean
the plasmid. These criteria must be met for the transposition frequency observed with this strain was
delivery system to be successfully used. In our recent 1.0 x 10-4 • This frequency was sufficient to yield
experience two strains of S. mutans were used suc- between 104 and 105 mutants per culture tube. The
cessfully, JHI00S and NGS, however, no attempts frequency of transposition observed with S. mutans
have been made to date with other strains. strain NGS in 6 independent, successful experiments
varied between O.S x 10--{i to 2.2 X 10-5 with a mean
Transposition frequencies frequency of 1.1 x 10-5 • These values typically
resulted in over 1000 mutants per tube.
It was our experience that the critical and most When the erythromycin-resistant mutants are
variable part of the experiment occurred during the quantitated it is also useful to determine the fre-
curing of the plasmid. It is suggested that multiple quency of kanamycin-resistant clones in the popula-
tubes using a range of dilutions (1: 1000, 1:SOO, tion. kanamycin-resistant clones are the product of
1:200, and 1: 100) of the culture be transferred to the the entire delivery vector being integrated into the
higher curing temperature. Usable pools of mutants chromosome (replicon fusion). We found that the
have been generated successfully at all of the dilu- percentage of kanamycin-resistant clones varied from
tions, yet the highest values obtained do not appear 3% to 17% with strain JH100S and between 3% and
to be consistent with any single dilution factor. One 70% with strain NGS. The average values obtained
must also be aware that the optimal temperature for from 6 independent experiments with each strain
efficient plasmid loss may vary between strains. were 10% and 20% with strains JH100S and NGS,
Although the repATs mutation results in non-per- respectively. Since these mutants can also be
missive replication at temperatures as low as 3S DC recovered, replicon fusions should also be included
[17] it is suggested that a range of temperatures from in mutagenic screens. In fact, AS34 (Table 1) is a
40-4S DC be utilized. Typically, in our laboratories, replicon fusion mutant.
Table 1. Phenotypes and genes of S. mutans mutants isolated using pTVI-0K-mediated Tn917 mutagenesis
AS5 L22 AS, MUT- (BLIS-) jhs Adenine biosynthesis [9, 13] U48882
AX adenine U39612
AS 17 AS jjh Protein secretion [13] U48883 U88582
AS25 AS dfp Pantothenate metabolism, [13] U48885
DNA biosynthesis"
AS34 AS oad Oxalacetate decarboxylase" This study
AS36 AS gluP Glutamate transporter" This study
AXI AX glutamate ied Isocitrate dehydrogenase [10, 13] U48886
U62799
AX3 AX arginine argD Arginine biosynthesis" [13] U48887
AX5 AX CMM- gbpC Glucan binding protein [18] D85031
This study
DM25 MUT- (BLIS-) lanA-IanB Lantibiotic synthesis This study
" The function of these gene products has not been confirmed biochemically, but is based on strong homologies to
genes isolated from other bacterial species.
Abbreviations and definitions: AS = acid-senstive, reduced or absent growth on pH 5.0 agar plates; AX = auxotrophic
for specified amino acid or, as for strain AX5, unable to grow on minimal medium (CMM) [5]; MUT = mutacin or
BLIS = Bacteriocin-like inhibitory substance negative phenotype is defective in its ability to inhibit growth of target
cells as determined by a deferred antagonism assay [9].
9
The variability in the transposition and replicon of a single transposon insertion. Restriction digestion
fusion frequencies is likely a reflection of the over- of the chromosomal DNA followed by Southern
or under-representation of individual clones in the hybridization with pTVI-OK as a probe will verify
population of transposants. The occurrence of so- the insertion of a single transposon. In all of the
called 'jack-pots' must always be considered when mutants examined by our laboratories to date only
using mutagenesis followed by growth in liquid single transposon insertions have been observed in
culture. Erythromycin-resistant clones that are the each of the mutants examined. Although this result
result of an early transposition event or which have may tempt one to bypass this procedure, it is strongly
higher growth rates than the rest of the population recommended that the Southern hybridization be
can be over-represented. Conversely, those clones performed with a variety of restriction digests to give
that occur as the resuH of a later transposition event, an indication of the expected fragment size to be
or are slow growers will be under-represented among recovered, especially to determine if the fragment is
the population. We determined that cultures dis- amenable to recovery by shot-gun cloning into
playing a transposition frequency and percentage of pUC18. For example, if Southern analysis indicates
kanamycin-resistant phenotype closest to the mean that a restriction fragment harboring the trans po son
values likely contained pools of mutants representing generated by a particular enzyme is found to be very
the most heterogeneous mix of mutants and were large (> 15 kb), an alternate enzyme or recovery
therefore considered to be the best pools to screen strategy should be considered.
for relevant phenotypes.
Gene recovery and nucleotide sequence analysis
Isolation of mutants displaying various phenotypes
Two different techniques were used successfully to
Randomness of the transposon insertion in the bac- recover chromosomal DNA flanking Tn917 inser-
terial chromosome has not been completely assessed, tions in the mutants. The first involved the use of
although, based on the experiences in our laborato- the recovery vector pTV21.<12TetM. This vector has
ries resulting in the successful isolation of a variety been used successfully to recover segments of S.
of mutants with different phenotypes, we believe the mutans chromosomal DNA ranging in size from
degree of randomness is sufficient to isolate several 2.5-9 kb. It is recommended that each of the restric-
independent mutants, each harboring single copies of tion enzymes suggested in the 'Procedures' section
the transposon. Three individual phenotypes have be used to digest the chromosomal DNA before
been isolated using this technique: the example religation. Chances of recovery will be increased due
described in the 'Procedures' section, acid sensitivity to the fact that smaller products are favored to self-
(AS), is characterized by reduced growth on solid ligate and the greater the number of enzymes used
media at pH 5; auxotrophy (AX) for one or more the more likely that a self-ligatable plasmid will be
amino acids as determined, initially, by the inability generated. An illustration of the scheme for utiliza-
of the mutant to grow on minimal medium devoid tion of this technique is shown in Figure 3. The
of amino acids (CMM) [5, 10, 13]; and loss of recovery vector technique has proven effective with
mutacin production as indicated by the mutant's each of the strains tested (NG8 and JHlO05). Even
inability to inhibit growth of a target strain of though the latter strain is poorly transformable,
Streptococcus rattus BHT-2 as measured in a several gene .fragments have been recovered using
deferred antagonism assay [9, 12]. this technique.
The screening for acid-sensitive mutants resulted A second technique that has proven to be valuable
in the highest frequency of recovered mutants, rep- is the construction of genomic libraries in pUC18 and
resenting an average of 0.28% of the total pool. This selection of clones with ampicillin and erythromycin
result was not unexpected since a wide variety of resistance in E. coli MCI061. The use of this strain
mutations are known to cause an acid-sensitive phe- is mandatory since it is able to allow expression and
notype in other bacteria [20]. The acid protective selection of the Erm gene from Tn917 that can be
mechanisms utilized by S. mutans appear to be toxic to other E. coli strains [13]. While we have
similar to other bacteria and include both constitu- successfully recovered clones from Erm-Amp selec-
tive and inducible systems that involve a large subset tion plates we have found that plating of the E. coli
of genes [20]. The auxotrophs and MUT- phenotypes transformation mixture on LB-Amp plates and then
were isolated at frequencies of 0.2% and 0.11 %, replica plating onto LB Erm plates results in a greater
respectively (Figure 4). recovery of desirable clones.
Upon recovery of clones of interest, the purified
Southern analysis and genetic backcross plasmid DNA is sequenced using the specified
primers. The resultant sequences are often putatively
An important step to be taken before attempts to identified by comparison to the Genbank database
recover and identify interrupted genes is the demon- using BLASTN for nucleotide sequences or
stration that the observed phenotype is truly the result BLASTX for deduced amino acid sequences. A list
10
+
Temperature shift to 45"C induces pTVI-OK loss:
At45°C~ l~
plasmid loss transposon •lOsert'Ion plasmid jnteJ:ratjon
F\
wild-type JHlOO5 geneA::Tn917
,epAV kan erm
Tn917
replicon fusion
ErmS,KanS ErmR,Kan R
99.9% 0.01%
~
Screening of
independent mutant pools
Reduced growth
pH 5 agar
~j:::----LOSS
3/1500 2/1750
otMutacin
t
Physiological and Biochemical Characterization
Figure 4. Schematic flow chart of the protocol used to mutagenize S. mutans with pTVI-OK. The plasmid is shown in
the replicative form at 30°C and in the subsequent generation of mutants with the respective frequencies of isolation
after plasmid curing at 45 °C. The number of mutants isolated following various selection protocols is also listed and
described in the text.
of S. mutans genes that we have recovered is pro- appropriate E. coli mutants with chromosomal
vided in Table 1. The genes whose function have not plasmid libraries constructed from the wild-type S.
been confirmed biochemically and physiologically mutans DNA has also proven successful in our hands
are indicated. These genes have strong homology to [9, 10].
the bacterial genes listed, however further experi- The use of pTVI-0K for the generation of S.
mentation is necessary to confirm their identity. mutans mutants and the subsequent recovery of the
If a gene fragment appears to be interesting, the genes is now well established. The plasmid has also
intact wild-type gene can be cloned from a genomic been used successfully with Group B streptococci
library constructed from S. mutans parent strain DNA [11] and Streptococcus pyogenes [15]. The utility of
using the recovered gene fragment as a probe. An this vector has only recently come to light, but will
alternate strategy that involves complementation of surely prove useful for a variety of other species.
11
new method of measuring pH under human dental Address for correspondence: Arnold S. Bleiweis,
plaque in situ. J Dent Res 59: 2157-2162. University of Florida College of Dentistry, Department of
24. Yamashita Y, Takehara T, Kuramitsu HK (1993). Oral Biology, P.O. Box 100424, Gainesville, FL3261O-
Molecular characterization of a Streptococcus mutans 0424, USA
mutant altered in environmental stress responses. J Phone: (352) 846-0784; Fax: (352) 392-3070
Bacteriol 175: 6220-6228. E-mail: bleiweis@dental.ufl.edu
Methods in Cell Science 20: 13-20 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. We describe the use of suicide vectors the recombination events, PCR analysis is performed
to generate site-specific mutations in group B on chromosomal DNA isolated from the recombinant
streptococcus. This is accomplished by cloning clones. Using this strategy, we have generated site-
gene-specific sequences into a temperature sensitive specific insertions in several capsule genes and have
plasmid and selecting for clones which have under- found that these insertions occurred as expected and
gone homologous recombination. The recombinan that the mutations result in loss of capsule expres-
clones can be easily isolated by selecting for clones sion. In this report, we specifically detail the con-
which have retained the antibiotic resistance markers struction of cpsB mutants by single and double
that are present on the vector or cloned into the cross-over recombination and demonstrate that the
gene-specific sequences. To confirm the fidelity of resulting strains are acapsular.
Abbreviations: Amp = Ampicillin; BSA =Bovine Serum Albumin; cat =Chloramphenicol Acetyl Transferase;
Cm = Chloramphenicol; CmR = Chloramphenicol Resistant; cps = GBS capsule gene; CSF = Cerebral Spinal
Fluid; GBS = Group B Streptococcus; HRP = Horse Radish Peroxidase; LB = Luria-Bertani; NFDM = Non
Fat Dry Milk; ori = origin of plasmid replication; PBS = Phosphate Buffered Saline; THA = Todd-Hewitt
Agar; THB = Todd-Hewitt Broth; TBS = Tris Buffered Saline; TTBS = Tween-20 Tris Buffered Saline;
XGal = 5-Bromo-4-chloro-3-indoy1- ~- D-galactopyranoside
In addition to the single cross-over mutations, we PCR reactions that produced products over 2.0 kb,
have constructed allelic exchange mutants by a the Expand long template PCR system was used
modification of the single cross-over recombination according to the manufacturers recommendations
technique. Basically, capsule gene sequences are (Boeringer Mannheim Corp} Indianapolis, IN.
cloned into p VE6007, then a kanamycin omega Cat. #1 681 834). dNTP nucleotides were pur-
cassette is cloned into the central portion of the chased from Sigma (Sigma Chemical,2 St. Louis,
capsule sequence. The plasmid is transformed into MO, Cat. #D-7295).
GBS and the strain is exposed to conditions that favor Oligonucleotide primers were produced in-
two separate recombination events (occurring on each house on an ABI (Perkin-Elmer Cetus Corp.,9
side of the kanamycin cassette). In this way, we can Foster City CA) DNA synthesizer with trityl-OFF
obtain double cross-over mutations and allelic at a 0.2 ~Mol scale. PCR was performed using a
exchange mutants in cps genes resulting in unen- Perkin-Elmer 2400 thermocycler (Perkin-Elmer
capsulated GBS. As an illustration of these tech- Cetus Corp.,9 Foster City CA).
niques, we present the details of cpsB mutagenesis C. Immunoblot materials
by single and double cross-over recombination 0.45 ~m, 82 mm nitrocellulose circles (Cat.
resulting in an acapsular phenotype. #20440) were purchased from S&S Inc.
(Schleicher and Schuell Inc.,lo Keene, NH). BSA
(Sigma ChemicaV St. Louis, MO, Cat. #A7030)
2. Materials was resuspended in TBS (20 mM Tris-HCI, 0.5 M
NaCI, pH 7.5) at 3% wt/vol. Non-fat dry milk
A. Bacterial strains and media (Carnation brand purchased at a local grocery
GBS were grown in TH broth (DIFCO store) was resuspended at 5% wt/vol in water.
Laboratories,1 Detroit, MI, Cat. #0492-07-8) or 3MM filter paper was purchased from Whatman
TH agar and E. coli was grown in LB broth Inc. ll (Maldstone, UK, Cat. #3030917). TTBS is
(Sigma Chemical,2 St. Louis, MO, Cat. #L-3152) TBS with 0.05% Tween-20 (Sigma ChemicaV
or LB agar. COH1 is a type III encapsulated GBS St. Louis, MO, Cat. #C0378). Primary rabbit
isolated from the CSF of an infected newborn [4]. antibody specific to type III GBS capsule conju-
DH5a [supE44 I1lacU169 (<1>80 lacZI1M15) gated to tetanus toxoid was used at a 1:30,000
hsdR17 recAl endAl gyrA96 thi-l relAl] (Life dilution in 3% BSA in TBS and was a generous
Technologies Inc. [GIBCO BRL],3 Gaithersburg, gift from Dr. Mike Wessels. Goat anti-rabbit IgG
MD) was used as the E. coli host for cloning conjugated to horse radish peroxidase (Bio-Rad
and propagation of p VE6007. Chloramphenicol Laboratories, Hercules/ 2 CA, Cat. #170-6463)
resistance (Sigma Chemical,2 St. Louis, MO, Cat. was used at 1:10,000 dilution in 3% BSA in TBS.
#C0378) in E. coli was selected for at 10 ~g/ml SuperSignal chemiluminescent substrate for HRP
and kanamycin resistance (Sigma ChemicaV St. was used according to manufacturers protocols
Louis, MO, Cat. #K4000) at 40 ~g/ml. For GBS, (Pierce Chemical CO.,13 Rockford, IL, Cat.
chloramphenicol was used at 10 ~g/ml and #34080). X-ray film was from Kodak (Eastman
kanamycin at 400 ~g/ml when appropriate. To Kodak, Rochester, Ny' 14).
degrade the cell wall of GBS, mutanolysin (Sigma Restriction Enzymes purchased from NEB:
Chemical/ St. Louis, MO, Cat. #M-9901) was - BamBI (Cat. #136S)
added to a final concentration of 0.25-0.5 U/~l. - BglII (Cat. #144S)
B. Molecular biology reagents - EcoRI (Cat. #101L)
Purification of DNA was performed using various - HindIII (Cat. #104S)
kits from Qiagen Inc. according to the manufac- - KpnI (Cat. #142S)
turers protocols (Qiagen Inc.,4 Santa Clarita, CA):
plasmids from E. coli were purified using the
QIAprep spin plasmid mini-prep kit (Cat. 3. Procedures
#27106), PCR products were purified using the
QIAquick PCR purification kit (Cat. #28104) and As stated above, we have created gene-specific
DNA fragments were purified from agarose gels mutants via a homologous recombination between an
using the QIAquick gel extraction kit (Cat. intragenic fragment cloned into a temperature-
#28704). For cloning PCR products, we used the sensitive plasmid and the chromosomal copy of the
T-vector, pT7Blue (Novagen Inc.,s Madison WI, gene. The basic steps for such a project involve;
Cat. #69835-1). Restriction enzymes (see list 1) generating a mutated form of the gene, 2) cloning
below), ligase (Cat. #202L) and Klenow poly- the altered gene into an appropriate vector, 3) transfer
merase (Cat. #210S) were purchased from New of the recombinant plasmid into the host cell
England Biolabs (New England Biolabs,6 Beverly, 4) setting up conditions that favor recombination
MA). Taq polymerase (Cat. #M1861) was pur- between vector and chromosome 5) isolation of the
chased from Promega Co. 7 (Madison, WI). For recombinant strain and 6) confirmation of the genetic
15
structure of integrant. These steps are outlined below ligase buffer and 200 units ligase in a total volume
and are illustrated by our derivation of the cpsB of 15 ~l. 5 ~l of the ligation mix is transformed
mutations. into DH5a and transformants grown on selective
A. Construction of the plasmid integration vector L-agar plates containing chloramphenicol (5
Mutations in any gene can be easily created by ~g/ml) and kanamycin (40 ~g/ml) at 30 DC.
using common molecular techniques such as B. Transformation of E. coli and GBS by electro-
cloning and peR to delete the 5' and 3' ends of poration
a gene. A typical reaction includes: 100 ng of E. coli was prepared and transformed according
chromosomal DNA from COHl, 25 pmols of each to standard protocols [3]. GBS were transformed
primer, Ix PCR buffer (50 mM KCl, 10 mM Tris- with at least 400 ng of plasmid DNA using the
HCl pH 9.0, 0.1 % Triton X-I00), 2.5 mM MgC1 2 , electroporation method described by Framson et
200 ~M dNTPs and 2.5 U Taq polymerase in a al. [1]. After electroporation, the cells were
100 ~l reaction. The PCR cycling protocol was: incubated at 30 DC for two hours in recovery broth
1 cycle of 94 DC for 5 minutes, 30 cycles of so that plasmid containing cells could express the
94 DC for 30 sec, 53 DC for 30 sec and 72 DC for cat gene. Transformants were selected by plating
1 min and finally 1 cycle of 72 DC for 15 min and cells on THA + Cm (10 ~g/ml) and growing cells
a 4 DC hold. The product is purified using the at 30 DC.
QIAquick PCR purification kit, and ligated to C. Conditions that favor in vivo recombination
pT7Blue. Ligations were performed according to between vector and chromosome DNA for single
the manufacturers protocol (50 ng vector, 2 ~l of cross-over mutagenesis
purified PCR product, Ix ligase buffer [50 mM We observed that specific growth conditions
Tris-KCl, 10 mM MgCl, 10 mM Dithiothreitol, affected optimal recovery of clones containing
1 mM ATP, 25 ~g/ml BSA, pH 7.5] and 200 U single cross-over mutations. After cloning the
of ligase, 3 ~l of the ligation mixture was trans- intragenic fragment of a gene into pVE6007 and
formed into DH5a, and grown on L-Agar with transformation into GBS, a chloramphenicol
100 ~g/ml ampicillin, 40 ~g/ml XGal. White resistant colony is grown overnight in liquid
colonies were grown in LB + 100 ug/ml ampi- culture at 30 DC with antibiotics. After a 1:50
cillin and the plasmids isolated using QIAprep dilution in fresh broth with antibiotics, the culture
DNA miniprep kit. The plasmids were screened is grown for 2-2.5 hrs at the permissive temper-
for the presence of the insert by cutting 5 ~l of ature of 30 DC in a shaking incubator at 200 rpm.
each DNA miniprep with a restriction enzyme The culture is then shifted to a shaking incubator
specific to the vector and unable to cut the insert at 200 rpm at the non-permissive temperature
fragment. Plasmids containing the PCR product (37 DC) and allowed to grow for an additional
are identified because they are larger than the 2-2.5 hours. The cultures are then serially diluted
original pT7Blue plasmid (which is approximately in pre-warmed (37 DC) PBS and spread on
2.9 kb). The insert is digested from the pT7Blue- pre-warmed TH agar plates containing chloram-
based plasmid and ligated into pVE6007, previ- phenicol (10 ~g/ml). We found that without
ously digested with the same restriction enzymes. dilution of the original culture, high background
Although there are several methods to directly growth occurs on the selective agar plates that
clone PCR products into a vector, we found that obscures the identification of true chloram-
cloning the PCR product first into pT7Blue vector phenicol resistant colonies. A duplicate set of
and then subcloning into pVE6007 was more samples were spread on TH agar plates without
reliable and less time consuming. The new any antibiotics to determine the number of total
plasmid derivative is then transformed into viable bacterial cells. After overnight incubation,
COH 1, and the presence of the plasmid verified the relative number of chloramphenicol resistant
by plasmid mini-prep analysis and agarose gel clones is compared to the total number of viable
electrophoresis. colonies to estimate the recombination frequency.
To construct the allelic replacement vector, a Typically, the frequency of Cmf clones (at 37 DC)
gene fragment is first cloned into pVE6007 vector representing the plasmid integrants is between
and then digested with an enzyme that cuts once 0.1 % to 0.001% of the total cell population.
within the cloned sequence. The kanamycin Chloramphenicol resistant colonies are streak
omega cassette is isolated by digesting pC 1V2 [5] purified and maintained at 37 DC, their chromo-
with BamHI and gel purifying the 2.3 kb band somal DNA isolated and analyzed to confirm
containing the cassette. If the ends of the vector plasmid integration into the chromosome at the
and the ends of the kanamycin cassette are not proper gene location (see below).
compatible, they are converted to blunt ends by D. Allelic replacement mutagenesis using a kana-
standard molecular techniques [3]. The kanamycin mycin omega cassette and the conditions to
cassette is ligated into the vector under the fol- select for double cross-over recombination
lowing conditions; 50 ng vector, 100 ng insert, Ix Allelic replacement recombinants are generally
16
more stable than the single cross-over recombi- recombination event induced by growth at room
nants. This is because single cross-over insertions temperature without antibiotics (as discussed
have duplicate homologous sequences in close above, growth of a single cross-over plasmid
proximity to each other (which can undergo integrant at the permissive temperature encour-
homologous excisional recombination) and ages excision). This second recombination event
secondly, strains with plasmid integrants can be can occur at either site A or site B and results in
exposed to conditions (such as growth at per- two possible outcomes. If recombination occurs
missive temperatures) that promotes plasmid on the same side that was used during plasmid
replication after excision from the chromosome. integration, the strain reverts to wild type and the
If excision occurs by homologous recombination, original plasmid is excised in its entirely (Figure
reversion to wild-type and restoration of the IB). However, if the second recombination occurs
original integrity of the chromosomal gene results. on the side opposite to the original recombination
By contrast, allelic replacement mutants don't event, an allelic exchange occurs and the omega
contain duplicate sequences in close proximity to fragment is left on the chromosome creating an
each other, and don't contain plasmid replicons, insertion mutation (Figure IB). Although excision
resulting in more stable mutations. should theoretically occur on either side of the
As mentioned above, the plasmid construct for kanamycin gene with equal probability, we have
allelic exchange mutagenesis was constructed by observed that excision occurs more frequently on
cloning the kanamycin omega fragment from the side of the original recombination site.
pCIV2 into the gene of interest on pVE6007, Following excision, the autonomously repli-
resulting in the kanamycin gene flanked by the cating plasmid (now without the kanamycin
cloned gene sequences. These flanking sequences cassette) can be eliminated by growing the cells
serve as potential homologous recombination sites at the non-permissive temperature without anti-
for cross-over recombinations on either side of the biotics. Recombinant clones are recovered by
kanamycin cassette during the cross-over events. plating cultures on THA + kanamycin (400
Double cross-over recombination transfers the Ilg/ml). Surviving clones are then screened for
cassette to the chromosome within the recipient chloramphenicol sensitivity (10 Ilg/ml). Kanr , Cms
gene (Figure IB). The first recombination event strains are strong candidates for clones in which
is obtained by a single cross-over recombination double recombination has occurred. Chromosomal
as described above. There are two possible sites DNA from the resulting clones is isolated and
for recombination upstream of the kanamycin analyzed (see below). Using this method, we have
gene (site A) and downstream of the kanamycin successfully inserted the kanamycin cassette into
gene (site B; Figure IB). The excision of the several capsule genes in the GBS chromosome.
integrated plasmid is promoted by a second
t
----(9
cat I
'-})(5
t
J
----~
cat I I I
'--gec_ J
recombination ~"o~bi~~
I I
I
A B A B A B A B
----c1TIJJ- -@_...-{r--r----.!---or --1r----.--.}- ~ -+-rfill:::J.-
\
Site A )
recombination /
/
,/
./
..-
~--
{ I '
cat. I
'----~
Figure 1. Diagram of single and double recombination events. In panel lA, insertion of a plasmid into the chromosome
is mediated by a single recombination event between the plasmid and the chromosome. In panel lB, allelic exchange
mutagenesis occurs vis a double recombination event. See text for details.
17
E. Mini-scale plasmid preparation from GBS cloned into pVE6007 (primers 3 and 4; Figure
GBS were grown overnight in TH broth + 0.2% 2A). By using a plasmid-specific primer together
glycine with the appropriate antibiotic. The with a chromosome-specific primer in a PCR
culture (approximately 4 ml) was harvested by reaction, a product is generated only if a single
centrifugation and resuspended in 250 III proto- cross-over integration event has occurred at the
plasting buffer (20% sucrose, 20 mM Tris-HCl pH correct location. For example, it is impossible for
7.0, 10 mM MgC1 2 , 0.5% Triton X-100). 10 III of primers 2 & 3 and/or 1 & 4 to produce a product
mutanolysin (10 units/ill) was added to the cells unless pHYI05 has integrated into the chromo-
and incubated at 37°C for 15 minutes. The some at the correct location and in the proper
culture was then frozen at -80°C for 15 minutes orientation (Figure 2A). For allelic exchange
and thawed at 37°C for one minute to aid in insertions (Figure 2B), the presence of the
bacteria lysis. From the QIAprep spin plasmid kanamycin omega cassette can be determined by
mini-prep kit, 250 III of resuspension solution I performing PCR using primers that flank the
was added, 500 III of lysis solution II and 700 III cassette. Allelic exchange mutations result in the
of neutralization solution III were added to the PCR product being larger by 2.3 kb (the size of
lysed GBS. The rest of the procedure involves a the kanamycin cassette) compared to the PCR
typical mini-prep as detailed in the Qiagen product produced when using the wild type chro-
protocol. mosomal DNA.
F. Isolation of chromosomal DNA from GBS H. Detection of type III capsule by colony
Chromosomal DNA was isolated by the method immunoblot analysis
described by Framson et al. [1]. To detect the presence of capsule on recombinant
G. Analysis of chromosomal DNA to determine strains, potential mutants are inoculated onto solid
recombination efficacy agar plates with the appropriate antibiotic.
Following isolation of chromosomal DNA from Following overnight growth, the bacteria are
a potential recombinant, the site and orientation transferred to nitrocellulose filters by placing a
of the integrated plasmid on the chromosome filter onto the agar plate. After one minute, the
must be confirmed. Southern analysis can be used filter is carefully lifted off the plate using gloved
but is often a tedious method especially if many fingers or forceps and placed colony side-up onto
samples need to be analyzed. A quick and easy Whatman 3MM filter paper that has been satu-
method to confirm the location of the plasmid site rated with TN buffer (0.02 M Tris-HC1, 0.5 M
is to use PCR analysis. The analysis requires only NaCl pH 7.5). When the entire blot is wet, it is
four primers; two that are plasmid specific and transferred (colony side up) to Whatman 3MM
flank the multiple cloning site in p VE6007 filter paper saturated with 70% ethanol. After one
(primers 1 and 2; Figure 2A), and two that are minute, the blot is removed and placed on
capsule locus specific and not part of the gene Whatman 3MM paper to dry for at least fifteen
------~tsori 2B.
2A.
cat ~ pHY'05 ~.
~I 'cpsB' I_ j
3_1 ><. c~sB
-2
+ recombination
-4
+
,
double
recombination
3-- _4
ts ori
3_: cpsB' 1+--2---e---~2...-1 'cpsB
I PCR amplify using
• -4
, primers 5 & 6
1 1
PCR using primers 2+3 and' +4.
Products made only if recombination 3~4
occurs at correct region of chromosome.
3-
+-2
'- _4 Allelic exchange peR product is larger than wild
type chromsomal copy because of the n fragment.
Figure 2. Analysis of recombinant chromosome structure by peR. Panel lA, insertion of plasmid into the chromosome
can be verified by performing peR using one primer that anneals to the vector (primers land 2) and a second primer
that anneals to the chromosome (primers 3 and 4). Therefore, peR products from a reaction using primers land 4 or
primers 2 and 3, are only possible if recombination has occurred. If a product is produced, this is evidence of recombi-
nation at the correct location. To test for an allelic exchange event, chromosome-specific peR primers are used. These
primers are specific to the target gene and flank the kanamycin cassette recombination site. When compared to the wild
type chromosome, an allelic exchange mutant will produce a larger product because of the kanamycin cassette. Small
arrows represent primers.
18
minutes. The blot can be used immediately or the chromosome, a PCR product is only possible if
placed in plastic wrap and stored at 4 QC. the vector has integrated into the chromosome at the
The dried blot is soaked in TBS for 10 minutes correct region and orientation (Figure 2). PCR
and transferred to 5% NFDM for 30 minutes to products of the correct size were produced from
block nonspecific binding sites on the membrane. HY I 02, but, as expected, the control reactions using
The blot is washed in TTBS for 5 minutes twice wild type COHI chromosomal DNA did not produce
and then exposed to the rabbit anti-type III GBS a product. The distinct PCR amplification product
capsule antibody (1 :30,000 dilution in TBS with was observed and was the predicted size, confirming
3% BSA). After one or two hours, the blot is that recombination occurred at the correct location
washed twice with TTBS for five minutes each. (data not shown).
The secondary antibody (goat anti-rabbit IgG To generate the plasmid construct for allelic
conjugated to HRP) is diluted I: 100,000 in TBS exchange mutagenesis in cpsB, we cloned the
with 3% BSA and added to the blot for one hour. kanamycin omega fragment from pCIV2 into
The blot is then washed three times with PBS with pHY105 to create pHYIl3 (Figure 3). After pHY1l3
5% NFDM (l37 mM NaCl, 2.7 KCI, 4.3 mM was transformed into COHI, a clone was exposed to
Na 2HP0 4·7H20, 1.4 mM KH 2P0 4, pH 7.3) and conditions that promote double homologous recom-
three times with PBS (5 minutes per wash). The bination. A potential mutant (KanT, CmS) clone was
blot is removed from the PBS wash, excess liquid identified and designated HY105. Chromosomal
is allowed to drain off and the blot is placed into DNA from this strain was isolated and analyzed (see
an open seal-a-meal bag. One ml of SuperSignal below).
chemiluminescent substrate for HRP is added so The potential allelic exchange mutant, HY105,
that the entire surface of the blot is covered with was subsequently isolated as described above.
substrate. Excess liquid is removed by touching Confirmation of the omega kanamycin cassette in
the edge of the blot with a paper towel. Air cpsB was performed using the primers 3 and 4 in
bubbles are removed, the bag sealed and the blot PCR amplification (Figure 2B). These primers
exposed to X-ray film for autoradiography. hybridized to the chromosomal region upstream and
downstream of the 'cpsB' sequences as predicted
(and are therefore not present on the plasmids
4. Results and discussion pHY105 or pHYIl3). Therefore, peR products can
only be obtained when using chromosomal DNA as
Plasmid integration and allelic replacement vectors a template for these primers. As compared to the
recombine into the chromosome by homologous amplification product that is generated when using
recombination the wild type COH1 chromosomal DNA as the
To produce the plasmid integration vector for template, the PCR product from HY105 is approxi-
mutating the cpsB gene, the intragenic portion of the mately 2.3 kb larger (data not shown), corresponding
cpsB gene was amplified from the COH1 chromo- to the size of the kanamycin cassette. These data
some using the primers #19 (5'-TTGCAACAG- confirmed that the allelic exchange mutation was
CTGGACTTTTCTATAGTC-3') and #20 (5'- successfully introduced into the wild type cpsB gene
CCTGCACCAACAATAAACCCGATTAG-3'). This of COHI.
peR reaction produced a 469 bp peR product that
contains only the intragenic portion of cpsB. This Capsule expression is abolished when cpsB is
PCR products lacks 100 nt from the 5' end of cpsB disrupted in the chromsome
and 120 nt from the 3' end. The 'cpsB' product was To determine if the mutations in clones HY102 and
cloned into pT7Blue to produce the plasmid pHY205 HY105 affected capsule synthesis on the bacterial
(Figure 3). The intragenic fragment was digested surface, immunoblot analysis was performed.
from pHY205 with KpnI and HindIII and ligated into Bacteria were streaked onto TH agar plates and incu-
p VE6007 that had been previously digested with the bated overnight. After transfer to nitrocellulose and
same restriction enzymes (Figure 3). The new probing with rabbit, anti-type III GBS capsule
plasmid derivative (pHY105) was transformed into antibody, we could not detect capsule from HY102
COH-I and clones were exposed to conditions that or HY105 in contrast to the strong signal observed
promote single cross-over homologous recombina- with the parental strain COH1 (Figure 4). These
tion (chloramphenicol resistance at 37 QC). A poten- results demonstrate that insertion mutations into cpsB
tial recombinant clone was designated HY102 and its inhibits wild type capsule expression.
chromosomal DNA isolated for further analysis (see How these mutations affects capsule gene expres-
below). sion is not clear. Because the kanamycin cassette
In an analytical PCR reaction, we used the primers contains translational and transcriptional stop signals,
2 and 3 and the HY102 chromosomal DNA as a it probably causes polar mutations in downstream
template as shown in Figure 2A. Since primer 2 genes. However, it is not clear what effect the single
anneals to the plasmid, and the primer 3 anneals to cross-over insertions have on downstream expres-
19
~[C~CS
\ peR amplify
\intragenic region
'.
vull
69 111 ECORV\ of cpsB 89 11
, 'cpsB'
Sph I l:llruW.l
Pstl
Sail
~~~f KRlll
pHY20S
pVE6007
Digest each with
Kpnl & HindIII
pHY105
Bam HI
Bgl II BamHi
digest ligate digest
I'..Qnl
r 6g1
pHY113
Figure 3. Schematic showing construction of vectors for recombination. See text for details.
20
Xiang Qin 1, 2, 4, Fang Teng 3,4, Yi XU 3,4, Kavindra V. Singh!' \ George M. Weinstock2,4 &
Barbara E. Murrayl, 2, 4
Division of Infectious Diseases, 1 Department of Medicine, 2 Department of Microbiology and Molecular Genetics,
3 Department of Biochemistry and Molecular Biology, 4 Center for the Study of Emerging and Re-emerging
Abstract. A series of methods was developed for constructed a vector for mutagenesis using small
targeted mutagenesis in enterococci. First, a trans- intragenic fragments of enterococcal genes to disrupt
poson mutagenesis system, miniyo-200 (myo), which targeted genes. This vector was modified from
was used previously to make insertion mutants in pBluescript SK (-) by cloning the kanamycin resis-
streptococci, was shown to be useful for generation tance determinant from myo into the Seal site
of mutants in enterococci. After mutagenesis of internal to the ampicillin resistance gene. It was then
cosmid clones carrying enterococcal DNA inserts in used to generate insertion mutants in Enterococcus
Escherichia coli with myo, we were able to isolate faecalis with an intragenic fragment as small as about
the mutants by phenotype or to screen for them by 500 bp. A third method, based on the conjugation
immunoblotting or comparison of restriction diges- system reported by Trieu-Cuot et al. [1], was devel-
tion patterns. Clones with myo insertions in targeted oped in order to circumvent difficulties in the elec-
enterococcal genes were then introduced into troporation of some enterococcal strains and to
enterococci by electroporation to generate targeted improve the efficiency with which targeted mutations
disruption mutations. Allelic replacement en masse, can be generated in enterococci. This system was
that is, electroporation of enterococci with DNA from capable of mobilizing both small plasmids and large
a pool of mutagenized cosmid clones, was shown to cosmids into enterococci by conjugation, and
be an efficient method to obtain mutations in genes produced disruption mutations by homologous
with detectable phenotypes in enterococci. We next recombination.
Abbreviations: Amp = ampicillin; BHI = brain-heart infusion; Cam = chloramphenicol; Erm = erythromycin;
Kan = kanamycin; IPTG = isopropyl thiogalactoside; Nal = nalidixic acid; Spc = spectinomycin; Tet =
tetracycline
mutagenesis. Thus, alternative ways to make targeted 3. Electroporation solution: 0.625 M sucrose,
enterococcal mutants are of interest. In this paper, we 1 mM MgCl 2 adjusted to pH 4.0 with 1 N HCI.
summarize the targeted mutagenesis methods for 4. ESP: 0.5 M EDTA, pH 9 to 9.5, 1% sodium
enterococci that were developed in our laboratory. lauroyl sarcosine, 50 ~g of proteinase K per
mi.
5. GTE: 50 mM glucose, 25 mM Tris and 10
2. Materials mM EDTA, pH 8.
6. LB medium: dissolve 5 g yeast extract, 10 g
A. Chemicals
tryptone, 5 g NaCI in 1 liter distilled water.
1. Agarose, No. 15510-019. 9
7. PCR buffer: 100 mM Tris.HCI (pH 8.3), 500
2. a 32P-dCTP, No. AA 0005. 1
mM KCI, 15 mM MgCl 2 and 0.01 % (w/v)
3. Ampicillin, No. A-9518Y
gelatin.
4. Bacto-agar, No. 0140-01. 6
8. PIV buffer: 1 M NaCI, 10 mM Tris.HCI (pH
5. Brain-heart infusion, No. 0037-01-6. 6
7.6).
6. Calf thymus DNA, No. D-1501. 15
9. SR medium: 109 tryptone, 5 g yeast extract,
7. Cesium chloride, No. BP21O-500. 7
200 g sucrose, 10 g glucose, 25 g gelatin,
8. Chloramphenicol, No. C-0378. 15
15 g agar in 1 liter distilled water; add
9. 4-chloro-1-naphthol, No. C-8990Y
MgCl 2 and CaCl2 to concentration of 2.5 mM,
10. dNTPs, No. U-1240.14
respectively.
11. Erythromycin, No. 104663. 4
10. SSC (20x): 3 M NaCI and 0.3 M sodium
12. Fusidic acid, No. F-0881Y
citrate, pH 7.0.
13. Gelatin, No. 0143-17-9. 6
11. SSPE (20x): 3 M NaCI, 0.2 M NaH 2P0 4,
14. Glycine, No. G46-500. 7
25 mM EDTA, adjust to pH 7.4 with ION
15. Hybond W membrane, No. RPN303B. 1
NaOH.
16. InstaGene™ kit, No. 732-6030. 3
12. TE buffer: 10 mM Tris.HCI (pH 7.4), 1 mM
17. Isopropyl thiogalactoside, No. BPI620-1. 7
EDTA (pH 8.0).
18. Kanamycin, No. 17924Y
B. Preparation of cosmid clones for mutagenesis
19. K1enow fragment of E. coli DNA polymerase
1. Transposon mutagenesis
I, No. 008404. 4
To make insertion mutations in E. coli for sub-
20. Low-melting-temperature agarose, No. 50123. 8
sequent use in construction of enterococcal
21. Nalidixic acid, No. 19436. 17
mutants by allelic replacement, the previously
22. NitroPlus ™ nitrocellulose filters, No.
described transposon miniyo-200 (myo) [19]
NOTHY 08250. 11
was chosen to mutagenize pLAFRx cosmid
23. pBluescript SK (_).16
clones in E. coli (Figure 1). The Kan resistance
24. pCR™ II, No. K2050-01. 10
determinant (aph( 3' )-IIIa) [19] in this element
25. Protein A-peroxidase, No. P-8651Y
is expressed in both Gram- positive and Gram-
26. Spectinomycin, No. S-9007Y
negative hosts. The cosmid vector pLAFRx is
27. Taq DNA polymerase, No. M-1861. 14
a derivative of pLAFR which contains the oriT
28. Tetracycline, No. 22105.17
and Tet resistance determinant of RK2 [5] and
29. Todd Hewitt, No. 4311736. 2
was used for construction of enterococcal
30. Tryptone, No. 0123-17-3. 6
genomic libraries.
31. Yeast extract, No. 0127-17-9. 6
a. Preparation of the donor strain: Transform
B. Equipment
the donor strain, CBK884 (pMGD5,
1. DNA thermal cycler, No. N801-0150.13
pXRD4043) [19], with the desired pLAFRx
2. Gene Pulser apparatus, No. 165-2075. 3
cosmid according to the method previously
3. Microtiter dish (Cell Wells™), No. 25850. 5
described [20]. Select transformants on
4. UltraLok tube, No. 3420-2539Y
LB/Tet (40 ~g/ml)/Kan (25 ~g/ml) agar
plates. The myo element which carries the
3. Procedures OKm-2 interposon (resistance to 25 ~g/ml
Kan in E. coli and 2000 ~g/ml Kan in
A. Preparation of stock solutions E. faecalis) is carried on a conjugative
1. BYGT medium: 19 g BHI, 5 g yeast extract, donor plasmid (pMGD5). The plasmid
2 g glucose, 100 ml 1 M Tris.HCI (pH 8.0) pXRD4043 provides yo transposase and
in 1 liter distilled water. encodes resistance to 40 ~g/ml Cam.
2. EC lysis solution: 6 mM Tris.HCI (pH 7.6), b. Transposon mutagenesis:
1 M NaCl, 100 mM EDTA (pH 7.5),0.5% 1) Grow a single transformant colony
Brij 58, 0.2% deoxycholate, 0.5% sodium (donor strain) in 3 ml of LB/Cam (40
lauroyl sarcosine, 20 ~g of RNase per ml, ~g/ml) with 0.5 mM IPTG for 3 h (to
1 mg of lysozyme per mi. OD660 = 0.3) at 37°C to induce trans-
23
= : resolvase gene
B : Target plasmid
c_______. . .) c )
cosmid
(Tet) 80
CBK884 (Kan/Cam)
TetiKan
~
080
l'PTG /cam
ii22222J ( )
o
LW49 (Nal)
conjugation CBK884 (cointegrate of target
~ plasmid and pMGD5)
! IPTG
TetiKan/Nal
LW49 (cosmid::myo)
Figure 1. miniyo-200 transposon mutagenesis. The larger rectangular squares labeled CBK884 represent mutagenesis
strain containing a conjugative donor plasmid pMGD5 (the shaded oval) with miniyo-200, and a plasmid, pXRD4043
(smaller open circle), with transposase gene. The elongated ovals represent the chromosome. L W49 is the recipient with
a source of inducible resolvase gene. Target plasmids (cosmids) containing a Tet resistance marker are represented with
a medium sized circle. The largest circle with two transposon symbols demonstrates the cointegrate of pMGD5 and
target plasmid.
posase production. At the same time, 2. Isolation of cosmid clones with transposon
grow the recipient strain, LW49 (NaY) insertions in desired genes
[19], in LB/Nal (20 ~g/ml) at 37°C (to a. Isolation of mutants by phenotype: If a
OD 660 = 0.3) targeted gene encodes a scorable phenotype
2) Mix 0.5 ml donor cells with 0.2 ml in E. coli, cosmids with insertions in the
recipient cells in an 18-mm diameter targeted gene can be isolated by the change
test tube, incubate for 30 min in a 37°C in phenotype. For example, pLAFRx
water bath without shaking. Then, add cosmid clones, pKV48 and pKV53, from
5 ml of prewarmed LB broth containing a genomic library of E. faecalis OG lRF
IPTG (0.5 mM), followed by incubation can complement pyrimidine and purine
for 3 h without agitation. Select excon- auxotrophs of E. coli, respectively (5). We
jugants on LB/Tet (40 ~g/ml)/Kan (25 transformed E. coli pyr or pur auxotrophs
~g/ml)/Nal (20 ~g/ml) plates. [5] with mutagenized pKV48 or pKV53
24
cosmids and scored for the loss of com- to the patterns of the original cosmid. This
plementation to identify the cosmids which approach is especially useful to identify
had insertions in responsible genes. We cosmid mutants with myo insertions in
used these phenotypes to illustrate the targeted genes when a scorable phenotype
utility of this system, other phenotypes can in E. coli and an antibody or serum are not
be used in a similar fashion. available. In a previous study, we mapped
1) Transform E. coli pyr or pur auxotrophs the gene encoding the immunoreactivity of
[5] en masse with DNA from pools pKV4 with MH-l serum to an 11 kb
of myo mutagenized exconjugants of HindIII fragment. We used this approach
pKV48 or pKV53 as described [20]. to identify cosmid mutants which had
2) Plate the transformants on LB/Tet/Kan insertions in this 11 kb HindIII fragment
plates, then transfer single colonies with after myo mutagenesis. For restriction
sterile toothpicks to both M63 minimal mapping:
agar medium [21] and LB/Tet (40 1) After mutagenesis of a cosmid clone
Ilg/ml)/Kan (25 Ilg/ml). Save the with myo as described above, pick
colonies that no longer complement the single colonies growing on LBlTet (40
relevant E. coli auxotrophs for subse- Ilg/ml)lKan (25 Ilg/ml)/Nal (20 Ilg/ml)
quent analysis and allelic replacement plates into 5 ml LB medium and
studies. incubate them overnight at 37 DC.
b. Immunoscreening: If serum or antibody is 2) Isolate cosmid DNA as previously
available for a specific targeted gene described [25].
product, immunoscreening can be used to 3) Digest the cosmid DNA with restriction
detect cosmid clones with insertions in the enzymes according to the instructions of
responsible gene, or in other genes that are the suppliers and separate DNA frag-
required for expression in E. coli. For ments on a 0.7% agarose gel by elec-
example, pKV 4 is a pLAFRx cosmid clone trophoresis.
from a genomic library of E. faecalis C. Preparation of constructs with small intragenic
OGIRF [22] which reacts with serum fragments for targeted mutagenesis
MH-l (from a human patient with entero- 1. Preparation of intragenic fragments and muta-
coccal endocarditis) [23]. Immuno- genesis constructs
screening was applied to identify mutants A mutagenesis vector pTEX4577 [23] was
which had lost their immunoreactivity with constructed by digesting pBluescript® SK (-)
MH-l serum. For immunoscreening: with the restriction endonuclease Seal,
1) After mutagenizing the desired cosmid followed by blunt-ending with Klenow
clone with myo as described above, pick fragment of E. coli DNA polymerase I and
single colonies growing on LB/Tet (40 ligation to the kanamycin resistance gene
Ilg/ml)/Kan (25 Ilg/ml)/Nal (20 Ilg/ml) (aph(3')-IIIa) of Gram-positive bacterial
plates into microtiter dish Cell Wells™ origin; this gene was released from pMGD4
and grow them overnight at 37 DC, [19] by BamHI digestion and blunt-ended by
inoculate the clones onto NitroPlus ™ Klenow fragment. Small fragments internal to
nitrocellulose filters which have been desired genes can be cloned into this vector
placed on LB agar with appropriate using the multiple cloning sites of pBluescript®
antibiotics (Tet, 40 Ilg/ml; Kan, 25 SK (-). Small internal fragments and muta-
Ilg/ml). genesis constructs can be generated as fol-
2) After about 6-10 h of incubation at lowing.
37 DC, remove the filters and use them a. Small internal fragments from subclones
for immunoscreening by the method can be obtained from subclones such as
previously described [24]. Use immune those that may have originally been gener-
serum as the primary antibody, protein ated for sequencing or other purposes. To
A-peroxidase to react with the primary make the mutagenesis constructs:
antibody and apply 4-chloro-l-naphthol 1) Digest subclones which contain internal
as a substrate for protein A-peroxidase fragments of desired genes with restric-
to produce visible color. tion endonucleases whose sites are
c. Restriction mapping: If the position of a present in the multiple cloning site of
targeted gene is known on a cosmid clone, the vector used. Digest pTEX4577 with
mutants with myo insertions in this region the same restriction endonuleases or
can be identified by restriction mapping other enzymes that produce compatible
and comparing the restriction enzyme ends. HindIII, EcoRV and BamBI
digestion patterns of mutagenized cosmids cannot be used because the fragment
25
containing the Kan resistance gene has digestion and clone it into EcoRI-
sites for these enzymes. digested mutagenesis vector pTEX4577.
In our experiments, pBluescript SK D. Electroporation of E. faecalis and selection
(-) subclones containing a 1.2 kb To generate allelic replacement in E faecalis,
fragment internal to the autolysin gene cosmids with myo insertions in target genes or
of E. faecalis OGIRF, and a 480 bp derivatives of pTEX4577 with internal fragments
fragment internal to the E. faecalis gene of targeted genes can be used to transform
encoding a homologue of P54 of E. OG lRF using a modification of the electropora-
faecium, which were originally gener- tion protocol as described [5].
ated for sequencing [26], were digested 1. Preparation of plasmid DNA
with Sad and KpnI, as was pTEX4577. Large scale preparation of cosmid or plasmid
2) Separate the fragments by agarose gel DNA from E. coli by equilibrium centrifuga-
electrophoresis and recover the frag- tion in CsCI-ethidium bromide gradients [24]
ments. is as follows:
3) Mix the the fragments with digested a. Preparation of bacterial cells: Inoculate a
pTEX4577, treat with DNA ligase, and 5 ml aliquot of an overnight E. coli culture
use the ligation mixture to transform E. into 500 ml of LB-broth with the appro-
coli DH5u. Select transformants on priate antibiotics. Incubate the culture at 37
LB/Kan (25 ~g/ml) plates. DC with shaking at 250 rpm for about 6 h.
b. Small internal fragments of target genes b. Isolation of cosmid or plasmid DNA:
can also be generated by PCR if the 1) Harvest E. coli cells by centrifugation
sequences are known. An intragenic efaA at 8,500 Xg for 15 min at 4 DC. Re-
DNA fragment (730 bp, coding for amino suspend the pellet in 20 ml of GTE
acids 24 to 254 of EfaA of E. faecalis) was buffer, then mix gently with 40 ml of
generated by PCR using a primer pair NaOH (0.2 N)/SDS (1 %) and chill on
(forward primer, 5'-CGT TAG CTG CTT ice for 5 min. Add 30 ml of KAc
GCG GGA ATC-3'; reverse primer, 5'- solution (5 M acetate, 3 M K+) and mix
CCA TAC TAC GTT TAT CGA CAC-3') gently. Chill the suspension on ice for
designed according to the previously pub- an additional 5 min.
lished sequence [27]. For PCR and gener- 2) Centrifuge the suspension for 30 min at
ation of mutagenesis constructs: 4,500 xg, collect the supernatant, add 90
1) Mix 1 ~l of forward primer (0.25 ml cold 95% ethanol.
~g/~l), 1 ~l of reverse primer (0.25 3) After chilling at -70 DC for 10 min,
~g/~l), 10 ~l of chromosomal DNA of pellet the mixture at 11,000 Xg for 20
OG lRF (2.0 ng/~l), 5 ~l of dNTP (2.5 min, dry the pellet under vacuum.
mM each), 5 ~l of lOx PCR buffer, 4) Resuspend the pellet in 7.5 ml of TE
1 ~l (1 unit of activity) of Tap DNA buffer. After the addition of 0.5 ml of
polymerase and 27 ~l of water (final ethidium bromide (10 mg/ml), measure
total volume, 50 ~l) in a PCR tube, the volume and add cesium chloride at
cover the top of the mixture with 1 g/ml followed by gentle shaking at
mineral oil. 37 DC for 10 min to dissolve the cesium
2) Heat the mixture at 94 DC for 5 min in chloride.
a DNA thermal cycler, and then start 5) Centrifuge at 25,000 xg (at room tem-
the PCR cycles (94 DC for 1 min, 56 DC perature) for 15 min to clear the
for 2 min, 72 DC for 3 min, total 35 mixture. Transfer the supernatant to
cycles). UltraLok tubes for quick seal and then
3) Purify the PCR product by precipitation centrifuge for 24 h at 230,000 Xg.
with 70% ethanol. 6) Remove the plasmid band (lower band)
4) Clone the PCR product into direct PCR with needle and syringe. Extract the
product cloning vector, pCR™ II by ethidium bromide with an equal volume
ligating 100 ng of PCR product with 50 of isoamyl alcohol three times.
ng of vector DNA overnight at 16 DC. 7) Precipitate the plasmid DNA by adding
Use the ligation mixture to transform E. two volumes of TE buffer and 2 times
coli DH5u as described [20] and select of the total volume of 95% ethanol.
transformants on LB/ Amp (50 ~g/ml) After placing the mixture at -20 DC for
plates. 2 h to overnight, harvest the plasmid
5) Isolate plasmid DNA as previously DNA by centrifugation for 15 min at
described [25]. Release the PCR 12,000 xg. Wash the DNA pellet with
fragment from pCR™ II by EcoRI 70% ethanol three times, vacuum-dry
26
including the E. faecalis strain BM4110 [1]. The gation using plasmid vector pTEX5235 with
mobilization of these vectors is due to transfer small intragenic fragments
functions provided by an integrated IncP plasmid a. Generating mutagenesis construct:
on the chromosome of donor E. coli S 17 -1 cells 1) Small internal fragments can be
[1]. We modified this conjugation system by obtained from subclones or PCR as
constructing vectors that carry the oriT from described above. Small inserts in the
pATl8 [1] and an antibiotic selection marker, but PCR product cloning vector pCR™n, or
which cannot replicate in enterococci. After other vectors, can be released using
transfer into enterococci, the conjugative plasmid restriction endonucleases whose restric-
carrying an intragenic fragment of an enterococcal tion sites are available in the multiple
gene, or enterococcal DNA with a transposon or cloning sites of pBluescript SK (-),
other insertion mutation, can integrate into the except KpnI and Xbal. In an experiment
enterococcal chromosome by homologous recom- designed to test the utility of this
bination and the resulting mutants can be selected system, we used BamHIIXhoI to digest
for antibiotic resistance. pTEX5235 and a pBluescript SK (-)
Two new vectors with oriT are shown in sub clone containing an internal frag-
Figure 2. pTEX5235 (pFTl) is a derivative of ment of an autolysin gene of E. faecalis
pBluescript SK (-) [31]. The spectinomycin OGIRF [26] for cloning.
resistance gene can be expressed in Gram- 2) Clone the fragment into pTEX5235 and
negative and Gram-positive bacteria [32]. The use the recombinant plasmid to trans-
oriT fragment ("" 760 bp) was amplified by PCR form E. coli S 17 -1 as described [20].
from pATl8 and cloned into the XmnI site of b. Conjugation:
pBluescript SK (-). The pTEX5235 vector cannot 1) Grow the donor, E. coli S 17 -1 con-
replicate in Gram positive bacteria but can be taining the mutagenesis construct, and
transferred to them by conjugation to make an enterococcus recipient (e.g., OGIRF)
mutants when an intragenic fragment of a Gram- in LB and BHI to about 108 cells per ml,
positive bacterial gene has been inserted into it. respectively. Mix 1 ml of the donor cell
The oriT sequence was also cloned into the culture with 1 ml of the recipient cell
cosmid vector pBeloBACll [26, 33] to generate culture. Pellet the cells by centrifuga-
pTEX5236 (pFT2) [31]. The pTEX5236 vector tion, wash them twice with BHI, and
can be used to clone large inserts and, after trans- then resuspend in 50 III BHI.
poson mutagenesis, the cosmid can be transferred 2) Spot the mixture on a nitrocellulose
from donor E. coli S 17 -1 to Gram-positive filter, and incubate (cells facing up)
bacteria to make targeted insertion mutations by on BHI agar overnight at 37°C as
double cross-over events. described [1].
1. Construction of E. faecalis mutants by conju- 3) Use BHI agar containing 25 Ilg/ml Kan
repE
Spc
oriT
\\RK2
pTEX5235 ~ pTEX5236
(4.8Kb) (7.8 Kb)
Ca
f1 origin
CoIE1 origin
~
MCS
BamHI
Figure 2. Two vectors with oriT of RK2. Plasmid pTEX5235 is a derivative of pBluescript SK (-). As a result of inserting
the spe gene in the Seal site in the bla gene, the plasmid no longer confers ampicillin resistance and the Kpnl and Xbal
sites are no longer unique. oriT was cloned into pBluescript SK (-) using the Xmnl site. MCS, multiple cloning sites of
pBluescript SK (-). Plasmid pTEX5236 is a derivative of the pBeloBACl1 vector. The plasmid has two unique cloning
sites, BamHI and HindIIl. The two Notl sites outside of BamHI and HindIIl sites can be used to excise the cloned insert.
Part of the laeZ coding region was delected when inserting the oriT sequence between the BamHI site and Notl site on
the left.
28
to select against E. coli S 17 -1, and 2000 each of two other primers which are comple-
Ilg/ml spectinomycin to select for the mentary to the 5' and 3' ends of the targeted
insertion of the plasmid into the gene may be used for PCR. A positive product
chromosome of OG lRF. The integration with one of the targeted gene primers can
of the plasmid into the chromosome by confirm the insertion and the orientation of
a single crossover is expected to disrupt mre. Sequencing of the PCR products will
the targeted autolysin gene. Save Spc determine the exact position and orientation of
resistant E. faecalis colonies for further the mre insertion.
characterization. a. Mini-preparation of bacterial DNA from E.
b. Generation of E. faecalis mutants by con- faecalis for PCR:
jugation using large mobilizable cosmids 1) Grow putative targeted mutants on
1) Mutagenize pLAFRx cosmid clones BHI agar with appropriate antibiotics
containing the desired genes with overnight at 37°C.
transposon mre, isolate the cosmids 2) Pick an isolated bacterial colony, resus-
with insertions in the desired genes as pend it in 1 ml of sterile water in a
described above. microcentrifuge tube and then pellet the
Cos mid pBEM219 (about 45 kb in cells at 8,000 to 11,000 Xg for 1 to 2
size) is a derivative of pLAFRx [5], min. Mix the pellet with 200 III of
which has the oriT from RK2. It also InstaGene matrix and incubate the
carries the pyr gene cluster from E. mixture at 56°C for 15-30 min. Vortex
faecalis OG lRF with an insertion of the the mixture at high speed for 10 sec and
Kan f transposon mre in the pyre gene. then place the tube in a 100°C heat
We used this cosmid to determine block or a boiling waterbath for 8 min.
whether insertion mutants in entero- After vortexing another 10 sec at high
cocci could be generated by conjugation speed, centrifuge the mixture at 8,000
using a large mobilizable cosmid via a to 11,000 Xg for 2-3 min. The super-
double cross-over event. pTEX5236 natant is ready for PCR (using 20 III the
containing the desired genes with resulting supernatant per 50 III PCR
transposon insertions, or other inser- reaction).
tions, may be used in a similar fashion, b. PCR: Perform PCR reaction as above
although use of mre with pTEX5236 except that the annealing temperature may
was not attempted because both vary according to the primers used.
pXRD4043 (in the donor strain, 2. Southern blots and hybridization
CBK884) and pBeloBAC11 were Southern blots and hybridization can be used
derived from the bacterial F factor. to map the positions of insertions in the
2) Transform E. coli S 17-1 with the muta- OG lRF chromosome using fragments from
genized cosmids (e.g., pBEM219) as cosmid clones containing the targeted genes as
described above. the probes, especially when the sequences of
3) Transfer the mutagenized cosmids con- the genes encoding the targeted products are
taining mro insertions in desired genes not known and PCR is not applicable.
from E. coli S 17 -1 to OG 1RF by filter a. Preparation of chromosomal DNA from E.
matings as described above. Select faecalis for Southern blot: The method for
insertion mutants of OGIRF on isolation of chromosomal DNA from
BHIIKan (2000 Ilg/ml). If a double putative enterococcal mutants is a modifi-
crossover occurs between the cosmid cation of the method described by Smith
and the chromosome of enterococci, the and Cantor [34].
desired gene is expected to be disrupted 1) Grow putative mutant clones overnight
by the transposon mre. in 5 ml of BHI at 37°C.
F. Characterization of mutant strains 2) Harvest the cells by centrifugation and
1. PCR characterization of enterococcal mutants resuspend the cells in 1 ml of PIV
PCR can be applied to verify the correct buffer. Mix a portion (0.6 ml) of this
insertion in E. faecalis using the genomic cell suspension with 0.6 ml of 1.6%
DNA from the putative enterococcal mutant low-melting-temperature agarose in
clones as templates. To determine the position water at 40 to 50°C and then pipette the
of the mre insertion, since the orientation of mixture into a plug mold and allow the
the mre insertion is not known, a primer (mre- agarose mixture to solidify at 4 °C for
R, 5'-GAT TTA GGA TAC ACG GAA TTT lO min.
CG-3') which is complementary to one end of 3) For lysis, place the plugs of each mutant
mre in combination, in separate reactions, with strain in lO ml of fresh EC lysis solution.
29
After incubation overnight at 37°C with (about 107 cpm per filter) with 0.2 ml
gentle shaking, replace the solution with calf thymus DNA (100 Ilg/ml) for 5 min
10 ml of ESP and then incubate the at 100°C, chill the probe on ice for
plugs overnight at 50°C with gentle 5 min, then add the probe to the
shaking. Wash the plugs three times for prehybridization pouch and continue
30 min each with 15 ml of TE buffer incubation at 42°C for 12 h.
and then store them at 4 0c. 5) After hybridization, wash the membrane
4) For digestion, place a small slice (5 x 5 with 2x SSPE and 0.1 % SDS with slow
mm) of an agarose plug in a microcen- shaking twice for 10 min at room
trifuge tube with 200 III of distilled temperature, followed by two 15 min
water followed by 25 III of reaction washes with O.lx SSPE and 0.1 % SDS
buffer and 2 III of the relevant restric- at 50°C. Air-dry and expose the
tion enzyme; incubate the reaction membrane to X-ray film.
mixture for 12 h at 37°C. Wash the
slices with 1 ml TE buffer for I hat 37
°C and then melt them at 55 to 65°C. 4. Results and discussion
Load the melted slices into the wells of
regular agarose gels for electrophoresis. We have shown that, following transposon mutage-
b. Southern blot and hybridization: Southern nesis in E. coli, it is possible to generate insertion
transfer of DNA from agarose gels to mem- mutants of E. faecalis by electroporation and homol-
branes by capillary action is performed as ogous recombination. Several approaches were used
described [24]. to isolate the transposon insertion mutants in E. coli.
1) Expose the DNA in the gel to UV light For the enterococcal genes that have detectable
for 80 sec and then denature the DNA phenotypes in E. coli, mutants could be easily
in the gel in 0.4 N NaOH for 20 min. isolated by their phenotypes. Disruption mutations in
Cut a Whatman 3 mm paper wick enterococcal pyrimidine and purine genes were
slightly longer than the solid gel support identified in this manner, which were then used to
(gel tray), wet it with transfer buffer generate E. faecalis OG lRF auxotrophs [5]. For ente-
(0.4 N NaOH) and then place it on top rococcal genes which do not encode detectable
of the gel tray in a glass baking dish phenotypes in E. coli, restriction mapping of the
filled with transfer buffer. Place the gel positions of the myo insertions were used to identify
on top of the 3 mm paper, then place a mutants with insertions in known genes, or genes for
piece of Hybond N+ membrane cut to which restriction maps are available [23]. For ente-
the size of the gel and pre-wet in 0.4 N rococcal genes for which antibodies directed against
NaOH on top of the gel and remove all their products are available, immunoscreening can be
air bubbles. Place another two pieces of a useful approach to isolate mutants with insertions
pre-wet Whatman 3 mm paper on top of in antigen-encoding genes or in loci that affect
the membrane, then a stack of paper expression of the antigen-encoding genes [23]. One
towels (4-7 cm high) cut to the size of potential problem with immunoscreening is that some
the gel over the 3 mm paper, a glass insertions in the antigen-encoding genes may only be
plate over the paper towels and with a immuno-diminished and may be difficult to detect.
500 g weight on top. Allow transfer to We encountered this problem when screening for
proceed overnight. mutants of one antigen gene in E. coli. We isolated
2) The next day, carefully remove the only one immunonegative mutation in the pKV 4
Hybond N+ membrane from the blotting cosmid after immunoscreening 69 myo insertion
apparatus, wash the membrane in 0.5 M derivatives, while 9 insertions were found in the 11
Tris HCI (pH 7.0) for 5 min and then kb HindIII fragment thought to encode this antigen
neutralize in 2x SSC (24) for 2 min. Dry when restriction mapping was used to screen 100
the membrane at 80°C for 2 h to fix derivatives. Seven of these 9 myo insertion deriva-
the DNA to the membrane. tives were immuno-diminished. For the genes which
3) Incubate the filter with DNA to be have scorable phenotypes in enterococci, allelic
probed in a sealable plastic pouch for replacement en masse also proved useful for the
12 h at 42°C in prehybridization solu- generation of enterococcal mutants after myo muta-
tion (50% formamide, 5x Denhardt's genesis in E. coli. It simplifies the procedures for
solution, 5x SSPE (24) , 0.1 % SDS, and allelic replacement, as demonstrated by the genera-
100 Ilg/ml heat-denatured calf thymus tion of a gelatinase mutant and a pyrimidine mutant
DNA). in E. faecalis OGIRF [5, 23]. The efficiency of
4) After prehybridization, heat-denature mutant generation by electroporating cosmid DNA
the probe labeled with a 32P-dCTP into E. faecalis OG IRF is listed in Table 2.
30
a OG lRF and SE34 both have low level kanamycin resistance. 25 Ilg/ml kanamycin was used to select against E. coli
S 17 -1 in this study.
Among the enterococcal mutants created by intro- a mutant was about 500 bp. Intragenic fragments of
ducing cosmid DNA with myo insertions, all were the targeted genes can be obtained by subcloning
generated by double crossovers between cosmid and (e.g., the autolysin gene and P54 gene) or by peR
chromosome. By Southern blot analysis of the (e.g., efaA). The vector that we used in this work was
chromosomal DNA using the cosmid as probe, we pTEX4577 (pBluescript/Kan'). However, if vectors
did not observe any mutants that arose from a single with other antibiotic resistant genes are needed for
crossover between the cosmid and the host chromo- selection, they can also be generated by similar
some. The chance of myo transposition in the approaches. One potential disadvantage of insertion-
chromosome is very low, since neither transposase duplication mutagenesis is- the possible excision of
nor resolvase are supplied in myo. the insertion due to the duplication. However, we
Another approach to generate insertion mutants in have not encountered this as a significant limitation.
E. faecalis by electroporation is to use insertion- Mutagenesis following conjugation into E. faecalis
duplication mutagenesis if the sequences of the genes OG lRF was an efficient alternative method for
are known. Autolysin [35], P54 [23] and efaA [23] targeted mutagenesis in enterococci. With this
mutants were generated by this method. The effi- system, we were able to generate an autolysin mutant
ciency of this method is shown in Table 2. The by transferring pTEX5235 carrying an intragenic
smallest intragenic fragment that we used to generate fragment of the autolysin gene and a pyre mutant
31
Small insert pTEX4578 P54 (480 bp) OGlRF 3/5 Ilg DNA 3/3
in pTEX4577 pTEX4581 autolysin gene (1.2 kb) OGlRF 28/5 Ilg DNA 3/3
pTEX5124 efaA (730 bp) OGlRF 30/5 Ilg DNA 2/3
• Efficiency was calculated by the colonies which appeared on antibiotic selection plates.
b Correct allelic replacement was verified by either phenotypes or Southern blot analysis.
nucleotide base sequence analysis of a spectinomycin 36. Matsushima P, Broughton MC, Turner JR, Baltz RH
adenyltransferase AAD(9) determinant from Entero- (1994). Conjugal transfer of cosmid DNA from
coccus faecalis. Antimicrob Agents Chemother 35: Escherichia coli to Saccharopolyspora spinosa:
1804-1810. Effects of chromosomal insertions on macrolide
33. Shizuya H, Birren B, Kim U-J, et al. (1992). Cloning A83543 production. Gene 146: 39-45.
and stable maintenance of 300-kilobase-pair fragments
of human DNA in Escherichia coli using an F-factor-
based vector. Proc Natl Acad Sci USA 89:
8794-8797.
34. Smith CL, Canter CR (1987). Purification, specific Address for correspondence: Barbara E. Murray, Center
fragmentation, and separation of large DNA mole- for the Study of Emerging and Re-emerging Pathogens,
cules. Methods Enzymol 155: 449-467. Division of Infectious Diseases, Department of Medicine,
35. Qin X, Singh KV, Weinstock GM, Murray BE (1998). University of Texas Medical School, 6431 Fannin Street,
Effect of interruption of the gene encoding autolysin Houston, Texas 77030, USA
of Enterococcus faecalis strain OG 1RF. Antimicrob Phone: (713)-500-6767; Fax: (713)-500-5495
Agents Chemother 42: 2883-2888. E-mail: iminfdis@heart.med.uth.tmc.edu
Methods in Cell Science 20: 35-50 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. A conditionally replicating lactococcal (RepA-). A set of special purpose pORI vectors are
vector system is described, based on pWV01, that is available: (i) pORI280, designed to mutate or delete
used for chromosomal integration in Lactococcus genes or to insert new genes in the chromosome in
lactis. The system consists of plasmids that are all such a way that no heterologous DNA or antibiotic
based on the broad-host-range lactococcal replicon resistance markers are left in the recombinant strain,
pWVOl which has been deprived of its gene here designated as silent gene replacement; (ii)
encoding the replication initiation protein RepA. pORIl9, suitable for random mutagenesis of the
These so-called pORI plasmids can only replicate if chromosome, and (iii) pORIl3, constructed to make
RepA is provided in trans. Special Escherichia coli, random single copy transcriptional fusions in the
Bacillus subtilis and L. lac tis helper strains, pro- chromosome to search for (environmentally regu-
ducing RepA in trans (RepA+), are used as interme- lated) promoters. Some of these vectors have also
diate hosts for the construction of pORI integration been succesfully applied for integration in E. coli and
plasmids. The presence of a lactococcal chromosomal B. subtilis. We strongly believe that the systems
DNA fragment in a pORI plasmid enables its chro- described here can be used in various other bacte-
mosomal integration by homologous recombination rial species.
in an L. lactis strain that does not produce RepA
Key words: Conditional replication, Gene replacement, Integration, Mutagenesis, Transcriptional fusions
constructed by making use of a thermosensitive rely on the presence in the vector of one or more
derivative of the lactococcal vector pWVOl. This DNA fragments with homology to the chromosome
vector replicates in the target organism at 28°C but of the target organism. In principle any heterologous
is lost at 37.5 0c. The use of a thermosensitive plasmid which is unable to replicate in the bacterium
replicon uncouples transformation and transposition of interest can be used for this purpose. This
events which results in an important improvement approach has also been successfully taken for L.
of transposition frequencies. High-frequency random lactis to obtain single and double cross-over inte-
transposition (> 0.5%), allowing for efficient random grations [17]. However, the nature of several
gene inactivation, has been demonstrated with (potential) applications of chromosomally modified
pGh:ISS] in L. lactis, Streptococcus thermophilus lactococcal strains demanded the development of
and E. faecalis [19]. The wide host range of pWVO 1- more sophisticated integration vectors, allowing to
based replicons, which replicate both in Gram- do the necessary steps of vector construction in a
negative and Gram-positive bacteria [9], offers the homologous strain. The broad host-range lactococcal
possibility to use this tool in a great variety of plasmid pWVOl proved to be an excellent substrate
bacteria. Its successful application will depend on the for the construction of such versatile integration
thermo sensitivity of the pWVOl replicon and the vectors. Two conditionally replicating vector systems
transposition activity of the ISS] element in the target have been developed: (i) the above mentioned tem-
organism. perature sensitive (Ts) pWVOl derivative pG+host,
The rec-dependent plasmid integration strategies which was developed in the laboratory of D. Ehrlich,
_ _orr repA
_ _c:========:J1 pWVOl
Icloning of repA behind the
t L. lactis promoter p23
repA
p23 1
C :=====::J
1 integration in E. coli,
B. subtilis and L. lactis
\
..
orr
orr
manipulation
integration in
rep'L. lactis
rep' L. lactis
Figure 1. Schematic representation of the Ori+ -system. Ori+ (black box): fragment containing the plus origin of
replication of pWVOl; RepA (white box): fragment encoding the replication initiation protein of pWVOl; rep+: RepA-
producing strain; p23: constitutive lactococcal promoter, also active in E. coli and B. subtilis; pORI: Ori+-vector that
does not contain lactococcal chromosomal DNA; pINT: Ori+-vector containing lactococcal chromosomal DNA; rep-:
strain that does not produce RepA; chI. DNA (wavy lines): chromosomal DNA; M: selectable marker.
37
Jouy en Josas, France [18]. The Ts-system has the 8. Table top centrifuge no. 581OR.7
important advantage that it uncouples the transfor- 9. Vacuum dryer SpeedVac SCllO-240 and
mation and integration events, which bypasses the Refrigerated Vapor Trap RVTlOO.17
need for high transformation frequencies in the target 10. Vortex IKA-Vibrofix VFl Electronic no.
organism. Nevertheless, in some applications it may 7102775. 14
have the disadvantage that the mutant strains have B. Supplies
to be kept at temperatures above 37.5 dc. For more 1. Centrifuge tubes 50 ml no. 227.261. 9
details on the Ts-system the reader is referred to the 2. Electroporation cuvettes no. 165-2086. 4
original literature [2, 18, 19]; (ii) a system in which 3. Microcentrifuge tubes, safe-lock (1.5 ml and
pWVOl-derivatives deprived of repA, the gene essen- 2 ml) no. 0030.120.086 and 0030. 120.094.z
tial for plasmid replication, are multiplied in strains 4. Pasteur capillary pipettes (W.U. Mainz) no.
which provide Rep A in trans and are integrated in 222001.14
strains which lack the repA gene of pWVOI [Ori+- 5. Petri dishes no. 631102.9
system; 15, 17]. Figure 1 summarises the character- 6. pORI13 + EC 1000. 11
istics of the Ori+-system. The pWVOI repA gene 7. pORI19 + EC 101. 11
under the control of the lactococcal promoter P Z3 ' 8. pORI280. 11
which is expressed in Gram-positive as well as in 9. pVE6007. 1Z
Gram-negative bacteria [30], was integrated into the 10. Research pipette tips 100 III no.
chromosomes of strains of E. coli, B. subtilis and L. 0030.036.000. 7
lactis [l0, 13-15]. These RepN strains produce the 11. Research pipette tips 1 ml no. 808518. 14
Rep A protein in trans, thus sustaining the replication C. Media and chemicals
of pWVO I-derivatives lacking repA, but still carrying 1. Acetic acid, glacial no. 63.13
the recognition site for the initiation of replication 2. Agar no. 4311849. z
(pORI plasmids). The Rep+ helper strains are used 3. CaClz no. 2382. J3
for the construction of pORI-derivatives carrying 4. Chloramphenicol no. C-0378. 18
chromosomal DNA of the target strain (pINT 5. Chloroform no. A3505E. 10
plasmids). Such pINT vectors fail to replicate in 6. Dimethylformamide no. 822275 13
strains without a functional repA (Rep- strains) and 7. Double distilled HzO (ddHzO).
can thus be used to direct integration of cloned DNA. 8. EDTA no. 2802145. 3
The Ori+ -system has the advantage that it operates 9. Erythromycin E-6376. 18
independently from temperature shifts. However, in 10. Ethanol no. 983. 13
some applications this system may suffer from the 11. Glucose no. 8342.13
disadvantage that relatively high transformation 12. Glycerol no. 4094.13
frequencies are required. 13. Glycine no. 190. J3
The pORI-vectors and strategies tailored for 14. HCl no. 317.13
generating silent gene replacements, random chro- 15. Isoamylalcohol no. 999.13
mosomal mutants and random chromosomal tran- 16. Lysozyme no. 5281. 13
scriptional fusions will be discussed here. A 17. M17 no. 1856-17. 6
combined strategy will also be addressed, in which 18. MgCl Z no. 5832.13
the Ts-plasmid is used as a helper plasmid in the 19. NaAc no. 6268Y
Ori+ -system, to benefit from the advantages of both 20. NaCl no. 6404. 13
systems. We strongly believe that the nature of the 21. NaOH no. 6498.13
conditionally replicating vectors described here 22. Phenol no. 206. 13
implies that they can be successfully used in various 23. Proteinase K no. 24568. 13
other species of bacteria. 24. RNase no. 109.193. 5
25. SDS no. 1667.289. 5
26. Sucrose no. 17714-0010. 1
2. Materials 27. Tris no. 708976. 5
28. Xgal no. OP-0020-10.8
A. Equipment
1. Balances no. 1409 and BA160P. 16
2. Gene pulser no. 165-2077 and Pulse con- 3. Procedures
troller no. 165-2098.4
3. Incubator (Heraeus) no. B5060EY Some of the procedures for the pORI-system involve
4. Microcentrifuge no. 5417C. 7 cloning steps in E. coli and general DNA techniques
5. pH meter (Hanna Instruments) no. 8520.13 which are described in standard handbooks [26]. Here
6. Pressure cooker canner (Presto) no. we describe only the L. lactis-specific procedures.
VL511080. 13
7. Research pipettes no. 3110. 7
38
Silent gene replacements. One of the procedures M 17, 17.1 g sucrose and 1. 9 g glycine in
which avoids the use of an antibiotic resistance 80 ml ddH 20, adjust the volume to 100 ml,
marker to mutate a gene of interest is a two-step sterilize as before and store at room temper-
procedure described by Hamilton et al. [4]. In ature in the dark. Add 2.5 ml 20% glucose
general, in the first step of homologous recombina- just prior to use. Note: the optimal amount
tion between the delivery vector and the chromosome of glycine in the medium differs among
a co integrate is formed which is selected for by lactococcal strains e.g. for MG 1363 it is
positive selection. The second step is usually based 1.9 g per 100 ml while for IL1403 its 1 g per
on negative selection and, depending on the nature 100 ml.
of the delivery vector, consists of a temperature shift 6. GSM17MCery recovery medium: dissolve
or a period of non-selective growth. Resolution of the 3.7 g M17 and 17.1 g sucrose in 80 ml
cointegrate results either in reversion to the parental ddHP, adjust the volume to 100 ml, sterilize
chromosomal structure or in gene replacement. and store as before. Add 2.5 ml 20% glucose,
Bioassays or analyses of the chromosome are 2 ml 1 M MgCI 2 , 200 fll 1 M CaCl2 and 500
required to distinguish between the two possibilities. fll erythromycin (10 flg/ml) just prior to use.
We developed a vector, pORI280 (Figure 2), which Note: the 50 ng/ml erythromycin in this
was designed for use in such a strategy (Figure 3). medium is required for induction of the
A. Preparation of media and solutions expression of the erythromycin resistance
1. GM17 growth medium: dissolve 3.7 g M17 gene. If selection for another antibiotic is
in 100 ml ddH 20, sterilize by heat treatment used, erythromycin should be omitted.
using a pressure cooker or autoclave (15 min 7. GSM 17E5 agar medium: as in 3 but add 10 g
120 DC) and store at room temperature in the
dark. Add 2.5 ml 20% glucose solution just
prior to use.
2. GM17E 5 growth medium: as in 1, add 50 fll
of an erythromycin solution (10 mg/ml)
before use.
3. GM17 and GM17E 5 agar medium: as in 1 and
2 but add 1.5 g agar before sterilization.
4. GM17 Xgal and GM17E 5Xgai agar medium:
as in 3 but add 200 fll 4% Xgal before use.
5. GSM17gly growth medium: dissolve 3.7 g
A gene X B
Integration
I
ffi
~
~ORI+ P
T A B A gene X B
Excision
II
~lhrOUgh A through B~
pORI280 wild type gene - replacement
Xbal ·.:~
5263 bps A B
Xmalll/
NOlli muUnl Ag~nt: X
Bam HI;
PSll ',
A,$ulI " next round of mutagenesIs
£coRl' :
Smal i!
Ncol,:
Sphl ! Figure 3. Scheme of a two-step procedure to obtain
8g/l1
gene-replacement recombination. A (grey box) and B
(hatched box): two fragments flanking the geneX to be
deleted from the chromosome and through which recom-
Figure 2. Plasmid map of pORI280 (5.3 kb). The restric- bination can take place. In step I depicted here, only
tion enzyme sites indicated are unique. Em', erythromycin recombination via A is visualized. The end result in II
resistance gene; lacZ, ~-galactosidase gene of E. coli would be the same if an integration would take place
expressed under control of lactococcal promoter P32 (p32); through B. P: promoter P,ere of pWVOl; ORI+ (open box):
T (open arrow), terminator of the lactococcal proteinase origin of replication of pWVO 1; lacZ (black arrow): ~
gene prtP; open arrow (ORI+), origin of replication of galactosidase gene of E. coli expressed under control of
lactococcal plasmid pWVOl; Prepe, promoter of the repC lactococcal promoter P32 ; Emr (open arrow): erythromycin
gene of plasmid pWVOl [16]. resistance gene; gene X: target gene; ~: deletion.
39
Random lactococcaJ
pORI 19 chromosomal D A
fragments
(500-1 ,500 bp)
Library ofpORI19
derivatives in E. coli
Screening for
a-complementation
t
Replication of pORl19
30°C by in trans pVE6007
produced RepA
~
Temperature shift:
- loss of PVE6007
- integration of pORI 19
Bank ofMG1363
ori+ pORI 19 integrants
Emr
Figure S. Scheme of using pORI19 for generating random chromosomal insertions in L. lactis. In the first step (I) a
library of pORI19 containing random lactococcal chromosomal DNA fragments is established in E. coli EC101 (RepN).
In the second step (II) L. lactis MG1363 (pVE6007) is transformed with the pORI19 library. The integration of the
pORI19 derivatives is effectuated by making use of the temperature-sensitive character of pVE6007 (RepAts). See text
for details.
ORI+ (black box): origin of replication of pWVOl; Em': erythromycin resistance gene; alacZ: gene encoding the a-
fragment of LacZ; grey boxes: random lactococcal chromosomal fragments; RepN: strain producing RepA of pWVOI;
repA (open box): gene encoding RepA of pWVOl; repA": gene encoding a temperature-sensitive RepA of pWVOI; em':
chloramphenicol resistance gene.
mutant competent cells according to the described under silent gene replacements,
protocol described under silent gene replace- paragraph C. Select transformants by growth
ments, paragraph B. at 30°C for at least 48 h on the same type of
3. Introduce pVE6007 in the mutant strain as agar plates that were used to identify the
43
ori+
Emr
ori+
Em r
IO~~' o
Replication of pORI 19
by in trans p VE6007
produced RepA
E. coli EC101
! Isolation of plasmid mixture
and transformation of
RepA+ strain
37°C
t~PA Selection Ernr
Loss ofpVE6007
Rescue ofpORI19 with insert
.+ Isolation of plasmid D A
Figure 6. Rescue of an integrated plasmid from an identified mutant using pVE6007 (RepA'S). See text for details.
ORI+ (black box): origin of replication of pWVOl; Em': erythromycin resistance gene; alacZ: gene encoding the
a-fragment of LacZ; grey boxe: lactococcal chromosomal DNA fragment; RepA+: strain producing RepA of pWVOl;
repA (open box): gene encoding RepA ofpWVOl; repA ts : gene encoding a temperature-sensative RepA ofpWVOl; cm':
chloramphenicol resistance gene.
44
mutant but include chloramphenicol (end con- 21. Dissolve the DNA in 20 ~l TE.
centration 5 ~g/ml). 22. Add 1 ~l RNase and incubate 10 min at
4. Select a transformant that has reverted to the 37°C.
wild-type phenotype and grow the strain 23. Store at -20°C.
overnight in 3 ml GM17E5C5 at 30°C.
5. Isolate plasmid DNA according to the Random transcriptional fusions. In most cases
'miniprep' procedure. promoter screening systems are plasmid-based.
6. Use mixture ofthe two plasmids to transform Multiple copies of a regulated promoter on a plasmid
a RepN strain, e.g., E. coli ECI01. may interfere with the regulation mechanism by
7. Select only for the presence of the Emr gene titration of regulatory proteins. In addition, plasmid
in pORI19 and incubate the plates at 37°C. copy numbers may vary. Therefore, we developed the
In this way p VE6007 is lost. integrative pORI13 transcriptional fusion vector
8. Isolate the pORI19 derivative that contains which allows to assess the expression of (regulated)
part of the gene of interest. This plasmid can genes in a single copy fusion with a reporter gene in
be used for sequencing with the standard pUC the chromosome. The strategies to obtain a library of
sequencing primers. pORI13 integrants and the rescue of the integration
D. 'Miniprep' plasmid DNA isolation procedure by plasmid from an integrant of interest are the same
means of the alkaline lysis method as described for random mutagenesis (Paragraphs B
1. Inoculate the strain in 3 ml GM17E 5C 5 and and C; Figures 5 and 6).
grow overnight at 30°C (standing culture). A. Preparation of media and solutions
2. Transfer 2 ml overnight culture to a 2 ml As described under silent gene replacements and
microcentrifuge tube and pellet the cells using random mutagenesis.
a microcentrifuge (14,000 rpm, 3 min). B. Random transcriptional fusions in MG 1363 using
3. Remove the supernatant. Note: it is important pORI13 [29]
to remove all traces of supernatant. 1. Insert a pool of chromosomal DNA fragments
4. Resuspend the cells in 200 ~l lysis solution in a suitable restriction enzyme recognition
to which lysozyme has been added (end con- site of the pORI13 mcs (Figure 7) and trans-
centration 5 mg/ml). form E. coli ECIOOO (RepN). This strain
5. Incubate 15 min at 50°C. lacks lacZ. Generate the fragments in a similar
6. Add 400 ~l SDS/NaOH to the solution and way as described for the random mutagenesis
mix well by inversion. The mixture should procedure paragraph B. It is not necessary to
become clear and should not be stored longer use small chromosomal fragments to obtain
than 5 min. transcriptional fusions after integration of the
7. Add 300 ~l 3 M NaAc pH5.2 and mix well plasmid in the chromosome, as large fragments
by inversion. also may result in transcriptional fusions so
8. Pellet the precipitate by centrifugation long as no transcriptional terminator is present
(14,000 rpm, 10 min). between the promoter and the reporter gene.
9. Pour the supernant into a clean 2 ml micro- The chromosomal fragment does not have to
centrifuge tube. carry a promoter sequence. If the fragment
10. Add 400 ~l phenol to the solution and vortex. carries only part of an open reading frame, the
11. Add 400 ~l Chl/IAA to the mixture and reporter gene in pORI13, after the integration
vortex. event, will be placed under the control of the
12. Separate the two phases by centrifugation promoter present in the upstream chromosomal
(14,000 rpm, 4 min). DNA.
13. Carefully transfer 650 ~l of the aqueous 2. Isolate the pORI13 library and follow the pro-
(DNA-containing) upper phase to a clean 2 cedures of the random mutagenesis (paragraph
ml microcentrifuge tube. Do not take any of B) to establish and stock a library of pORI13
the interphase and/or phenol phase. integrants.
14. Add 1.3 ml ice cold 96% ethanol to the DNA C. Screening for environmentally regulated genes
solution and vortex. and rescue of the integration plasmid
15. Pellet the DNA by centrifugation (14,000 1. Thaw an aliquot of the L. lactis library of
rpm, 10 min). integrants and plate appropriate dilutions onto
16. Remove the ethanol using a Pasteur pipette. X-gal containing plates. Apply an environ-
17. Rinse the pellet with 1 ml ice cold 70% mental condition that will activate a certain set
ethanol. of genes.
18. Centrifuge 1 min at 14,000 rpm. 2. Select blue colonies and transfer them to
19. Remove the ethanol using a Pasteur pipette. GM17E 5Xgai plates and incubate at 30°C.
20. Dry the pellet using a vacuum dryer. 3. Identify colonies that are white or light blue
under 'normal' conditions (GMI7E5Xgal at
45
pORI 13
5097 bps
Figure 7. Plasmid map ofpORIl3 (5.1 kb). ORI+ (open arrow): plus origin of replication ofpWV01; Em': erythromycin
resistance gene; T (open arrow): terminator of prtP; lacZ, promoterless E. coli ~-galactosidase gene fused to the ribosome
binding site (RBS) and translational start codon of lactococcal orf-32 (shown in detail). Stop codons are indicated by
asterisks. Unique restriction enzyme recognition sites present in the multiple cloning site (MCS) are shown.
30°C) and that are blue under 'stress' condi- which is recognized in Gram-positive and Gram-
tions. negative bacteria. Strains containing an integrated
4. Excise and rescue the integration plasmid from copy of pORI280 are erythromycin resistent (Em')
the integraIit of interest as described before and stain blue on agar plates containing X-gal. After
(random mutagenesis, paragraph C). excision of the plasmid from the chromosome by
homologous recombination through one of the
flanking regions, the lacZ reporter and Em resistance
4. Results and discussion genes (and Ori+ -fragment) are lost. Consequently,
colonies which arise from cells in which this process
Silent gene replacements. The vector pORI280 has occurred stain white on non-selective agar plates
(Figure 2) has been constructed to replace genes in containing X-gal, a readily scorable phenotype
a two-step strategy and allows the isolation of among the majority of blue colonies which retained
mutants which do not carry a selectable marker the integrated plasmid. Resolution of the integrated
(Figure 3). The mcs of pORI280 contains several plasmid either restores the wild-type gene or results
unique restriction enzyme recognition sites that allow in a gene replacement. Standard DNA analysis
insertion of chromosomal DNA fragments that flank techniques or, if available, an activity assay can be
the gene to be deleted or mutated. For L. lac tis a used to discriminate between the two possibilities.
minimum size of 500-bp for each flanking region is To illustrate the feasibility of the strategy, inte-
recommended. It has been observed that recombina- gration studies using the pepX X-prolyl-dipeptidyl
tion frequencies in L. lactis increase when the sizes amino peptidase) gene region of L. lac tis MG 1363
of the chromosomal fragments increase, although no will be described. We selected this region because
further stimulation of recombination was observed in previous gene-replacement studies we observed
for fragments larger than 2.5-kb [2] . To detect that recombination frequencies in pepX of this strain
resolution of plasmids integrated in genes for which are relatively low with values as low as 10-6 per gen-
no simply detectable phenotypic difference exists eration. Two 1.5-kb fragments of pepX were cloned
between the wild-type and mutant copy, the E. coli in pORI280. Fragment A carried the promoter and
lacZ reporter gene is present in pORI280. The lacZ the 5' -end of the gene and fragment B the 3' -end. In
gene is under control of the lactococcal promoter P32 the final construct, pORI280-pepX, pepX lacks an
46
internal 716-bp fragment. pORI280-pepX was used bouring pWV01-like plasmids, as these would
to transform L. lac tis MG 1363 by the two-step provide Rep A in trans. In such a case, a plasmid
strategy outlined in Figure 2. Colonies were screened curing step prior to the use of pORI280 will be
for LacZ activity but not for PepX activity during this necessary. Adjustment of the vector for use in some
procedure to mimic the case in which a bioassay is bacterial species may be required, such as insertion
lacking. We first determined through which fragment of another selectable marker and replacement (of the
pORI280-pepX had integrated. Southern blots were promoter) of the reporter gene. A pORI-vector with
used to analyse 28 transformants: nineteen had inte- a tetracycline resistance marker, pORI240, is avail-
grated through fragment A (MG1363::pepX-A) and able. Other adjustments can easily be achieved by
nine through fragment B (MG1363::pepX-B). One virtue of the modular design of the vectors. The pORI
transformant of each type was taken and grown for plasmids have the advantage over other nonreplica-
35 generations under nonselective conditions to allow tive vectors that they allow cloning of the target gene
resolution of the cointegrate structure. Approximately (fragment) in Gram-positive or Gram-negative back-
20,000 colony forming units (4,000 cfu per 0 15 cm grounds. This may minimise cloning problems. If
plate) of each culture were plated onto agar medium such problems should persist, construction of a
containing X-gal. Five LacZ- colonies were detected homologous RepA + background may be considered.
among the MG1363::pepX-A colonies and nine The availability of a repA expression cassette is
among those derived from MG 1363::pepX-B. All convenient for this purpose. A drawback of the
LacZ- colonies were EmS and by Southern blot method is that transformation frequencies are
analysis (data not shown) it was shown that the required which are sufficiently high to obtain at least
numbers of gene-replacements were one and three for a few integrants. Therefore, it may be necessary to
the MG1363::pepX-A and MG1363::pepX-B cultures, optimise transformation protocols for poorly trans-
respectively. The mutant nature of the colonies was formable strains.
confirmed by the PepX plate assay. Taken together, The pORI280 system uses lacZ expression to
the frequencies by which gene replacements visualize recombination events in the second step of
were generated in the MG 1363::pepX-A and the procedure by a simple blue/white screening of
MG1363::pepX-B cultures ranged between 1.4 x 10-6 colonies. As this system is based on negative selec-
and 4.2 x 10-6 per generation. Recombination through tion, it may result in extensive screening of colonies
the promoter-containing fragment A was consistently if recombination frequencies in the target gene are
more efficient than through the promoterless very low « 10-6 per generation). The reporter gene
fragment B. We also observed this phenomenon in may be replaced by a gene which allows positive
other replacement studies and it is in agreement with selection, if available. Nevertheless, the pORI280-
results described earlier by Biswas et al. [2]. system was found to be highly efficient in all gene
The pORI280-system has been used in our group replacements so far. The fact that no antibiotic resis-
to replace over ten different chromosomal genes. The tance marker is left on the chromosome of a mutated
recombination frequencies observed for these genes strain makes the strain not only more desirable for
range from approximately 10-4 to 10-5 per generation. applied purposes but also leaves open the possibility
We noted that recombination in the second step of to introduce more desired mutations (or genes) in the
the procedure could be stimulated about five to ten strain, which is of special interest for metabolic
times by increasing the growth temperature to 37°C, pathway engineering purposes.
a temperature that induces a heat-shock response in
L. lactis. The system has also been successfully Random mutagenesis. A pORI-system suitable for
applied to combine several different gene replace- generating random chromosomal insertions makes
ments in a single strain. For instance, a strain was use of pORI19 (Figure 4). This vector contains an
constructed from which seven different peptidase Em' gene as selectable marker and the pUCl9 lacZa
genes were removed [20, M. A. Hellendoorn, unpub- gene and mcs in which random chromosomal DNA
lished data]. During this work it was observed that fragments can be inserted. To inactivate as many
the promoter PrepC of pORI280 is active and can drive genes as possible upon integration of the plasmid
the transcription of genes located downstream of the bank, a few requirements with respect to the plasmid
plasmid insertion site (Figure 3). This is an impor- bank have to be met: (i) the inserts in the plasmid
tant feature if the gene to be removed from the should be relatively small. A gene will be inactivated
chromosome is located upstream of (an) essential only when the fragment used for the homologous
gene(s) in an operon structure. Thus, polar effects due recombination is internal to the gene. Since the
to the insertion of the pORI vector can be avoided by average size of genes is about 1 to 1.5 kb, the cloned
employing the transcriptional activity of PrepC [14]. chromosomal DNA fragments should preferentially
The pORI280-system has also been successfully be smaller. However, recombination frequencies of
used in B. subtilis [14]. It is very likely that the fragments smaller than 500 bp are very low in L.
vectors can be used in many other bacterial hosts. An lactis. Therefore, DNA fragmentation conditions
exception may be some (lactococcal) strains har- should generate fragments in the range of 500 to
47
1,500 bp. This can either be done by using a proper integration event by using a Ts version of a pWVOl
DNA restriction enzyme or by DNA shearing tech- derivative which enables the replication of both the
niques; (ii) ligation of the random chromosomal Ts and the pORI plasmid at the permissive tempera-
fragments in the integration vector should be effi- ture (see above). Therefore, MG 1363 containing the
cient. For this purpose lacZa is present in the vector pWVOl Ts-variant pVE6007 (Cm') was transformed
which allows rapid assessment of the cloning effi- with the pORI19 plasmid bank (Figure 5). A subse-
ciency by testing part of the ligation mixture for a quent temperature shift from 30°C to 37 °C causes
complementation in the RepA+ derivative of E. coli loss of pVE6007 and integration of the pORI19
JMIOI, strain ECIOI (Figure 5). derivatives at the sites on the chromosome from
For fragmentation of chromosomal DNA of L. which the inserts originated. An l-~g sample of the
lactis MG 1363 the restriction enzyme Alul was pORI19 chromosomal DNA bank was used to
selected. Several partial Alul digests were made in transform L. lactis MG1363 (pVE6007). The trans-
which most fragments ranged in size from 100-1,500 formation mixture was incubated at 30°C for 90 min
bp. These digest were pooled and the mixture was in the presence of 50 ng of Em per ml to induce
ligated into the dephosphorylated Sma I site of expression of the Em' gene. Subsequently, the Em
pORI19 and used to transform E. coli EC101. More concentration was increased to 5 ~g/ml and incuba-
than 90% of the transformants were white or pale tion was continued at 30°C for a further 90 min to
blue on agar plates containing Xgal. All of the white ensure proper replication of the plasmid bank in L.
colonies analysed contained inserts, as did several lactis prior to the temperature shift. Plating the bank
of the blue and pale blue colonies, indicating that at this point and incubating overnight at the non-
in-frame insertions had occured in those cases. By permissive temperature for pVE6007 (37°C) did not
PCR, the estimated average insert size in pORI19 of cure the plasmid, as 40% of the colonies at this stage
100 randomly picked colonies was 650 bp. The were Em'Cm' and thus harbored both plasmids. To
number of colonies required for 99% certainty that ensure total curing of pVE6007, it was necessary to
all 0.65-kb fragments of the L. lactis MG 1363 chro- incubate the transformation mixture at 37°C for at
mosome had been cloned is 18,000. The plasmid least 3 h following the 3 h period at 30°C before
bank was isolated from approximately 30,000 E. coli plating on GM17 Em agar plates and incubation
colonies without further propagation of the cells. overnight at 37°C. Overnight incubation at 30 °C
Alternatively, the ligation mixture can be intro- following this 6-h treatment reduced the percentage
duced in a RepA + helper strain of one of the other of colonies harboring both replicating plasmids to
bacterial species to isolate the pORI19 plasmid bank. 10%, while at 37°C all Em' colonies, recovered at a
E. coli EC101 can then be merely used to assess the frequency of 104/~g of DNA, were cm'. The chromo-
cloning efficiency. One reason for following the latter somal DNAs of several integrants were analysed in
approach is that in a homologous system (in this case Southern hybridizations and these indicated that
a RepN L. lactis) a pORI19 library would probably pORI19 had integrated at different sites [10].
be more complete since it is likely that many lacto- To assess the feasibility of the system to generate
coccal DNA fragments can not be cloned in E. coli. stable random chromosomal mutations in L. lactis,
However, we reasoned that a high percentage of those the bank of integrants was screened for a mutation in
fragments which are unclonable in E. coli probably the cell wall hydrolysing system. The target gene,
contain (strong) promoters or complete (lethal) genes. acmA, was selected because: (i) it is nonessential;
Such fragments will not result in mutants since the (ii) a simple bioassay is available, and (iii) the gene
cloned fragments need to be internal to a transcrip- is monocistronic and of average size (1.3 kb). The
tional unit to result in a mutant phenotype. Therefore, size of the target fragment is even smaller if it is
constructing the library in the RepA + E. coli strain taken into account that the three repeats located in
may increase the percentage of plasmids containing the C terminus of the hydrolase can be removed
an internal gene fragment, which would enhance the without loss of enzyme activity. Therefore, integra-
efficiency of the library to generate mutations. tion within the first 700 bp of the gene is required
In the next step, the pORI19 plasmid bank was in order to inactivate it. About 5000 L. lactis trans-
used to transform the RepA- L. lactis strain MG1363. formants were spread onto glucose-M17 plates in
Under optimal conditions this resulted in approxi- which autoclaved Micrococcus lysodeikticus cells had
mately 103 Em' colonies per ~g of pORI 19 plasmid been included, allowing approximately 40 colonies
bank DNA. Southern hybridization analysis of per plate. One transformant without a halo was
chromosomal DNAs of several of the transformants detected and analysis of its chromosomal DNA
revealed that all carried an integrated copy of pORI19 learned that, indeed, pORI19 had integrated in an
at different locations. A library of pORI19 integrants internal 5'-fragment of acmA.
in which a maximum number of different genes are The pORI19 system was further tested by selec-
inactivated should contain at least 18,000 colonies, tion of mutants in the maltose metabolic pathway,
if not more. To increase the number of integrants, the as isolation of stable Mal- mutants had previously
transformation event can be separated from the been shown to be unsuccessful using other insertional
48
methods. The bank of lactococcal integrants was non-essential ones and to genes that allow detection
plated on maltose indicator plates with a density of of phenotypic negatives. Another disadvantage of the
approximately 1000 colonies per plate. Following 24 method is that rather high transformation frequencies
hours of incubation at 30°C, I in 10,000 colonies are required if the Ts-helper plasmid can not be used,
was found to be unable to ferment maltose (approx. which may be the case in a number of bacterial
30,000 colonies were screened). One of the L. lactis species. Critically important is also the construction
Mal- colonies was streaked for single colonies on of a proper pORI19 library in one of the RepA+
maltose selective agar and after 3 days of incubation helper strains. However, cloning efficiencies can be
at 30°C, all of the colonies were still white, indi- easily assessed by the availabilty of the E. coli helper
cating that the mutation was stable. A culture of this strain EClOl. Other important advantages of the
strain maintained its maltose-negative phenotype method are that: (i) stability of the mutants is high
even after incubation overnight at 30 °C in GMl7 because of the absence of residual activity of trans-
without Em and subsequent plating on maltose selec- posases or (Ts) replication proteins; (ii) screening of
tive agar. mutants can be performed at optimal growth tem-
The maltose-negative strain was used to develop peratures; (iii) the integration plasmid can be readily
a strategy for the easy isolation of the integrated recovered by a simple and rapid procedure, and (iv)
plasmid from a defined mutant. Such a strategy the availability of RepA+ L. lactis, B. subtilis, and
would allow to rapidly identify the disrupted gene E. coli helper strains minimizes difficulties (lethality
(Figure 6). L. lactis Mal- was endowed with and deletions) in cloning of the targeted gene in
pVE6007 (RepNS) and plated on maltose indicator incompatible host backgrounds.
agar containing Cm and Em. After 24 h of incuba-
tion at 30°C all transformants were still unable to Random transcriptional fusions. An Ori+-integration
ferment maltose, whereas after 48 h approximately vector, pORI13, was developed to screen for
8% were faintly yellow, indicating that cells in these (environmentally regulated) gene expression signals
colonies had reverted to wild type. After colony in L. lactis and to assay for transcriptional activity of
purification it was found that 20% had regained genes in single copy. The plasmid carries a pro-
maltose-fermenting ability, most probably by precise moterless E. coli lacZ gene preceded by a start codon,
excision of the integrated plasmid. A Mal+ L. lactis a lactococcal ribosome binding site, and a mcs. It
single colony isolate contained two plasmids, contains translational stop codons in all three reading
pVE6007 and pORIl9 containing an insert (pMAL). frames upstream of or overlapping the start codon
To rescue pMAL, the plasmid mixture was used to of lacZ to prevent translational fusions (Figure 7).
transform E. coli EC101 (RepN) at 37°C with selec- Plasmid pORI13 did not produce detectable p-galac-
tion for Emf only, resulting in the separation of tosidase activity when replicating in L. lactis RepA +
pVE6007 and pMAL. Upon introduction of pMAL helper strains.
into L. lactis MG 1363 (RepA-), all integrants were To produce chromosomal transcriptional fusions
maltose negative, as expected. Southern hybridisa- using pORI13 there are no restrictions with respect
tion analysis of five transformants revealed that to the size of the fragment and, also, promoter
pMAL had integrated at the same site on the lacto- elements do not have to be present on the cloned
coccal chromosome as in L. lactis Mal-. chromosomal fragment. Any fragment of a tran-
The nucleotide sequence of the AluI insert in scriptional unit cloned upstream of lacZ will produce
pMAL was determined by making use of the standard a transcriptional fusion after integration of pORI13,
pUC19 sequencing primers. A continuous open provided that the transcriptional terminator of that
reading frame was present on this 564-bp fragment, unit is lacking. Several partial Sau3A digests of L.
and its deduced amino acid sequence of 189 amino lactis MGl363 chromosomal DNA, with the majority
acids showed high homology to ATP-binding proteins of the fragments ranging in size from 1 to 10 kb, were
of several sugar uptake systems, among which the pooled and the mixture was ligated into the alkaline
inner membrane MalK proteins of the maltose uptake phosphatase-treated BamHI restriction enzyme site
systems of Enterobacter aerogenes and E. coli. These of pORI13. The ligation mixture was used to trans-
results suggested that, indeed, a gene involved in the form E. coli EC1000 (RepN), a derivative of strain
maltose metabolic pathway of L. lactis had been MC1000 (l1lacZ). More than 10,000 transformants
targeted and that part of it had been cloned [10]. The were collected from agar plates and their plasmid
5' - and 3' -end of the gene may now be cloned by DNA content was isolated. The resulting pORI13
restriction of the chromosomal DNA of the Mal- plasmid DNA bank was used for integration in the
integrant with suitable enzymes and recovering the chromosome of L. lactis MG 1363 by employing the
integration plasmid with additional flanking DNA in same strategy as described for pORI19, with
one of the RepA + helper strains. pVE6007 as a means to uncouple transformation and
Although the method described here is efficient in integration events. The system was tested by plating
generating chromosomal insertions, the isolation of the transformants, immediately after the temperature
genes is, like most insertional methods, restricted to shifts, onto selective agar plates containing 0.3 M
49
diacetylactis 18-16 after introduction of Tn919. FEMS other gram-positIve bacteria. J Bacteriol 178:
Microbio1 Lett 81: 135-140. 931-935.
6. Holo H, Nes IF (1989). High-frequency transforma- 20. Mierau I, Kunji ERS, Leenhouts KJ, Hellendoom MA,
tion by electroporation of Lactococcus lactis subsp. Haandrikman AJ, Poolman B, Konings WN, Venema
cremoris grown with glycine in osmotically stabilized G, Kok J (1996). Multiple-peptidase mutants of
media. Appl Environ Microbiol 55: 3119-3123. Lactococcus lactis are severely impaired in their
7. Israelsen H, Hansen EB (1993). Insertion of trans- ability to grow in milk. J Bacteriol 178: 2794-2803.
poson Tn917 derivatives into the Lactococcus lactis 21. Polzin KM, Shimizu-Kadota M (1987). Identification
subsp. Lactis chromosome. Appl Environ Microbiol of a new insertion element, similar to gram-negative
59: 21-26. IS26, on the lactose plasmid of Streptococcus lactis
8. Israelsen H, Madsen SM, Vrang A, Hansen EB, ML3. J Bacteriol 169: 5481-5488.
Johansen E (1995). Cloning and partial characterisa- 22. Renault P, Heslot H (1987). Selection of
tion of regulated promoters from Lactococcus lac tis Streptococcus lactis mutants defective in malolactic
Tn917-lacZ integrants with the new promoter probe fermentation. Appl Environ Microbiol 53: 320-324.
vector, pAK80. Appl Environ Microbio 61: 2540- 23. Romero DA, Klaenhammer TR (1990). Character-
2547. isation of insertion sequence IS946, an iso-ISSI
9. Kok J, van der Vossen JMBM, Venema G (1984). element, isolated from the conjugative lactococcal
Construction of plasmid cloning vectors for lactic plasmid pTR2030. J Bacteriol 172: 4151-4160.
streptococci which also replicate in Bacillus subtilis 24. Romero DA, Klaenhammer TR (1992). IS946-
and Escherichia coli. Appl Environ Microbiol 48: mediated integration of heterologous DNA into the
726-731. genome of Lactococcus lactis subsp. lactis. Appl
10. Law J, Buist G, Haandrikman A, Kok J, Venema G, Environ Microbiol 58: 699-702.
Leenhouts K (1995). A system to generate chromo- 25. Romero DA, Klaenhammer TR (1993). Transposable
somal mutations in Lactococcus lac tis which allows elements in lactococci: A review. J Dairy Sci 76:
fast analysis of targeted genes. J Bacteriol 177: 1-19.
7011-7018. 26. Sambrook J, Fritsch EF, Maniatis T (eds) (1989).
11. Le Bourgeois P, Lautier, Mata M, Ritzenthaler P Molecular cloning. A laboratory manual, 2nd edn.
(1992). New tools for the physical and genetic New York: Cold Spring Harbor Laboratory Press.
mapping of Lactococcus strains. Gene 111: 109-114. 27. Sanders JW, Leenhouts K, Burghoom J, Brands JR,
12. Le Bourgeois P, Lautier M, van den Berghe L, Gasson Venema G, Kok J (1998). A chloride-inducible acid
MJ, Ritzenthaler P (1995). Physical and genetic map resistance mechanism in Lactococcus lactis and its
of the Lactococcus lactis subsp. cremoris MG 1363 regulon. Mol Microbiol 27: 299-310.
chromosome: Comparison with that of Lactococcus 28. Sanders JW, Leenhouts KJ, Haandrikman AJ, Venema
lactis subsp. IL1403 reveals a large genome inversion. G, Kok J (1995). Stress response in Lactococcus
J Bacteriol 177: 2840-2850. lactis: Cloning, expression analysis, and mutation of
13. Leenhouts K, Bolhuis A, Venema G, Kok J (1998). the lactococcal superoxide dismutase gene. J Bacteriol
Construction of a food-grade multiple-copy integra- 177: 5254-5260.
tion system for Lactococcus lactis. Appl Microbiol 29. Sanders JW, Venema G, Kok J, Leenhouts K (1998).
Biotechnol 49: 417-423. Identification of a sodium chloride-regulated promoter
14. Leenhouts K, Buist G, Bolhuis A, Berge A ten, Kiel in Lactococcus lactis by a single copy chromosomal
J, Mierau I, Dabrowska M, Ven8ema G, Kok J (1996). fusion with a reporter gene. Mol Gen Genet 257:
A general system for generating unlabelled gene 681-685.
replacements in bacterial chromosomes. Mol Gen 30. Van der Vossen JMBM, van der Lelie D, Venema G
Genet 253: 217-224. (1987). Isolation and characterisation of Lactococcus
15. Leenhouts KJ, Kok J, Venema G (1991). Lactococcal lactis subsp. cremoris Wg2-specific promoters. Appl
plasmid pWV01 as an integration vector for lacto- Environ Microbiol 53: 2452-2457.
cocci. Appl Environ Microbiol 57: 2562-2567. 31. Youngman P (1993). Transposons and their applica-
16. Leenhouts KJ, Tolner B, Bron S, Kok J, Venema G, tions. In: Sonenshein AL, Hoch JA, Losick R (eds)
Seegers JFML (1991). Nucleotide sequence and Bacillus subtilis and other gram-positive bacteria, pp
characterisation of the broad-host-range lactococcal 585-596. Washington, DC: American Society for
plasmid pWV01. Plasmid 26: 55-66. Microbiology.
17. Leenhouts KJ, Venema G (1993). Lactococcal plasmid
vectors. In: Hardy KG (ed), Plasmids. A practical
approach, 2nd edn, pp 65-94. New York: Oxford
University Press. Address for correspondence: Dr J. Kok, Department of
18. Maguin E, Duwat P, Hege T, Ehrlich SD, Gruss A Genetics, Biomolecular Sciences and Biotechnology
(1992). New thermosensitive plasmid for gram- Institute, University of Groningen, Kerklaan 30, NL-9751
positive bacteria. J Bacteriol 174: 5633-5638. NN Haren, The Netherlands
19. Maguin E, Prevot H, Ehrlich SD, Gruss A (1996). Phone: +31503632111; Fax: +31503632348
Efficient insertional mutagenesis in lactococci and E-mail: kokj@biol.rug.nl
Methods in Cell Science 20: 51-57 (1998)
© 1998 Kluwer Academic Publishers.
Abstract. Two new shuttle-suicide plasmid vectors, forcing integration of the plasmid after introduction
pWM245 and p71NT, capable of site-specific inte- into S. pyogenes at the phage TI2 bacterial attach-
gration have been developed for gene insertion or ment site (attB) , a serine tRNA gene. This tRNA
allelic replacement of inactivated genes in strepto- gene is conserved in a number of streptococcal
cocci. These vectors contain the integrase gene species, thus these integration vectors may serve
(int) and phage attachment site (attP) from the tem- as broad host-range insertion vectors. The phage
perate bacteriophage T12 of Streptococcus pyogenes. excisionase gene (xis) is not present in these vectors,
Additionally, these plasmids contain the ermB thus, integration is highly stable and permanent.
gene specifying resistance to erythromycin in both These vectors will provide important new tools for
Escherichia coli and streptococci. The plasmid origin genetic analysis of group A and possibly related
of replication, however, is only functional in E. coli, streptococci.
int and attP as well as a selectable marker for standard conditions. Erythromycin is prepared as a
erythromycin resistance. This plasmid was found to 20 mg/ml stock solution in 95% ethanol and added
integrate into attB reproducibly and at a high fre- to the media after autoclaving at a concentration of
quency, and its potential as a cloning vector for site- 3 ~g/ml for S. pyogenes and 300 ~g/ml for E. coli.
specific integration of new genes into S. pyogenes The 0.5 M sucrose solution is prepared by dis-
was immediately apparent. Integration vectors that solving 171.2 g sucrose in distilled water to a final
use bacteriophage-derived int genes for such inte- volume of 1 liter. Sterilize by autoclaving and store
gration have been constructed for use in a number at 4°C. TE is 10 mM Tris-HCl, 1 mM EDTA, pH
of bacteria including, for example, E. coli [8], S. 8.0. TEN is 10 mM Tris-HCl, 1 mM EDTA, 50 mM
aureus [6, 15], lactococcus [7, 13, 18] and lacto- NaCl, pH 8.0. GES for lysis of S. pyogenes is
bacillus [1, 10, 11] species, mycobacterial species prepared by combining 60 g guanidium thiocyanate,
[16], and various streptomyces species [12, 19, 30]. 20 ml 0.5 M EDTA, pH 8, and 20 ml distilled water.
In this report, we describe two integration vectors Heat the mixture at 65°C until the guanidium thio-
for S. pyogenes and possibly other streptococcal cyanate is completely dissolved. Allow the solution
species. to cool to room temperature and add 5 ml of 10%
(v/v) sarkosyl. Adjust the final volume to 100 ml.
Filter the solution through a 0.45 ~m filter (Nalgene)
2. Materials and store at room temperature [25].
1. Todd-Hewitt dehydrated broth, Cat. No. 0492- Plasmid construction and DNA purification. Each
17-6.' of the integration vectors described in this paper
2. Bacto-Yeast Extract, Cat. No. 0127-01-7.' possesses unique restriction endonuclease sites for
3. Bacto-Agar, Cat. No. 0140-01.' cloning for insert DNA. A standard reference (or
4. Bacto-Tryptone, Cat. No. 0123-01.' the manufacturer's instructions) should be consulted
5. Normal, pooled, sterile-filtered horse serum, Cat. for methods using these sites for the cloning of the
No. S2-0109. 2 DNA of interest [27]. In any electroporation protocol,
6. Erythromycin, Cat. No. E-6376. 3 the purity of the transforming DNA will effect the
7. Lysozyme, Cat. No. L-2879. 3 number of transformants obtained. We routinely
8. N-Lauroyl sarcosine (sarkosyl), Cat. No. L- perform electrotransformations with plasmid DNA
5125. 3 prepared with a commercially available kit and fol-
9. Sodium chloride, Cat. No. BP358-1O. 3 lowing the manufacturer's protocol (QIAprep-spin,
10. Sucrose, Cat. No. S-9378. 3 Qiagen, Chatsworth, CA). Standard plasmid prepa-
11. Tris base. 3 ration protocols that employ a cesium chloride
12. Electroporation cuvettes plus apparatus, Cat. No. density gradient for purification may also be used to
610. 4 maximize electrotransformation efficiencies [27].
13. Genius DNA labeling kit, Cat. No. 1175033. 5 No matter what method of plasmid isolation is used,
14. Ethylenediaminetetraccetate, dis odium salt the transforming DNA must be in the supercoiled,
(EDTA), Cat. No. BPI20-500. 6 circular form. The energy used by phage integrases
15. Isopropanol, Cat. No. A464-4. 6 for integration is derived from the supercoiling of the
16. Isoamyl alcohol, Cat. No. A393-500. 6 plasmid (or phage genome in natural infections) [33].
17. Bio-Rad E. coli Pulser™, Cat. No. 165-2102.7 Linear DNA will not be a substrate for site-specific
integration using this system, and methods of plasmid
purification that give preparations with significant
3. Procedures amounts of linear or nicked DNA will result in
decreased electrotransformation efficiencies.
Media and reagents. Strains of S. pyogenes are grown
in Todd-Hewitt broth supplemented with yeast extract Electrotransformation. The described protocol for
(THY). Per liter, dissolve 30 g Todd-Hewitt dehy- electrotransformation has been optimized for S.
drated broth (Difco Laboratories) and 2 g Bacto yeast pyogenes NZ131, a highly electrocompetent type
extract in distilled or deionized water (THY broth). M49 laboratory strain [29]. For other strains of S.
THY-HS broth is THY broth supplemented with pyogenes, modifications may be necessary, especially
50 mIll of heat-inactivated horse serum (65°C for in the incubation time to achieve the early loga-
30 min). Strains of E. coli are propagated in B broth rithmic phase of growth. We employ a 0.5 M sucrose
(per liter: combine 109 Bacto-tryptone, 5 g Bacto- solution for electrotransformation; other investigators
yeast extract, and 5 g NaCl, and adjust the pH to 7 have used alternative solutions resulting in essentially
by the addition of approximately 2.2 ml of 1 M the same transformation efficiencies [20]. Whether
NaOH [17]). For solid media, 15 g Bacto-agar are a given strain will transform well with a given buffer
added per liter to both media for streptococci and E. may have to be determined experimentally. The
coli. All media are sterilized by autoclaving using competence for electrotransformation of some strains
53
is also increased by the addition of cell wall weak- of mutanolysin, and place the mixture at 37°C for
ening agents such as glycine or threonine [5, 9]; addi- 30 min. Lyse the cells by adding 0.5 ml GES. Mix
tionally, the amount of hyaluronic acid in the capsule by repeated inversion of the tube and cool the
of group A streptococci can influence electrocom- mixture on ice for 5 min. Lysis should be complete
petence [29]. A complete review of these issues has at this point. Add 0.25 ml cold 7.5 M ammonium
been presented previously by our laboratory [20]. acetate, mix by repeated inversion, and incubate on
Inoculate 2-5 ml of THY broth with an isolated ice for 10 min. Add 0.5 ml chloroform:isoamyl
colony of NZ131 and incubate overnight at 37°C. alcohol (24: 1) and mix thoroughly until a complete
Add 0.4 ml of this overnight culture to 20 ml of pre- emulsion is achieved. Separate the phases by cen-
warmed THY-HS broth and grow the cells at 37°C trifugation (14,000 Xg for 10 min). A thin, white
for 3 hours to an A600 between 0.2 and 0.25 (early interface should separate the phases. Collect the
logarithmic phase). Harvest the cells by centrifuga- upper phase and estimate its volume. Add 0.6
tion at 7600 xg for 5 min at 4 °C (8000 rpm in a volumes of isopropanol and mix by inversion for at
Sorval SS34 roto). Resuspend the cell pellet in 1 ml least one minute. Typically, a fibrous mass of DNA
of ice-cold, sterile 0.5 M sucrose prepared in distilled is visible at this point. Collect the DNA by centrifu-
water by vortex mixing, and transfer to a 1.5 ml snap- gation (7,000 xg for 1 minute). If the DNA is not a
capped microcentrifuge tube. Harvest the cells by visible mass after the addition of the isopropanol,
centrifugation for 15 seconds at 14,000 xg (full speed then centrifugation should be performed at 14,000 Xg
in most standard desktop microcentrifuges). Remove for 5 min. Wash the pellet five times with 0.5 ml
the supernatant by vacuum aspiration and wash the 70% ethanol using 7,000 Xg for 1 minute. Completely
cells three more times using the same conditions. remove any traces of the supernatant by vacuum aspi-
Minimize the time the cells are not kept on ice. ration and dry the DNA pellet in vacuo for 5 min.
Finally, resuspend the cells in 100 J.lI of cold 0.5 M Dissolve the DNA in 100 J.lI sterile, distilled water
sucrose. Add the integration plasmid (1 J.lg) to the or TE.
cells in no more than a 10 J.lI volume. Transfer the The DNA probe for attB, cloned on pWM130
mixture to a BTX Electroporation Cuvette Plus™ or [23], was prepared using the Boehringer Mannheim
equivalent cuvette with a 1 mm gap. Using a Bio- Genius ™ digoxigenin-dUPT (DIG-dUTP) labeling
Rad E. coli Pulser apparatus, expose the cells to a kit following the manufacturer's protocols. Agarose
single electrical pulse of 1.8 kV. In this unit, the gel electrophoresis, Southern DNA transfer to nylon
capacitance is pre-set to 25 J.lF, and the pulse con- membrane, and hybridization to DIG-dUTP DNA
troller is pre-set to 200 11 resistance. A safety inter- probes are conducted according to standard protocols
locking system is in place on this Bio-Rad unit to [27].
prevent accidental operator exposure to high voltage;
no attempt should be made to defeat this system.
Alternatively, a cuvette with 2 mm electrode gap may 4. Results and discussion
be used, and the voltage should be set to 2.5 kY. After
the electric pulse, immediately place the cells on ice Site-specific integration vectors. The construction of
and cool for 5 min. Add 0.9 ml of THY-HS to the pWM139, containing the integrase and phage attach-
cells and incubate for 2 hours at 37°C. Plate 0.1 ml ment site of bacteriophage TI2, has been described
aliquots on THY agar plates supplemented with the previously (Figure 1A). The essential features of this
appropriate antibiotic. Colonies are usually visible vector and its derivatives are a selectable antibiotic
after 24 hours of incubation, and typically, electro- resistance marker that is expressed both in E. coli and
transformation with 1 Ilg of integration vector streptococci, an origin of replication that is functional
plasmid DNA will result in > 103 transformants. in E. coli but not in streptococci, and the presence
of the TI2 int and aUP. Importantly, the phage xis is
Isolation of chromosomal DNA and screening for not present on this plasmid so that integration is not
plasmid integration. Streptococcal chromosomal reversible. The region from upstream of the Cia I
DNA is prepared by the method of Pitcher et al. [25]. restriction endonuclease site contains the lacZ
Inoculate 20 ml of THY broth with an isolated colony promoter; passage of the plasmid in E. coli results
of the S. pyogenes strain of interest and incubate in the isolation of clones containing the plasmid with
overnight at 37°C. Pellet the cells by centrifugation spontaneous deletions of this region. The promoter
(1000 xg for 15 min) and remove the supernatant by and the coding region for int as well as aUP are unaf-
vacuum aspiration. Resuspend the cell pellet in 1 ml fected by this deletion. We hypothesize that the lacZ
TEN, transfer the suspension to a 1.5 ml microcen- promoter causes overexpression of the phage gene
trifuge tube and again pellet the cells by centrifuga- product in the E. coli, and that this overproduction
tion (14,000 Xg or full speed in the microcentrifuge is toxic to the bacterium. The spontaneous derivative
for 1 minute). Suspend the pellet in 100 J.ll of freshly of pWM139, pWM245 (Figure lB), is stable in E.
prepared lyzozyme (50 mg/ml in TE) plus 50 units coli and able to integrate efficiently into the S.
pyogenes genome at TI2 attB. This plasmid is
54
A. M13 -40
T7
c. Apal
Bsp20
Aatll
Xbal
(Aval)
Xhol
EcoRI
(Kpnl)
(A val)
Smal
Clal
(Hindlll)
BamHI
(Sstl)
BspEI
Mlul
Nsil
B.
D. Sp6
M13 Rev
Sacl
Figure 1. Integration vectors derived from bacteriophage TI2. Shown are four plasmids containing the genetic elements
derived from bacteriophage TI2 that are responsible for mediating site-specific recombination with the S. pyogenes
chromosome. These plasmids contain the origin of replication from pUCI8 and thus can be maintained in E. coli. However,
this origin does not function in streptococci, forcing the plasmids to integrate into the bacterial attachment site (attB), a
serine tRNA that exists as a single copy in the bacterial chromosome. The antibiotic resistance gene is functional in both
E. coli and streptococci. The construction of plasmid pWM139 (4378 bp, panel A) and its capacity to integrate into the
bacterial chromosome have been previously reported [23]. Upon passage in E. coli DH5aF', pWM139 spontaneously
deletes about 100 bases of the plasmid backbone region upstream of the integrase gene to give pWM245 (4270 bp,
panel B). This deletion results in the loss of the E. coli lacZ promoter as well as several unique restriction endonuclease
cleavage sites. In order to make a more versatile integrative cloning vector, p7lNT was constructed (4374 bp, panel C).
This plasmid has a multiple cloning site (MCS) in a functional copy of the E. coli lacZ gene, allowing blue-white screening
of recombinants as well as the phage TI2 integrase and attachment site and the ermB gene to select for erythromycin
resistance. The MCS also contains the sequences complementary to the standard commercially available M13-40, T7,
Sp6, and M13 reverse sequencing primers. pWM130 (3257 bp, panel D) is derived from the PCR product cloning vector,
pT7Blue, and contains the serine tRNA gene from the S. pyogenes chromosome that serves as attB. This plasmid is used
to make probles for DNA hybridization to attB and is not for introduction into streptococci since it contains a functional
~-lactamase gene, encoding ampicillin resistance.
somewhat limited in its usefulness because it contains pyogenes that lacks a functional copy of the group
relatively few unique cloning sites and lacks blue- A streptokinase gene (ska) [A. Nordstrand, manu-
white screening of cloned inserts. An improved script in preparation], Finally, plasmid pWM130,
vector with these characteristics is p7INT (Figure containing the serine tRNA that serves as attB and
IC), This plasmid has a multiple cloning site present its surrounding region from the S. pyogenes chro-
in the promoter of lacZ containing a number of mosome, is useful as a probe for determining whether
unique restriction endonuclease cleavage sites that integration has occurred. Insertion of either a phage
interrupt lacZ expression when used for cloning DNA genome or an integration vector into the phage Tl2
inserts. Additionally, there is no instability of this attB of S. pyogenes is detected by hybridization of
plasmid in our hands as was the case with pWM139. HindUI digested chromosomal DNA. attB is con-
Both of these vectors use erythromycin resistance tained on one HindUI fragment when it is unoccu-
as the selectable marker. These vectors and their pied by a phage or integrative plasmid (Figure 2, lane
derivatives will be useful tools for allelic replace- 2). However, integration of a plasmid into attB alters
ment. One such construct has been used to introduce this pattern and creates two hybridizing HindIII
a functional and expressed streptokinase gene from fragments generated by the plasmid-associated
Streptococcus equisimilis (skc) into a strain of S. restriction endonuclease sites (lane 3).
55
1 2
mycobacterial phage integration vectors [16]. We
have detected the presence of this gene by both
polymerase chain reaction and DNA hybridization in
Streptococcus agalactiae, S. mutans, S. salivarius, S.
downei, S. gordonii, S. macacae, S. sanguis, S. oratis,
S. mitis, S. equisimilis, S. pneumoniae, Lactococcus
lactis, and Enterococcus faecatis [22]. The integra-
tion vectors should theoretically be functional in any
of these species that contain the appropriate target
sequence. Several factors will determine whether
they may be used successfully in practice, including
competence for transformation or electrotransforma-
tion, presence of required accessory factors, and
sequence fidelity of the attB core region. Published
2.2 protocols for introducing plasmid DNA into a
number of these bacteria have been reported in the
2.0 literature [20], but the actual frequency of a partic-
ular isolate of interest may deviate considerably from
the published values, and it may well be that some
isolates, especially natural isolates, will prove to be
difficult or impossible to transform. Additionally,
factors influencing integration may determine the
success or failure in non-group A streptococcal
species. As discussed above, the host-encoded inte-
gration host factor (lHF), a DNA binding protein, is
required for integration for phage-derived systems.
While a requirement for IHF has not been rigorously
demonstrated for bacteriophage TI2 integration, con-
sensus sequences for IHG binding homologous to the
0.6 sequences from E. coli are present in the appropriate
positions in the TI2 attP region [23]. Variations in
IHF and its associated target sequence in non-group
Figure 2. Integration ofpWM245 into the phage TI2 attB A species could influence the efficiency of integra-
site. Insertion of pWM245 into attB of S. pyogenes NZ131 tion of the phage T12-derived vectors. Finally,
was detected by hybridization of HindIII digested chro- single base changes in the core region of the attB
mosomal to a probe specific for the serine tRNA that homologues in the non-group A streptococci could
serves as the phage TI2 attachment site. The phage TI2 decrease the efficiency of integration significantly
attB is contained on one HindIII fragment of approxi- [33]. We have sequenced the attB region from S.
mately 5,000 bp when it is unoccupied by either a phage mutans, S. agalactiae, S. downei, and E. faecalis, and
or integrative plasmid (lane 2). However, integration of
have found it be identical to the sequence from S.
the plasmid into attB alters this pattern and creates two
hybridizing HindIII fragments of approximately 5,100 bp pyogenes in all cases [W. M. McShan, unpublished
and 1,300 bp generated by the plasmid-associated restric- results]. Therefore, one would anticipate that these
tion endonuclease sites (lane 3). HindUI digested bacte- organisms would be a suitable hosts for these vectors.
riophage Lambda DNA was used as a molecular weight No sequence information is yet available for any of
standard (lane 1). The size of the Lambda fragments are the other species. In one preliminary experiment,
labeled in kilobases. transformants were obtained with pWM245 in a
strain of S. macacae and in the naturally competent
Because of the instability in pWM139, this S. gordonii Challis strain. Both of these species
plasmid will not be available for release. The contain a region in their genomes that hybridizes with
two other vectors, pWM245 and p7INT, as well as the probe for the phage TI2 attB, and one anticipates
pWM130 will be made available to the scientific that these regions will also have DNA sequences that
community upon request. are identical to the one from S. pyogenes.
Only one copy of the attachment site for TI2
Applications and possible limitations. The serine exists in the S. pyogenes genome [23]. Alteration of
tRNA that serves as the bacterial attachment site the 'core' sequence of a phage attachment site by
appears to be conserved among a number of strepto- only one nucleotide results in a 100- to 1000-fold
coccal and related species, and thus it may be reduction in integration frequency [14,28,33]. Given
that these phage-derived vectors may be useful as that electroporation is a relatively inefficient process
wide host-range vectors as has been done with and that the plasmid cannot replicate independently
56
in streptococci suggests that the chances for even 5. Boehringer Mannheim Biochemicals, P.O. Box 50414,
having two copies of the plasmid present in a cell Indianapolis, IN 46250, USA
before integration is highly unlikely. Thus, (1) inte- 6. Fisher Scientific, 711 Forbes Ave., Pittsburgh, P A
gration at the target site is highly favored, and (2) 15219-4785, USA
7. Bio-Rad Laboratories, 2000 Alfred Nobel Drive,
tandem duplication by the plasmid at the attB site is
Hercules, CA 94547, USA
unlikely. However, it is crucial to determine whether
a resident phage is occupying the TI2 attB in a given
strain of S. pyogenes before proceeding with any
References
experiments. It should not be assumed that because
a strain does not contain the gene for the
1. Auvray F, Coddeville M, Ritzenthaler P, Dupont L
erythrogenic toxin (speA) that no phage occupies (1997). Plasmid integration in a wide range of bacteria
attB. We have recently reported that it is possible mediated by the integrase of Lactobacillus delbrueckii
to isolate phages that share int and attP with phage bacteriophage mv4. J Bacteriol 179: 1837-1845.
TI2, but do not have speA [21]. Tandem insertions 2. Ball CA, Johnson RC (1991). Efficient excision of
of the plasmid along an integrated phage are possible phage A from the Escherichia coli chromosome
because the sequence of attB is not altered by requires the Fis protein. J Bacteriol 173: 4027-
integration, and such events may give unexpected 4031.
patterns upon DNA hybridization. Further, integra- 3. Barletta RG, Michalek SM, Curtiss III R (1988).
tion of plasmid DNA in tandem with a resident phage Analysis of the virulence of Streptococcus mutans
serotype c gtfA mutants in the rat model system. Infect
may decrease the stability of integration because
Immun 56: 322-330.
of the presence of the phage excisionase gene.
4. Campbell AM (1992). Chromosomal insertion sites
Homologous recombination between the flanking for phages and plasmids. J Bacteriol174: 7495-7499.
regions of an inactivated chromosomal gene and the 5. Caparon MG, Scott JR (1991). Genetic manipulation
plasmid borne replacement allele could theoretically of pathogenic streptococci. Meth Enzymol 204: 556-
occur before integration; however, such an event 586.
would only occur at very low frequency. Analysis of 6. Carroll D, Kehoe MA, Cavanagh D, Coleman DC
putative transformants for integration at the bacte- (1995). Novel organization of the site-specific inte-
rial attachment site easily confirms the desired event. gration and excision recombination functions of the
The chromosomal DNA sequence containing attB is Straphylococcus aureus serotype F virulence-con-
contained on one HindUI fragment when it is unoc- verting phages <1>13 and <1>42. Mol Microbiol 16:
877-893.
cupied by either a phage or integrative plasmid
7. Christiansen B, Johnsen MG, Stenby E, Vogensen FK,
(Figure 2, lane 2). However, integration of pWM245
Hammer K (1994). Characterization of the lactococcal
or the other integration vectors into attB alters this temperate phage TP901-1 and its site-specific inte-
pattern and creates two HindUI fragments that gration. J Bacteriol 176: 1069-1076.
hybridized with the attB probe because of the dupli- 8. Diederich L, Rasmussen LJ, Messer W (1992). New
cation in aftp (Figure 2, lane 3). This alteration in cloning vectors for integration in the Lamba attach-
restriction pattern is diagnostic for integration and ment site attB of the Escherichia coli chromosome.
should be used to confirm may putative transfor- Plasmid 28: 14-24.
mants. Further, if eliminating homologous recombi- 9. Dunny GM, Lee LN, LeBlanc DJ (1991). Improved
nation is a particular concern for a given experiment, electroporation and cloning vector system for Gram-
a recA strain of S. pyogenes could be employed [31]. positive bacteria. Appl Environ Micobiol 57: 1194-
1201.
10. Dupont L, Boizet-Bonhoure B, Coddeville M,
Auvray F, Ritzenthaler P (1995). Characterization of
Acknowledgments genetic elements required for site-specific integration
of Lactobacillus delbrueckii subsp. bulgaricus bacte-
This work was supported by Public Health Service riophage mv4 and construction of an integration-
grants AI19304 and AI38406 from the National proficient vector form Lactobacillus plantarum. J
Institutes of Health. Bacteriol 177: 586-595.
11. Fremaux C, Antoni GLD, Raya RR, Klaenhammer
TR (1993). Genetic organization and sequence of the
region encoding integrative functions from Lacto-
Notes on suppliers bacillus gasseri temperate bacteriophage <1>adh. Gene
126: 61-66.
1. Difco Laboratories, Detroit, MI 48232-7058, USA 12. Gabriel K, Schmid H, Schmidt U, Rausch H (1995).
2. Lampire Biological Laboratories, P.O. Box 270, The actinophage RP3 DNa integrates site-specifically
Pipersville, PA 18947, USA into the putative tRNA Arg (AGC) gene of Streptomyces
3. Sigma Chemical, P.O. Box 14508, St Louis, MO rimosus. Nucleic Acids Res 23: 58-63.
63178, USA 13. van der Guchte M, Daly C, Fitzgerald GF, Arendt
4. BTX, 11199-A Sorrento Valley Rd., San Diego, CA EK (1994). Identification of int and attP on the
92121-1334, USA genome of lactococcal bacteriophage Tuc2009 and
57
their use for site-specific plasmid integration in the 24. Perry D, Nilsen LJ, Kuramitsu HK (1985). Mapping
chromosome of Tuc2009-resistant Lactococcus lactis of a cloned glucosyltransferase gene in Streptococcus
MG1363. Appl Environ Microbiol 60: 2324-2329. mutans. Infect Immun 50: 130-135.
14. Kim S, Landy A (1992). Lambda Int protein bridges 25. Pitcher DG, Saunders NA, Owen RJ (1989). Rapid
between higher order complexes at two distant chro- extraction of bacterial genomic DNA with guanidium
mosomalloci attL and attR. Science 256: 198-203. thiocyanate. Lett Appl Micro 8: 151-156.
15. Lee CY, Buranen SL, Ye ZH (1991). Construction of 26. Ptashne M (1992). A genetic switch: phage Lambda
single-copy integration vectors for Straphylococcus and higher organisms, 2nd edn. Cambridge, MA:
aureus. Gene 103: 101-105. Blackwell.
16. Lee MH, Pascopella L, Jacobs Jr WR, Hatfull GF 27. Sambrook J, Fritsch EF, Maniatis T (1989). Molecular
(1991). Site-specific integration of mycobacteriophage cloning: a laboratory manual, 2nd edn. Cold Spring
L5: integration-proficient vectors for Mycobacterium Harbor, NY: Cold Spring Harbor Laboratory Press.
smegma tis, Mycobacterium tuberculosis, and bacille 28. Shimada K, Weisberg RA, Gottesman ME (1972).
Calmette-Guerin. Proc Natl Acad Sci USA 88: Prophage lambda at unusual chromosomal locations.
3111-3115. I. Location of the secondary attachment sites and the
17. Lennox ES (1955). Transdunction of linked genetic properties of the lysogens. J Mol BioI 63: 483-503.
characters of the host by bacteriophage Pl. Virology 29. SimonD, Ferretti II (1991). Electrotransformation of
1: 190-206. Streptococcus pyogenes with plasmid and linear DNA.
18. Lillehaug D, Nes IF, Birkeland NK (1997). A highly FEMS Microbiol Lett 82: 219-224.
efficient and stable system for site-specific integration 30. Sladkova lA, Orekhov AV (1994). Actinomycete
of genes and plasmids into the phage phiLC3 attach- plasmid and integrative vectors based on DNA of
ment site (attB) of the Lactococcus lactis chromo- the temperate Phi C31 actinophage, determining
some. Gene 188: 129-136. limitation of lytic development of phage Phi C31, not
19. Matsuura M, Noguchi T, Yamaguchi D, Aida T, dependent on repressor. Antibiot Khimioter 39: 3-11.
Asayama M, Takahashi H, Shirai M (1996). The sre 31. Tao L, Hollingshead SK, Suvorov AN, Ferretti II,
gene (ORF469) encodes a site-specific recombinase McShan WM (1995). Construction of a Streptococcus
responsible for integration of the R4 phage genome. pyogenes recA mutant via insertional inactivation, and
J Bacteriol 178: 3374-3376. cloning and sequencing of the complete recA gene.
20. McLaughlin RE, Ferretti II (1995). Electrotrans- Gene 162: 59-62.
formation of streptococci. In: Nickoloff JA (ed), 32. Tao L, LeBlanc DJ, Ferretti II (1992). Novel strepto-
Electroporation protocols for microorganisms, vol. 47, coccal-integration shuttle vectors for gene cloning and
pp 185-193. Totowa, NJ: Humana Press. inactivation. Gene 120: 105-110.
21. McShan WM, Ferretti II (1997). Genetic diversity in 33. Weisberg RA, Landy A (1983). Site-specific recom-
temperate bacteriophages of Streptococcus pyogenes: bination in phage lambda. In: Roger JWR, Hendrix
Identification of a second attachment site for phages W, Stahl W, Weisberg RA (eds), Lambda II, pp
carrying the erythrogenic toxin A gene. J Bacteriol 211-250. Cold Spring Harbor Press, NY: Cold Spring
179: 6509-6511. Harbor.
22. McShan WM, Ferretti II (1997). Genetic studies
of erythrogenic toxin carrying temperate bacterio-
phages of Streptococcus pyogenes. Adv Exp Med 418: Address for correspondence: Dr Joseph J. Ferretti,
971-974. Department of Microbiology and Immunology, The
23. McShan WM, Tang YF, Ferretti II (1997). University of Oklahoma Health & Science Center,
Bacteriophage T12 of Streptococcus pyogenes inte- Oklahoma City, OK 73190, USA
grates into the gene encoding a serine tRNA. Mol Phone: (405) 271 2133; Fax: (405) 271 0000
Micobiol 23: 719-728. E-mail: joe-ferretti@uokhsc.edu
Methods in Cell Science 20: 59-64 (1998).
© 1998 Kluwer Academic Publishers.
Lin Tao*
Department of Oral Biology, School of Dentistry, University of Missouri-Kansas City, Kansas City, Missouri, USA
(*Present address: Department of Oral Biology, College of Dentistry, University of Illinois, Chicago, Illinois, USA)
Abstract. Five streptococcal integration vectors replication. If these plasmids carry a fragment of
containing different antibiotic resistance-encoding streptococcal DNA they can specifically integrate
genes capable of expressing in both Streptococcus sp. into the streptococcal chromosome via Campbell-
and Escherichia coli are introduced. These plasmids like, single crossover homologous recombination.
can replicate in E. coli, but not in streptococci, Methods are described to use these vectors for gene
because the plasmids lack a streptococcal origin of inactivation and cloning in streptococci.
1. Introduction 2. Materials
2 ml ice-cold TSS medium. Distribute the cells in a glass test tube. Incubate the culture at 37 DC
into multiple microcentrifuge tubes. Add 1-5 for 16 h. Transfer the culture into 100 ml
III plasmid DNA to 100 III competent E. coli prewarmed THB broth. Incubate the culture at
cells, and incubate at 4 DC for 30 min. Add 0.9 37 DC for 4 to 5 h to reach mid-exponential phase.
TSS or LB broth, and transfer to a glass test Add 1,000 U penicillin G to the culture, and
tube. Incubate the culture at 37 DC for 1 h with continue to incubate for 2 h. Harvest the cells by
vigorous shaking. Plate the cells on LB centrifugation (7,000 Xg, 10 min), and wash the
selective agar plates. cells with deionized water. Suspend the cells in
2. Transformation of streptococcal cells 5 ml lysis buffer supplemented with 5 mg
a. Natural transformation lysozyme and 50 Ilg RNase, and incubate at
Inoculate a single colony of a streptococcal 37 DC for 1 h. Add 0.5 ml10% SDS and incubate
strain, such as Streptococcus gordonii at 55 DC for 20 min. Add 0.6 ml 3 M sodium
Challis, into 2 ml THBS medium in a glass acetate, 3 ml phenol and 2 ml chloroform to the
test tube. Incubate the culture at 37 DC for viscous lysate. Gently mix the lysate, and cen-
16 h. Transfer 0.1 ml of the culture into trifuge at 15,000 Xg for 10 min. Transfer the upper
4 ml prewarmed THBS medium. Incubate phase to a new tube and repeat the procedure with
the diluted culture at 37 DC for 2 h to 5 ml chloroform. Transfer the upper phase again
achieve competence. In the case of to a new tube and add 10 ml 95% ice-cold
Streptococcus mutans or Streptococcus ethanol. Centrifuge at 15,000 Xg for 10 min. Wash
milleri, the incubation time for achieving the pellet with 10 ml 70% ethanol. Dry the pellet
competence is 3 to 3.5 h [13-15, 18]. After for 30 min at room temperature, and dissolve it in
adding 1-5 III plasmid DNA to the com- 200 III TE buffer.
petent cells, incubate the culture at 37 DC D. Inactivate streptococcal genes with an integration
for 1 h. Spread the cells on the THB selec- plasmid
tive agar plates. 1. Inactivation by insertion duplication (Figure 2)
b. Electrotransformation. a. Digest a cloned streptococcal gene with the
Inoculate a single colony of a streptococcal restriction enzyme Sau3AI, and purify the
strain that cannot be transformed naturally, internal gene fragment in the agarose gel
such as Streptococcus pyogenes NZ131 with the GeneClean kit after performing
[16], into 2 ml THBS medium in a glass electrophoresis.
test tube. Add glycine to the broth at dif- b. Digest the streptococcal integration vector
ferent concentrations (0.3 to 1.5%) if other pVA891 with BamHI that produces the
streptococcal species are used [6]. Incubate same cohesive ends as Sau3AI. Remove
the culture at 37 DC for 16 h. Transfer 1 ml the enzyme and buffering salt by the
culture into 40 ml of prewarmed THBS GeneClean kit.
medium in a glass bottle and continue to c. Ligate the internal gene fragment and the
incubate for 3 h. Harvest the cells by cen- plasmid with T4 ligase.
trifugation (7,000 Xg, 5 min, 4 DC) and d. Transform the ligation mixture into E. coli
resuspend the cells in 1 ml ice-cold sterile JM 109, and select for Cm resistant trans-
0.5 M sucrose solution. Transfer to a 1.5- formants.
ml microcentrifuge tube, washing twice by
resuspension in 1 ml 0.5 M sucrose, and
pelleting for 30 seconds in a microcen- Sau3AI Sau3AI
trifuge. Resuspend the cells in 200 III ,zAzmzJ"'Q4
1
>
0.5 M sucrose, and distribute into 5 micro- X gene Restriction enzyme
centrifuge tubes with 40 III each. After digestion
mixing with 1 or 2 III DNA solution, I'ZZZZZL.I
transfer the cell suspension to a chilled
Ligation
Gene Pulser cuvette (4 millimeter electrode
gap) (Bio-Rad), and expose to one electric
pulse with the Bio-Rad Gene Pulser (peak
voltage, 2.5 kV; capacitance, 25 F; pulse
C"\BamHI
x+ XTransfonnation
,
controller, 200 Q). Immediately dilute the
culture into 1 ml THBS and incubate for
~ wt strain 1
pVA891
2 h at 37 DC. Spread the cells on the THB ~ EmR cmR
selective agar plates. Inactivation of X gene
C. Isolating genomic DNA from streptococci by insertion duplication
Inoculate a single colony of a streptococcal strain,
such as S. gordonii Challis, into 2 ml THB broth Figure 2. Gene inactivation by insertion duplication.
62
e. Screen for a recombinant plasmid that mini prep with the Qiagen Kit and by
carries the correct insert among the trans- restriction enzyme analysis.
formants by performing mini prep with the g. Linearize the recombinant plasmid with
Qiagen kit, restriction enzyme digestion, Scal that cuts a unique site in the pUC18
and agarose gel electrophoresis. ApR gene but does not cut pSFI51.
f. Transform the recombinant plasmid into the h. Transform the streptococcal strain with the
streptococcal strain either by natural trans- linear DNA in which the integration vector
formation or electroporation. is inside the cloned streptococcal gene.
g. Incubate the transformed culture at 37 DC i. Incubate the transformed culture at 37 DC
in a candle jar for 24 to 48 h on the THB in a candle jar for 24 to 48 h on the THB
agar plates containing Em (20 Ilg/ml) to agar plates containing Km (350 Ilg/ml) to
select for transformants. select for transformants.
2. Inactivation by allelic exchange (Figure 3) 3. Confirm the plasmid insertion
a. Digest the cloned streptococcal gene with a. Southern hybridization
one or two restriction enzymes that cut Integration of a plasmid into a bacterial
inside the gene but not the cloning vector chromosome can shift the electrophoretic
pUCI8. position of the target gene. This can be
b. Digest the streptococcal integration vector detected by Southern hybridization with the
pSF151 with the same restnctIOn labeled integration vector and the cloned
enzyme(s) that cuts a unique site in the streptococcal gene [9, 20].
vector. b. Phenotypic analysis
c. Purify the two digested DNA fragments Inactivation of a functional gene may cause
from agarose gel with the GeneClean kit, a physiological, biochemical and/or mor-
and ligate them with T4 ligase. phological change in the mutant. This may
d. Transform the ligation mixture into E. coli be detectable by phenotypic analysis. For
JMI09. example, the S. mutans dexA mutant gen-
e. Plate the transformed cells onto LB agar erated by insertion of pVA891 showed an
plates containing both Ap (60 Ilg/ml) and altered colonial morphology on sucrose agar
Km (50 Ilg/ml) to select for transformants and diminished dextranase activity [5].
that carry the recombinant plasmid. E. Clone the inactivated gene (Figure 4)
f. Screen for the recombinant plasmid in 1. Identify a pVA891 insertion mutant (Figure
which the integration vector has inserted 2) that displays an altered expression of a
into the cloned streptococcal gene by specific gene (e.g., X gene).
2. Isolate the chromosomal DNA from this
Ooned streptococcal X gene ~ mutant.
Ap"~ ..Lin! ~PSF151 3. Digest the DNA with HindIII that cuts a
unique site on the left of the inserted plasmid.
1
the cloning strategy described above (Figure 4), a
small gene may be readily cloned in one step.
X Mutant Digest with HindUI; However, if a gene is large, one cloning step may not
ligate and transform obtain the complete gene. A second round of chro-
mosomal walking may be required with a different
~. E. coli. restriction enzyme, or the cloned gene fragment can
on \1
\Cm Em\ be used as a probe to obtain the complete gene via
chromosomal walking [20].
Confirmation of the integration of these vectors
x+ X Transformation into the chromosome of a streptococcal strain can be
accomplished by Southern-blot hybridization [9, 20].
!
Vllllijl
Abstract. Competence in genetic transformation is ated by the use of synthetic competence stimulating
well known in oral streptococci as well as in the peptides. These peptides are small, stable and inex-
pathogenic pneumococcus. But the application of this pensive molecules inducing competence under a
genetic phenomenon as a tool to study the biology wide variety of conditions in strains belonging to the
and the virulence of these organisms has been same pherotype. Their use circumvents many of the
restricted to a limited number of strains, both by the limitations to the expression of transformability in
stringency of the conditions required for competence oral streptococci and the pneumococcus, and expands
development, and by the fact that wild types or the opportunities for application of tools of molec-
encapsulated pneumococci transform poorly, or not ular genetics to many strains of the species without
at all. Many of these limitations have been allevi- prior genetic manipulation.
Key words: Competence, Competence stimulating peptide, Oral streptococci, Pheromone, Pneumococcus,
Transformation
1. Introduction 2. Materials
OD 55o = 0.2, and stored at -80°C after adjusting to 0.8-0.9. The competent cells can be prepared in this
10% glycerol. way for each experiment or by using frozen cells
(frozen during their competence peak and stored at
Competent cells -80°C in 10% glycerol until use).
Many strains, wild as well as collection strains, of
the species Streptococcus intermedius, Streptococcus DNA
constellatus, Streptococcus anginosus, Streptococcus Both chromosomal and plasmid DNA can be used
mitis, Streptococcus oralis, Streptococcus crista, S. in transformation experiments. Most commonly,
sanguis and S. gordonii become competent sponta- antibiotic resistance is chosen as a marker that can
neously and produce CSP in quantities sufficient to be selected readily in rich media (examples include
transform without adding CSP from other sources. streptomycin [9] or novobiocin [20] resistance).
Table 1 shows collection strains in which natural Streptococci are transformable with many different
competence has been demonstrated and the sequence plasmids [1,23], but chromosomal DNA, especially
of a CSP has been found. Other strains belonging to chromosomal DNA from the same species, gives a
these species have to be induced to competence by higher transformation yield than plasmids.
adding CSP, either from culture filtrate or as chem- For use of chromosomal DNA, streptomycin resis-
ically synthesized material [9, 19]. tant mutants from oral streptococci can be obtained
Among the pneumococci rough strains have tra- by plating a sensitive strain on blood agar plates
ditionally been use in transformation experiments, (5% blood) containing 500 mg/l streptomycin. When
but with the availability of synthetic CSP, transfor- direct plating does not yield mutants, it may be
mation also has been achieved in many capsulated helpful to seed onto blood agar plates and allow 3-6
strains [28]. Various laboratories have reported dif- hours growth before transferring the agar to agar
ferent critical cell densities (2 x 106 to 108 cells/ml) bottoms (4 ml Brain Heart Infusion agar with, Difco, l
for the appearance of competence in pneumococci 600 Jlg/ml streptomycin) giving a final concentration
[see reference 4] and other oral streptococci. Com- of 100 mg/l streptomycin. DNA has to be present
petence occurs usually during exponential growth when the cells are induced to competence, but as
and the optimal cell density has to be determined for streptococci usually do not have any DNase, DNA
each strain and medium. can be added before the cells reach the critical cell
For oral streptococci competent cells can be density. A concentration of DNA during the experi-
prepared by a lO-fold subculture in prewarmed THS ments of 1 Jlg/ml is usually above saturation.
of an 18 hour THS culture (OD 625 = 0.8). Incubation
is continued for 150 to 210 min until OD 625 reaches
3. Procedures
Table 1. Collections strains of the mitis and anginosus Naturally transformable streptococci
group naturally competent and reference for sequence of
the competence stimulating peptide
Occurrence of natural competence in vitro was first
Species Collection strain Reference
described in S. pneumoniae by Dawson [8]. Later the
for CSP phenomenon was described in different species of
oral streptococci, such as S. sanguis, S. gordonii,
S. pneumoniae Rx 16 Streptococcus mutans, and Streptococcus milleri
S. mitis NCTC" 12661 19 group [6, 9, 21, 25]. Natural competence in strepto-
cocci arises at a specific stage of growth in culture,
S. oraUs NCTC 11427 19
but does not always develop spontaneously in all
S. gordonii NCTC 7865 18 strains of a competent species. By the use of culture
NCTC 3165 19
filtrates from competent cells, competence can be
NCTC 7868 18
induced even outside the period of spontaneous com-
S. sanguis NCTC 7863 13 petence. Strains induced to competence by the same
S. crista NCTC 12479 18 competence factor belong to the same transformation
S. anginosus NCTC 10713 19 group or same pherotype [11, 12, 19]. In naturally
S. intermedius NCDO 2227
b 19 competent streptococci the competence regulation
S. constellatus NCTC 11325 19 operon consists of three genes, comC encoding the
CSP precursor, comD a histidine kinase and comE
S. milleri NCTC 10708 19
which encodes its cognate response regulator [5, 16,
a NCTC, National Collection of Type Culture, 61 Colindal
17, 27, 28]. CSP is secreted and processed by a
Avenue, London NW9 5HT, England. secretion apparatus consisting of ComA and ComB
b NCDO, National Collection of Food Bacteria, Agri- [15, 17,33]. By screening strains with PCR, using
cultural and Food Research Counsil, Institute of Food primers complementary to highly conserved genes
Research, Norwich, United Kingdom. flanking the comC gene [18, 27], it has been possible
67
to characterize the CSP from oral streptococci growth. In addition, competence can be induced by
belonging to the anginosus- and the mitis-groups. synthetic CSP of the same transformation group.
The comC gene has been found in all species of the Competence can be achieved by growing the
two groups with the exception of one (Streptococcus strains in THS until OD 625 (about 3 x 106 cells/ml)
parasanguis), and the strains have been naturally (see above, competent cells). The peak of compe-
transformable [19]. tence for many strains is reached after 90 to 120 min
Table 2 shows members of streptococcal groups of incubation, but can occur later.
for which evidence for natural transformation has Before the competence peak, after 60 min growth
been demonstrated and the presence of the comC CSP (200 ng/ml) (in excess to avoid elimination by
gene has been established. When comC gene is CFI) and str-R DNA (20 Ilg/ml) are added. Treatment
present CSP can be synthesized for use in transfor- with DNase (10 Ilg/ml) when the DNA uptake has
mation experiments. taken place is optional.
Further incubation allows for integration and
Induction of competence expression of new alleles, and their selection, for
example by the use of blood agar plates with 100
Competence can be induced either by the use of mg/l streptomycin.
sterile culture filtrates or by the use of synthetic CSP. When selection for transformants by this proce-
The use of sterile culture filtrates requires knowledge dure does not succeed, the transferring of seeded
of the peak of competence in the donor as well as the blood agar plates (2-4 h growth) onto agarbottoms
recipient strain and the use of an appropriate trans- containing streptomycin (final concentration 100
formation medium. In S. sanguis and S. gordonii, mg/l) can be used to ensure the phenotypic expres-
both the donor strain of CSP as well as the induced sion of streptomycin resistance [2].
strain may produce a competence factor inactivator
(CFI) during exponential growth. The CFI's destroy Competence induction by synthetic CSP in
the activity of CSP, but may be eliminated by heating S. pneumoniae
of the culture filtrate [10]. Strains descended from the pneumococal strain Rx
In pneumococcus, competence terminates after a [29] both respond readily to CSP stimulation and
short time, independent of such external factors [7]. release large amounts of the peptide into culture
fluids during competence. A simple procedure for
Competence induction by synthetic CSP in oral transformation of these strains uses frozen cell stocks
streptococci prepared from cultures grown to OD 55o = 0.2,
The majority of laboratory strains as well as wild adjusted to 10% glycerol, and stored at -80°C. For
strains of the mitis and milleri groups are trans- competence induction with CSP, stock cells are
formable. They release CSP during exponential diluted 100-fold in the transformation medium
(CTM) adjusted to a pH below 7 by addition of HCL
to 5 mM. Growth proceeds with a doubling time of
Table 2. Naturally competent streptococci and the approximately 40 min at 37°C. When the culture
presence of the come gene" reaches approximately OD55o = 0.06, peptide is added
Species Natural to induce competence. The culture is chilled to 0 °c
Phylogenetic come
group competence gene and 100 ng/ml CSP is added. Portions of the cold
culture are distributed into tubes containing donor
Mitis group: S. pneumoniae + + DNA, and warmed to 37°C for induction (20 min),
S. mitis + +
transformation (5 min), and expression of new alleles
(30-90 min, depending on the marker). Plating on
S. oralis + +
suitable selective media completes the process.
S. gordonii + + While cells also respond well to the stimulating
S. sanguis + + peptide at lower densities, this density was chosen
S. crista + + for routine work as the highest density at which
maximal competence is induced in 100% of the cells.
Mutans group: S. mutansb + The chilling step is not required for competence
Anginosus group: S. anginosus + + induction, but it is included for convenience when
S. intermedius +
multiple transformation assay reactions are desired.
+
Less CSP is usually sufficient, but an excess does not
S. constellatus + +
reduce competence.
a In the salivarius group, pyogenic group and bovis group
neither natural transformation nor the come gene has been
found [19].
b In S. mutans natural transformation occurs in the sero-
transformable streptococci. J Gen Microbiol 31: Two allelic variants of the peptide pheromone. J
125-133. Bacteriol 178: 6087-6090.
23. Pearce BJ, Naughton AM, Masure HR (1994). Peptide 29. Ravin AW (1959). Reciprocal capsular transforma-
permeases modulate transformation in Streptococcus tions of pneumococci. J Bacteriol 77: 296-309.
pneumoniae. Mol Microbiol 12: 881-892. 30. Tomasz A, Hotchkiss PD (1964). Regulation of the
24. Perry D, Slade HD (1966). Effects of filtrates from transformability of pneumococcal cultures by macro-
transformable and non transformable streptococci on molecular products. Proc Natl Acad Sci USA 51:
the transformation of streptococci. J Bacteriol 91: 480--487.
2216-2222. 31. Tomasz A (1965). Control of the competent state in
25. Perry D, Kuramitsu HK (1981). Genetic transforma- pneumococcus by a hormone-like product: an example
tion of Streptococcus mutans. Infect Immun 31: of a new type of regulatory mechanism in bacteria.
1295-1297. Nature 208: 155-159.
26. Peterson JM, Guild WR (1968). Fractionated strands 32. Westergren G, Emilson CG (1983). Prevalence of
of bacterial DNA. III. Transformation efficiencies and transformable Streptococcus mutans in human dental
rates of phenotypic expression. J Bacteriol 96: 1991- plaque. Infect Immun 41: 1386-1388.
1999. 33. Zhou L, Hui FM, Morrison DA (1995). Competence
27. Pestova EV, Hiivarstein LS, Morrison DA (1996). for genetic transformation in Streptococcus pneu-
Regulation of transformability in Streptococcus pneu- moniae: organization of a regulatory locus with
moniae by an auto-induced peptide pheromone and homology to two lactococcin A secretion genes. Gene
two-component regulatory system. Mol Microbiol21: 153: 25-31.
853-862.
28. Pozzi G, Masala L, Iannelli F, Manganelli R, Address for correspondence: Peter Gaustad, Microbio-
Hiivarstein LS, Piccoli L, Simon D, Morrison DA logical Institute, National Hospital, 0027 Oslo, Norway
(1996). Competence for genetic transformation in Phone: 47.22869510; Fax: 47.22869490
encapulated strains of Streptococcus pneumoniae. E-mail: peter.gaustad@labmed.uio.no
Methods in Cell Science 20: 71-78 (1998)
© 1998 Kluwer Academic Publishers.
Abstract. Conjugation is a common mode of genetic coccal-Escherichia coli shuttle plasmid pTRK28.
transfer among the lactic acid bacteria. In an effort In this work we will discuss our use of pTRK28-
to exploit conjugation as a means of lactococcal mediated cointegration to identify, clone, and char-
strain development, we have characterized the acterize the pRSOI transfer regions including the
transfer regions of pRSOI by insertional mutagenesis conjugative origin of transfer.
via IS946-mediated cointegration with the lacto-
broader implications for pTRK28 cointegration-based (a total of 100 ng to 500 ng DNA), and the
mutagenesis as a tool for analysis of conjugative cell/DNA solution mixed gently and transferred
elements in LAB. to a coldO.l-cm electroporation cuvette (No. 165-
2099 6). The electroporation is conducted at a
voltage of 1.7 kV/cm and a capacitance setting of
2. Materials 100 ohms, and the cells are immediately trans-
ferred to a tube containing 0.5 ml of M17 broth
A. Bacterial strains and media. Escherichia coli containing 0.5 M sucrose and incubated for 2 to
strains were grown in Luria broth (LB) medium 3 hours at 30 DC. The cells are then plated on
(No. 0414-07-3') [15] with agitation. Selective GM17 plates containing the appropriate anti-
media for E. coli strains contained tetracycline biotic and incubated overnight at 30 DC.
(No. T-3383 2 ) at 15 )lg/ml or chloramphenicol Typically, transformants arise within 24 to 48
(No. C-0378 2 ) at 30 )lg/ml. L. lactis strains were hours. E. coli transformation was performed as
grown in GM17 (M17 medium [No. 1856-17-4'] described by Dower et al. [4] in a O.I-cm cuvette
[19] containing 0.5% glucose [No. G-6152 2], at a voltage of 1.7 kV /cm and a capacitance
at 30 DC without agitation. L. lactis NCK168 setting of 100 ohms.
(pTRK28, pRSOl) is resistant to erythromycin C. Swab matings. To rapidly screen for transfer
due to a resistance gene within pTRK28. L. lactis phenotype, an overnight culture of L. lac tis
MMS370 is resistant to streptomycin, and L. MMS370 strains containing a pTRK28::pRSOl
lactis LM2345 is resistant to spectinomycin and cointegrate plasmid (Ermr) is streaked onto
rifampicin. Selective media for L. lactis strains GM17 medium using sterile cotton swabs and
contained antibiotics at the following concentra- cross-streaked with an overnight culture of the
tions: erythromycin (No. E-6376 2 ), 10 )lg/ml; recipient strain L. lactis LM2345 (Spc" Rif').
chloramphenicol, 5 )lg/ml; rifampicin (No. R- Following overnight growth at 30 DC, the streak
3501 2 ), 50 )lg/ml; streptomycin (No. S-65012), intersection growth is collected using a sterile
600 )lg/ml; spectinomycin (No. S-9007 2 ), 300 cotton swab and swabbed onto the surface of
)lg/ml. All plating media contained 1.5% Bacto- a selective GM17 agar plate containing ery-
agar (No. 0140-02-9'). Milk-agar plates contained thromycin, spectinomycin and rifampicin. As
5% skim milk powder (No. 902887 3 ), 1% glucose controls, isolated (initial) portions of the donor
(No. G-6152 2), and 1.5% Bacto-agar. and recipient streaks are also swabbed onto
B. Reagents for DNA manipulation. Restriction selective GM17 agar. Strains possessing high
endonucleases, T4 DNA ligase (No. 15224-0174 ), frequency transfer (Trahigh ) phenotypes typically
the Klenow fragment of DNA polymerase 1 produced a confluent lawn on selection plates,
(No. 18012-021 4 ), and calf intestinal phosphatase while stains defective in transfer ability produced
(No. 18009-0127 4) were purchased from Gibco- few or no colonies. With the pRS01 element, we
BRL Life Technologies Inc., Gaithersburg, MD, have found that swab matings are a rough indi-
and used as described by the manufacturers. cator of transfer frequency, thereby allowing us
to gauge potential frequencies and reduce the
number of plates needed to determine transfer fre-
3. Procedures quencies in subsequent plate matings. This was
helpful, as the potential transfer frequency for
A. Cloning and DNA manipulation. Plasmid isola- pTRK28::pRSOl cointegrates can range from 1
tion from L. lac tis was performed as described by to <1 X 10-9 transconjugants per input donor cell.
0' Sullivan et al. [9]. Rapid plasmid isolation In general, donor strains which produced a con-
from E. coli was accomplished using the alkaline fluent lawn of growth on selection plates
lysis method [2]. Large scale plasmid isolation commonly possessed transfer frequencies of
from E. coli was performed according to the PEG 1 to 1 x 10-4 transconjugants per input donor
precipitation procedure [2]. DNA fragments were cell. In contrast, donor strains which produced
isolated from agarose gels using the GeneClean isolated colonies or no growth at all on the swab
II kit (No. 1001-4005) as indicated by the transconjugant plates typically possessed transfer
manufacturer. frequencies from 1 x 10-5 to 1 x 10-8 or < 1 x 10-9
B. Transformation. Electroporation of E. coli or L. transconjugants per input donor cell, respectively.
lactis was performed using a Bio-Rad Gene For a visual depiction of a lactococcal swab
Pulser apparatus (No. 165-2105 6). L. lactis cells mating we refer the reader to the review by Steele
grown in GM17 to an optical density of 0.5 and McKay [18].
(A = 600 nm) are harvested at 4000 x g and D. General plate mating procedure
washed twice in an equal volume of sterile cold 1. Inoculate 10 ml of GM17 containing the
distilled water. Cells are then suspended in 1/50th appropriate antibiotic (erythromycin for L.
the original volume, purified plasmid is added lactis MMS30 [pTRK28::pRSOl]) with a 2%
73
inoculation from an overnight culture of donor inserts is obtained by subtracting the junction
cells. Inoculate 10 ml of GM17 containing the pTRK28 DNA (total of 4.4 kb including the
appropriate antibiotic (spectinomycin and IS946 sequence) from the novel PvuII-PvuII
rifampicin for L. lactis LM2345) with a 2% junction fragments. Further localization of the
inoculation from an overnight culture of recip- pTRK28 insertion site can be achieved by using
ient cells. additional restriction sites internal to pTRK28
2. Incubate cultures for 6 hours at 30°C without (SphI, BamBI, HindIII, XbaI, EcoRI, Seal; Figure
shaking. 1).
3. Pellet cells by centrifugation, remove super- Mapping of pTRK28 insertion sites within
natant, and resuspend donor and recipient cells pTRK28::pRSOl cointegrates. Plasmid pRSOl is
in residual GM17 broth (typically around 150 cleaved into five distinct fragments by a PvuII,
to 300 III total volume). Sphl restriction digest (fragments A, B, C, D, E;
4. For determination of input donor and recip- Figure 2). Disruption of one of the five pRSOl
ient cell numbers, dilute a small aliquot of fragments by insertion of pTRK28 results in the
concentrated donor and recipient cultures generation of two novel PvuII-PvuII (or PvuII-
in phosphate-buffered saline (PBS) and plate Sphl) junction fragments and two Pvull-Sphl
dilutions of 10-7 to 10-9 (in triplicate) on fragments internal to the pTRK28 plasmid (see
GM17 agar containing the appropriate antibi- Figure 1). Localization of pTRK28 inserts in
otic. Incubate the plates at 30°C overnight. pRSOl is obtained by subtracting the junction of
5. For each mating pair, mix 75 III of recipient pTRK28 DNA (4.4-kb, see above) from the novel
and 75 III of donor cells (from step 3), vortex, PvuII-PvuII or Pvull-Sphl junction fragments.
and spread 100 ilion a single milk-agar plate. Verification of the pTRK28 insertions is achieved
Different ratios of donor and recipient cells by Southern hybridization analysis using pTRK28
may also be employed (typically one may use plasmid as a DNA probe (data not shown).
1:1, 1:5 and 1:10 donor to recipient ratios). Confirmation of the location of pTRK28 inserts
Spread 50 III of recipient and 50 III of donor(s) within pRSOl fragments C, D, and E (Figure 2)
separately on milk-agar plates as controls. is accomplished by additional BamHI or Xbal
Incubate mating plates overnight at 30°C. restriction digest.
6. Add 1 ml sterile PBS to milk agar plates, care- E. Subcloning of Tra region DNA into E. coli.
fully scrape cells off the plate surface, collect The plasmid pTRK28 contains the E. coli plasmid
the cells with a pipette (500 to 700 Ill), and pACYC184 origin of replication in addition to
place them in a sterile microfuge tube. tetracycline and chloramphenicol resistance
7. Dilute the mating mixture, and then plate genes. The presence of pACYC184 within
it onto selective medium (in matings with pTRK28 enables the subcloning of DNA flanking
L. lactis MMS370 [pTRK28::pRS01] and any pTRK28 cointegrate insertion, in either direc-
L. lactis LM2345 this is medium con- tion, by restriction, self-ligation and transforma-
taining erythromycin, spectinomycin and tion into E. coli. As indicated in Figure 1,
rifampicin). Dilute the mating mixture pTRK28 contains several unique sites on either
depending on the expected transfer frequency side of the pACYC184 origin of replication for
as determined from prior swab matings. use in this sub cloning strategy.
If frequencies between < 10-9 to 10-7 are To isolate Tra 1 and Tra 2 region DNA, coin-
expected, plate all cells from the mating tegrate plasmid pM2035 is cleaved with Sphl,
mixture onto six or more selection plates. self-ligated and transformed into E. coli DH5a.
If frequencies between 10-7 to 10-5 are The resulting plasmid, pE351 contains approxi-
expected, plate triplicates at dilutions of 10-2 mately 17.4 kb of pRSOl DNA spanning the
to 10-3 , if frequencies between 10-4 to 1 are Tra 1 and Tra 2 regions (Figure 2). To isolate the
expected, plate triplicates at dilutions of 10-3 Tra 3 and Tra 4 region DNA, plasmid pM1001
to 10-5 • Plate 100 III of undiluted controls is digested with Seal or Xbal, and the reaction
directly onto selection plates. products self-ligated and transformed into E. coli
8. Incubate 3 to 4 days at 30°C to ensure DH5a. The resulting plasmids, pE231 and
growth and expression of transconjugant pE560, each contain approximately 16 kb of
phenotypes. pRSOl DNA comprising the Tra3 and Tra4 region
9. Calculate transfer frequencies as number DNA, respectively (Figure 2).
of transconjugants per input donor or input
recipient. .
D. General mapping of pTRK28 insertions. Plasmid
pTRK28 possesses two internal Pvull restriction
sites each approximately 3.6-kb from the IS946
sequence (Figure 1). Localization of pTRK28
74
..... - - - - - - - -
...
EeoRl
Seal
6phl
BamHl
Hind1ll ---------
Xbal
pRSOl
I Error 16946 pRSOl
....
16946 Rep/Cop
I...... kb
~
~ pACYCIB4 ...
4.4
""
(E. col!)
Pvu11 Pvu11
/
"" /
/
" /
"
pTRK28
/
insertion
Figure 1. Mapping of pTRK28 insertions within pRSOl. Internal Pvull sites which reside 4.4-kb from the ends of
IS946 facilitated the mapping of pTRK28 insertion sites within pRSOI in pTRK28::pRSOI cointegrates. Specific restric-
tion of pTRK28::pRSOI cointegrate plasmids followed by self-ligation and transformation into E. coli DH5a allows for
the subcloning of pRSOl DNA flanking pTRK28 insertions. Rep/Cop, replication region for maintenance in lactococci;
Tetf, tetracycline resistance determinant from pACYC184; Ori, pACYC184 origin of replication; Cmf, chloramphenicol
resistance determinant from pACYC184; Ermf, erythromycin resistance determinant (for lactococci).
0 0 0
0
0 0 ~
.q-
~ q
q ~ ~ 0\ co
. . => .. ..= . . . . . . =. .
C 0\ B 0 A tot> E tot> D .q-
0
::l
4
::l
.~ ..
::l ::l
• .-
::l
> >
~
Il.
Q. >
Il. Il. >
Il. (fl
Il.
0
~ 0
C'I ~ 0
0\ ~
.q-
::I: .q-
E
tV
.....
.-
INV
tV
ID (fl
~ O'IO'I~O'IO'IO'I~~
0 0 0 0 ........ 0 0 0
WC7\~WWWOIl:loW
C>\OWUl"'~~\OO'I
•••••••••••••••••••••••••••••••••••
UlUI ........ WWUlUl
00000000
UlUIUIUI~UlO'I .... WUlUlUlUlUl~UlUlW~
.... 0 0 0 0 .... 0 0 0 0 .... 0 .... 0 0 0 0 0 0
UI UlUI .... ~ .... ~ UI
0000000 0
.... ~ .... OC>~C>O'I .... o~...:Iw~...:Iw ....... OO'l .... C>O'I\O\O .... W UI O'IW owo ... w
UI ...... UI 0'1 00'1'" ... C> 00'1 .... 0 C> 0'1 ...:I C> UI 0'1 W ~ C> \0...:1 0'1 W ... ~ ~ w ~ ........ ...
pE351'.----------------~,
pE231.'----------------~
pE560
Figure 2. Plasmid pTRK28 insertions within the 49 pTRK28::pRSOl cointegrate plasmids. Individual cointegrate plasmids
are denoted at the site of pTRK28 insertion into pRSOl by the final four numbers of the pM-series (e.g. pM1001 to
pM6078) cointegrate plasmids. Assignment of Trahigh , . , 1 - 1 x 1O-3/donor cell; Tramed , . , 1 x 10-4 - 1 x lO-{)/donor
cell; Tra1ow , . , 1 x 10-7 - 1 x 1O-9/donor cell; and Tra-, ., < 1 x 1O-9/donor cell phenotypes are based on calculated
transfer frequencies. Subclones of pRSOl generated in E. coli DH5a are indicated below the figure. Plasmids pE560 and
pE231, containing the Tra3 and Tra4 regions, respectively, were derived from cointegrate plasmid pM1001. Plasmid
pE351, containing regions Tral and Tra2, was derived from cointegrate plasmid pM2035.
functions are present, in trans. We hypothesized Seal-Stul fragment did not complement Tra3C
that complementation of the pRSOl transfer region mutants for transfer. Neither plasmid pLE14 nor
insertions with the cognate Tra region DNA would pLE16 were mobilized by pTRK28::pRSOI cointe-
reveal the pRSOl oriTby subsequent mobilization of grates, indicating the pRSOl oriT does not reside
the complementing plasmid. within Tra3C.
To complement insertions within Tra3, three dif- To complement Tral region insertions, a 7.5-kb
ferent subclones (pLE14, pLEl6, pLE33; Figure 3B) Pstl fragment encompassing the Tral region was
were generated in the E. eoli-L. laetis shuttle plasmid cloned from plasmid pE351 (Figure 2) into pLEl,
pLEl [S]. Plasmid pLE33, which possessed a 6.S-kb resulting in the plasmid pLEl2 (Figure 3A). All Tral
Seal-Stul fragment encompassing both Tra3A and insertion mutations were complemented for transfer
Tra3B, complemented all Tra3A and Tra3B insertions with pLEl2 in trans. In addition, mobilization of
for transfer. Mobilization of the complementing pLE12 vector was observed with all matings (Figure
plasmid, pLE33, was not observed in any mating, 3A). The mobilization of plasmid pLE12 indicated
suggesting that the 6.S-kb Seal-Stul fragment does the 7.5-kb Pstl fragment contained a cis-acting oriT.
not encode the pRSOl oriT. Plasmid pLEl4, which The complete sequence of the Tral and Tra2 region
was generated by subcloning a 9.5-kb Hgal fragment was generated, and subsequent sequence analysis
from pE231 and contained all of Tra3C, function- revealed six substantial open reading frames, [tre,
ally complemented Tra3C insertional mutants (Figure ltrD, ltrE, ltrEEl, ltrA, and ltrEE2, in the same ori-
3B). In contrast, pLEl6, which contained a 5.2-kb entation (Figure 3A). In order to localize the oriT,
76
Complementation of 'Mobilization by
Tra insertions pRSO)
-pLE12X ND +
-------pLE12S ND +
A
-------------pLE12K +
---------------------pLE12
+ +
".,ltrBE2 J' ItrA l~tBEl
ItrE ltrD ItrC
~ i~~ll:;::::::::::::::;::ll-r~'~ • ~ ~ 7 . 5 kb
~ ,7:. ~ ~ __ "
",
, Tral Tra2
Tra3 Tra4
p RS 0 1 LI---'-'--N!I!NlNIHIlit-=---:=--; ~~_ _-ll 48.4 kb
"
r
~, j
;:r.; Complementation of Mobilization by
I I
'-I]
) )})l\( (k)
................ • •• ••• • •
t t t t t t
- - - - - - - - - - - pLE33 +
----------pLE16
-------------pLE14 +
Figure 3. Complementation and mobilization analysis of the Tra 1 and Tra3 regions. Transfer phenotype of pTRK28 inser-
tions is depicted by the symbol: A, Trahigh ; . , Tramed , . , Tra1ow ; . , Tra-. (A): Genetic map of the Tral and Tra2 regions
of pRSOI with accompanying results of pLEl2-derivative complementation and mobilization analysis. Plasmid pLEl2
contains the complete Tral region. Plasmids pLEI2K, pLEl2S and pLEl2X are deletion derivatives of pLE12 gener-
ated through Kpnl, Seal, and Xbal cleavages, respectively, and self-ligation. aMobilization analysis of pLEl2 and pLE12-
based derivatives was accomplished using a Trahigh strain L. lac tis DM2036 [8] (L. laetis MMS370 possessing a Tra+
pTRK28::pRSOl cointegrate). (B): Genetic map of the Tra3 region with accompanying results of complementation and
mobilization analysis. Plasmids pLE33, pLEI4, and pLEl6 were generated by subcloning from plasmid pE23l as described
in the text. ND, not determined.
we analyzed a series of pLE12 deletion derivatives the fact that pRSOI is cleaved by Pvull into DNA
for mobilization in the presence of a Tra+ cointegrate fragments which are easily separated by gel elec-
plasmid. As shown in Figure 3A, all derivatives trophoresis. Since other conjugal elements may not
possessing the small OA-kb PstI-XbaI fragment, be cleaved by PvuII, other sites internal to the two
which contains the intergenic region between ItrD Pvull sites in pTRK28 could be used in conjunction
and ItrE, were mobilized by a Tra+ pTRK28::pRSOI with Pvull to map cointegrate junctions.
cointegrate. Sequence analysis of the intergenic The success of this method relies on the formation
region between ItrD and ItrE revealed clusters of of stable conintegrates between the target conjugal
inverted and direct repeat structures similar to other element and the IS-containing vector within the
bacterial oriT regions [7,21]. recipient. Typically, resolution of cointegrate
In this work we demonstrate the use of IS946- plasmids occurs through homologous recombination
mediated cointegration between the shuttle plasmid between duplicated IS-elements. We have screened
pTRK28 and the conjugal element pRSOI as a numerous pTRK28::pRSOI cointegrates within the
method for insertional mutagenesis of pRSOl. While Rec- lactococcal strain MMS370 and have not
we have not used pTRK28 cointegration to map other observed resolution of the constituent plasmids. In
conjugal elements, this method was quite reliable contrast, Anderson and McKay identified several
for analysis of pRSOl. The resolution of mapping types of resolution products derived from cointe-
the pTRK28 insertions using PvuII-SphI restriction grates formed between the lactococcal plasmid
analysis (Figure 2) has proven to be quite accurate. pSK08 and pRSOI in the Rec+ lactococcal strain
To date, we have sequenced the exact site of pTRK28 LM2301 [1]. Thus, one possible way to circumvent
insertion within 23 cointegrate plasmids, and found cointegrate plasmid resolution may be to generate
that all insertions were within O.S-kb of the location cointegrates within a Rec- recipient strain.
predicted by restriction mapping (of the 60-kb coin- The mating procedures described here are standard
tegrate plasmid). This accuracy was facilitated by for lactococcal matings. We have found that the
77
cocci for use in dairy fermentations. Appl Environ conjugation of Streptococcus lactis ML3. J Bacteriol
Microbiol52: 1001-1007. 146: 937-944.
17. Sing WD, Klaenhammer TR (1993). A strategy for 21. Wilkins B, Lanka E (1993). DNA processing and
rotation of different bacteriophage defenses in a lac- replication during plasmid transfer between Gram-
tococcal single-strain starter culture system. Appl negative bacteria. In: D Clewell (ed), Bacterial
Environ Microbiol 59: 365-372. conjugation, pp 105-129. New York: Plenum Press.
18. Steele JL, McKay LL (1989). Conjugal transfer of
genetic material in Lactococci: a review. J Dairy Sci
72: 3388-3397.
19. Terzaghi BE, Sandine WE (1975). Improved medium Address for correspondence: Larry L. McKay, Department
for lactic streptococci and their bacteriophages. Appl of Food Science and Nutrition, University of Minnesota,
Microbiol 29: 807-813. 1334 Eckles Avenue, St Paul, MN 55108, USA
20. Walsh PM, McKay LL (1981). Recombinant plasmid Phone: (612) 624-3090; Fax: (612) 625-5272
associated with cell aggregation and high-frequency E-mail: lmckay@che2.che.umn.edu
Methods in Cell Science 20: 79-84 (1998).
© 1998 Kluwer Academic Publishers ..
Abstract. We present two methods for electropora- insertional inactivation of a chromosomal gene
tion for the gram positive bacterium Enterococcus involved in binding substance formation as well as
faecalis that can also be used as guidelines for work for the expression of Aggregation Substance in
with other gram positive species. We demonstrate the strains with different chromosomal backgrounds. The
use and the advantages of this technique for investi- influence of defects in lipoteichoic acid synthesis and
gating genes, both chromosomal and plasmid-linked, the expression of Aggregation Substance on viru-
encoding surface structures. Electroporation was used lence was shown in a rabbit endocarditis model.
to deliver constructs created on shuttle vectors for
Key words: Aggregation substance, Electroporation, Enterococcal binding substance, Enterococcus faecalis,
Virulence
c recipient
• cCFIO
D 0 aggregation substance
binding substance
l..
• prgZ
Figure 1. Model of the sex pheromone plasmid transfer system in Enterococcus faecalis.
on protoplast transformation [19] but was soon using this method to study the surface structures EBS
replaced by the simpler method of electroporation. and AS in E. faecalis. No single method for electro-
We present two methods used for electroporation in poration exists which is uniformly effective, for
this gram positive species, and results obtained by streptococci and lactococci as well as enterococci.
SI
We present, therefore, two methods that are used in 3. Chill the culture on ice; harvest the cells and
our laboratory. Results may vary and depend on the wash the cells in 1/3 volume of chilled EP
strains used, uncharacterized clinical isolates may be buffer.
difficult to transform. 4. Harvest the cells and resuspend in 11100 of the
original volume. Aliquot in 40 fll portions and
store at -70 to -SO °C.
2. Material 5. For electroporation, thaw the cells on ice for
a few minutes. Add 10-20 fll of DNA in H 20
A. Electroporation (0.3-1 flg) and incubate for 5 min. Add
Method I: [6, 7] mixture to chilled electroporation cuvette and
- Medium compositions electroporate immediately, using the settings
- M9-YE: 25 flF, 200 Q and 2500 V. After the pulse
• Yeast extract 3 gil (#0127-01-7, Difcd). resuspend the cells and place them on ice for
• Casamino acids (#0230-17-3, Difcd) 10 gil. 5 min. Incubate at 37°C for 120 min. Spread
• lOx M9 salts* all chemicals from Fisher the cells on selective plates.
Scientific 2 (gram/liter: Na 2HP0 4, 60, S373- Method II:
500. 1. Grow cells for 25 h in THB, 0.5 M sucrose
• KH 2P04, 30, P3S2-500; NaCl, 5, S640-3. containing glycine. The amount of glycine
• NH 4CI, 10, A661-500) 1/10 volume. depends on the strain used. Examples:
• 20% glucose (DI6-500)* 11100 volume. OG1SSp: 5%, OGIRF: 3%, JH2-2: 6%. For
• 1 M MgS04 (M65-500)* 1/500 volume. example for 5% (10 ml): 2.5 ml 4x THB,
• 1 M CaCl 2 (C77-500)* 1/10000 volume. 2.5 ml 20% glycine, 5 ml 1 M sucrose
• 20% glycine (G46-500). 2. Wash the cells in chilled EP buffer in 100, 50
* Add after autoclaving. and 10% of the original culture volume.
Method II: Modified from [4] and [9] 3. Resuspend in 1/100 of the original culture
- 1 M sucrose. volume, aliquot and store at -70 to -SO °C.
- 20% glycine. 4. Follow step 5 in method I.
- 4x Todd Hewitt Broth (#0492-17-6, Difco1). B. Endocarditis
- Selective plates THB containing 0.5 M sucrose. Intraventricular Injection model (American Dutch
- GenePulser™ and Pulse Controller (165-2076 Belted):
and 165-209S, Biorad3 ), 0.2 mm cuvettes 1. Grow test organism to a density of approxi-
(#4307 -000-593, Eppendorf'). mately 108 CFU/ml.
B. Endocarditis 2. Use 25 mg/kg ketamine to calm the rabbits, do
- American Dutch Belted Rabbits (Birchwood not anesthetize.
Farm, Wisconsin). 3. Turn rabbit on its back, front legs over the
- New Zealand White rabbits (Birchwood head.
Farm, Wisconsin). 4. Go up two or three ribs on the left side of the
- Syringes, IS gage needle, catheter outside rabbit.
diameter 1.27 mm (No. 427420, Becton 5. Insert syringe filled with 1 ml of the test
Dickenson5). organism and a IS gauge needle.
- Ketamine (NDC 57319-291-02, Phoenix 6. Wait until blood comes pumping into the
Pharmaceutical 6), xylazine (NDC 57319-210- syringe and inject organisms, retreat needle.
26, Phoenix Pharmaceutica16). 7. Monitor the rabbits for two days, euthanize
and evaluate results.
Transaortic Catherization (New Zealand White):
3. Procedures 1. Anesthetize rabbits with ketamine (25 mg/kg)
and xylazine (20 mg/kg).
A. Electroporation 2. Shave the throat of the rabbit. Expose the left
Method I: carotid artery, and stop the blood flow with
1. Grow cells for 12 to 15 h in BYGT or M9-YE two threads knotted around the artery approx-
medium plus glycine. We recommend a range imately 2 cm apart. The next part should be
between 0.5 and 6.0%. Measure the A660 in carried out by two persons. Lift the artery up
comparison to a culture grown without on the threads, slit open the artery with a
glycine. Use the culture with a growth reduc- scalpel or razor blade and insert the catheter
tion between 70 and 90%. into the artery. Push the catheter into the heart.
2. Dilute the culture in fresh medium (containing Close the catheter by introducing a bend and
the same concentration of glycine) to a A660 use a thread to keep it in place.
between 0.05 and O.OS. Incubate the cells for 3. Keep the rabbits anesthetized for two hours.
60 min at 37°C. 4. Remove the catheter, seal off the artery with
82
the thread in place and close the opening with this clone is shown in Figure 2A. Since the negative
sutures. control elements of pCFlO are lacking in this
5. Inject 2 ml of the test organism in a marginal construct, expression of the aggregation substance
ear vein. Bacterial cell density of the injected is constitutive. Insertion of Tn5 (INY 4515 and
solution should be around 109 • INY 4561) confirmed the functions of the surface
6. Monitor the rabbits over the course of three proteins Sec10 (prgA) and Asc10 (prgB) [14, 15].
days. Sacrifice the rabbits on day three and The availability of a transformation system
assess the results. allowed further characterization of the Tn916
insertions in the INY3000 chromosome. The four
insertions were separated genetically by protoplast
4. Results and discussion transformation.
None of the individual Tn916 insertions showed
Transfer frequencies observed for electroporation a phenotype; no diminished recipient ability was
are shown for Method I in Table 1. It is generally detected [18]. This suggested that multiple loci were
advisable to propagate plasmids in E. coli before involved in synthesis of EBS. We have begun to
transforming them into Enterococcus. When the analyze each of the loci separately. Work on one of
electrocompetent cells are prepared, we recommend the loci in strain INY3039 greatly relied on an
to keep the cells chilled during the whole washing efficient transformation system. The INY3039 locus
and centrifugation procedure. Prechill the tubes used is shown in Figure 2B. The transposon was used as
for storage of the aliquots. selective marker for marker rescue of EcoRI-cut
Although the cells can be used immediately after INY3039 chromosomal DNA in E. coli. Sequence
the washing procedure, freezing them for 1 h before analysis showed that the transposon inserted in the
use can increase the efficiency. The cells should be intergenic region between two divergently tran-
put into cuvettes only shortly before the electropo- scribed genes, ebsA and ebsB [1]. Further transposon
ration and resuspended immediately after the pulse, mutagenesis of the cloned wild-type region with Tn5
since it was found, that long contact with the metal revealed that an insertion in the reading frame for
electrodes decreases transformation efficiency. ebsC abolished the ability of the fragment to com-
Optimal time constants for the described methods are plement the INY3000 phenotype. Sequence similar-
between 4.0 and 4.6 ms. The DNA used should be ities for ebsC showed homology to a negative
resuspended preferentially in H 20 or a low salt regulatory protein. This led to formulation of a
buffer. Too high amounts of DNA, poor quality or model suggesting that in wild-type E. faecalis ebsC
high salt concentrations in the buffer can lead to a negatively regulated expression of ebsA or ebsB.
short-circuit with time constants below 1 ms, drasti- Overexpression of one of the latter genes from a
cally reducing the probability of recovering trans- promoter within Tn916 resulted in a disruption or
formants. Dilution of the cells 1: 1 with 10% cold degradation of wild-type EBS. To test the model,
glycerol can help to avoid short-circuits. ebsC was inactivated in the vector pGHost in E. coli
The locus for AS on pCFI0 was first character- by deleting an internal fragment of the gene and
ized by transposon mutagenesis. Two adjacent EcoRI replacing it with a chloramphenicol resistance marker
restriction fragments containing the positive control flanked on both sides by about 1 kb of the wild-type
region and the structural genes for surface exclusion E. faecalis chromosomal sequences. This construct
and AS are cloned in a shuttle vector [3]. A map of was then transformed using electroporation in the
wild-type strain OG 1SSp and the cells were screened
Table 1. Transformation frequencies of different bacte-
for the double crossover event. A mutant strain
rial strains using the described method I carrying the resistance gene in the disrupted ebsC
gene was isolated. Plasmid transfer experiments
Organism Plasmid Transformants/Ilg revealed a diminished ability of the mutant strain to
DNA act as recipient in matings [2]. These results are
remarkable, since the original Tn916 insertion in that
E. faecalis OGlRF pDL414 5 x 104 locus did not show a phenotype. It is also important
E. faecalis OGlRF pWM401 1.2 x 105 to note that the allelic replacement experiment that
E. faecalis OG1SSp pDL414 1.6 x 104 confirmed the model involved a gene (ebsC) not
E. faecalis JH2-2 pDL414 2.5 x 104 targeted by the original Tn916 insertion. This is an
E. faecalis UV202 pDL414 4 x 103
important consideration in using Tn916 for random
s. pyogenes DW1009* pDL414 1.6 x 103
s. agalactiae H36B mutagenesis.
(type Ib) pDL414 4.8 x 103 Investigating the contribution of the two surface
S. sanguis FW213 pDL414 1.4 x 104 structures lipoteichoic acid and AS to virulence also
became possible. Rabbit endocarditis was chosen as
* No glycine for preparation of competent cells. Modified a model system. The combination of strain comprised
from [7]. in these experiments were the binding substance
83
plasmid
~ ~ EcoRI
EcoRI
~ E~"'/
1
kb
Figure 2A. The positive control region in pCFlO. Cloning of the adjacent EcoRI fragments in a shuttle vector lead to
the constitutive expression of the prgB gene resulting in a clumpy phenotype. Insertion of Tn5 in the genes prgA
(INY4515) and prgB (INY4561) confirmed their function, surface exclusion and aggregation substance, respectively.
lkb
EeoRl
.--------
EcoRl
.
(INY3000IINY3039) ~ cat~
I Y "Z-
~----------------
ebsA ebsS ~~ ebsC .. arfD
Figure 2B. The ebs locus in E. faecalis. Location of the initial Tn916 insertion separated from INY3000 into INY3039.
Insertional inactivation of the ebsC gene in wild-type OG 1SSp.
mutant INY3000 (EBS-) with the cloning vector wild-type. The highest grade of virulence however,
pWM401 (AS-) as negative control, the wild-type was reached when both lipoteichoic acid and AS
strain OGlSSp (EBS+) with pWM401 and INY3000 were expressed in strain OGlSSp:pINY1801 (EBS+,
or OGlSSp containing the plasmid pINY1801(AS+), AS+). All animals challenged with this strain died and
constitutively expressing the otherwise inducible showed destruction of tissue in heart and lung. These
Aggregation Substance in the cloning vector results demonstrate that not only the Aggregation
pWM401. All strains were transformed by electro- Substance on the pheromone plasmids has a major
poration. All combinations of the two surface influence on the virulence of E. faecalis, but the
structures (EBS- AS-; EBS- AS+; EBS+ AS- and EBS+ overall architecture of the cell wall is a deciding
AS+) were therefore available for use in this model factor for establishment of an infection. While the
system. The experiments involved intraventricular presence of the Aggregation Substance could at least
injection as well as catheterization for the assessment partially overcome the inability of INY3000 to
of the virulence of the chosen strains (Table 2). induce virulence, the altered lipoteichoic acid expres-
In both models the binding substance mutant sion in this strain clearly makes that strain avirulent
INY3000 showed no signs of morbidity or inflam- either by abolishing the ability to survive and to
mation, whereas the wild-type strain OG I SSp establish in the host, or by changing the cell wall
showed moderate virulence [16]. This shows clearly structure such, that other chromosomally-encoded
that the existence of an intact lipoteichoic acid adhesins are not properly displayed on the cell
structure is necessary for virulence of E. faecalis. The surface.
lack of virulence of the strain INY3000 could be Our examples show that the development of
overcome by the presence of the constitutive AS- electroporation allows better in-depth analysis of the
expressing plasmid pINY1801. The symptoms biology of bacteria species not naturally competent
induced by this strain resemble the plasmid-free for transformation. As the analysis of the transposon
Table 2. Influence of enterococcal binding substance (EBS) and Aggregation Substance (AS) on the virulence of E.
faecalis in rabbit endocarditis, intraventricular injection model EBS-: INY3000; EBS+: OGISSp; AS-: pWM401; AS+:
pINY1801. Modified from [16]
insertion in INY3039 demonstrates, transposon posItlve bacteria. Appl Environ Microbiol 57:
mutagenesis is useful for initial analysis, however the 1194-1201.
phenotypic effects shown might not reflect the situ- 8. Dunny GM, Leonard BA, Hedberg PJ (1995).
ation correctly. Insertional inactivation provides a Pheromone-inducible conjugation in Enterococcus
faecalis: Interbacterial and host-parasite chemical
more accurate tool and with the use of electropora-
communication. J. Bacteriol 177: 871-876.
tion this becomes available for many gram positive 9. Fiedler S, Wirth R (1991). Transformation of
species. Enterococcus faecalis and Enterococcus faecium by
elecrtroporation. In: Dunny GM, Cleary PP, McKay
LL (eds), Genetics and molecular biology of strepto-
Acknowledgments cocci, lactococci and enterococci, p 301. Washington
DC: ASM Press.
The work described in this paper was supported by 10. Galli D, Lottspeich F, Wirth R (1990). Sequence
NIH grants HL51987 and GM49530. analysis of Enterococcus faecalis aggregation sub-
stance encoded by the sex pheromone plasmid pAD 1.
Mol Microbiol 4: 895-904.
11. Galli D, Wirth R (1991). Comparative analysis of
Notes on suppliers
Enterococcus faecalis sex pheromone plasmids
identifies a single homologous DNA region which
1. Difco Laboratories Inc., P.O. Box 331058, Detroit, MI
codes for aggregation substance. J Bacteriol 173:
48232, USA
3029-3033.
2. Fisher Scientific Corp., 711 Forbes Ave., Pittsburgh,
12. Hirt H, Wanner G, Galli D, Wirth R (1993).
PA 15219, USA
Biochemical, immunological and ultrastructural
3. Bio-Rad Laboratories, Life Science Group, 2000
characterization of aggregation substances encoded by
Alfred Nobel Dr., Hercules, CA 94547, USA
Enterococcusfaecalis sex-pheromone plasmids. Eur J
4. Eppendorf Scientific Inc., 6524 Seybold Rd.,
Biochem 211: 711-716.
Madison, WI 53719, USA
13. Jett BD, Huycke MM, Gilmore MS (1994). Virulence
5. Becton Dickinson, 7 Loveton Circle, Sparks,
of enterococci. Clin Microbiol Rev 7: 462-478.
MD21152, USA
14. Kao SM, Olmsted SB, Viksnins AS, Gallo JC, Dunny
6. Phoenix Pharmaceutical Inc., 4621 Easton Rd., St.
GM (1991). Molecular and genetic analysis of a
Joseph, MO 64503, USA
region of plasmid pCF10 containing positive control
genes and structural genes encoding surface proteins
involved in pheromone-inducible conjugation in
References Enterococcus faecalis. J Bacteriol 173: 7650-7664.
15. Olmsted SB, Kao SM, van Putte LJ, Gallo JC, Dunny
1. Bensing BA, Dunny GM (1993). Cloning and mole- GM (1991). Role of the pheromone-inducible surface
cular analysis of genes affecting expression of binding protein Asc lOin mating aggregate formation and
substance, the recipient-encoded receptor(s) mediating conjugal transfer of the Enterococcus faecalis plasmid
mating aggregate formation in Enterococcus faecalis. pCF1O. J Bacteriol 173: 7665-7672.
J Bacteriol 175: 7421-7429. 16. Schlievert PM, Gahr PJ, Assimacopoulos AP, et al.
2. Chen Y (1997). Genetic analysis of genes involved (1998). Aggregation and binding substances enhance
in Enterococcus faecalis binding sustance formation. pathogenicity in rabbit models of Enterococcus
Masters Thesis, University of Minnesota. faecalis endocarditis. Infect Immun 66: 218-223.
3. Christie PJ, Kao S-M, Adsit JC, Dunny GM (1988). 17. Tailor SA, Bailey EM, Rybak MJ (1993).
Cloning and expression of genes encoding Enterococcus, an emerging pathogen. Ann
pheromone-inducible antigens of Enterococcus Pharmacother 27: 1231-1242.
(Streptococcus)faecalis. J Bacteriol170: 5161-5168. 18. Trotter KM, Dunny GM (1990). Mutants of
4. Cruz-Rodz AL, Gilmore MS (1991). Electroporation Enterococcus faecalis deficient as recipients in mating
of glycine-treated Enterococcus faecalis. In: Dunny with donors carrying pheromone-inducible plasmids.
GM, Cleary PP, McKay LL (eds), Genetics and Plasmid 24: 57-67.
molecular biology of streptococci, lactococci and 19. Wirth R, An FA, Clewell DB (1986). Highly efficient
enterococci, p 300. Washington DC: ASM Press. protoplast transformation system for Streptococcus
5. Dunny GM, Brown BL, Clewell DB (1978). Induced faecalis and a new Escherichia coli-Streptococcus
cell aggregation and mating in Streptococcus faecalis: shuttle vector. J Bacteriol 165: 831-836.
Evidence for a bacterial sex pheromone. Proc N atl
Acad Sci USA 75: 3479-3483.
6. Dunny GM (1991). Electroporation of Enterococci,
Streptococci and Bacilli. In: Dunny GM, Cleary PP, Address for correspondence: Gary Dunny, Department of
McKay LL (eds), Genetics and molecular biology of Microbiology, Medical School, Box 196 UMHC, 1460
streptococci, lactococci and enterococci, p 302. Mayo Memorial Building, 420 Delaware Street SE,
Washington DC: ASM Press. Minneapolis, MN 55455, USA
7. Dunny GM, Lee LN, LeBlanc DJ (1991). Improved Phone: 612-625-9930; Fax: 612-626-0623
electroporation and cloning vector system for gram- E-mail: gary-d@biosci.cbs.umn.edu
Methods in Cell Science 20: 85-93 (1998).
© 1998 Kluwer Academic Publishers.
Dental Research, University of Rochester, Rochester, New York, USA; 3 Department de Biochimie (Sciences),
Universite Laval, Quebec, Canada; 4 Department of Medicine (Infectious Diseases), University of Texas Health
Science· Center at San Antonio, San Antonio, Texas, USA
Abstract. Streptococcus mutans and Streptococcus published procedures for the transformation of any
sobrinus, members of the mutans group of strepto- strain of S. sobrinus. This report describes two
cocci, are the major etiologic agents of human dental methods for the transfer of plasmid vectors, and
caries. Several properties of these two bacterial recombinant DNA molecules, to S. sobrinus. The
species have been proposed as virulence traits, and first method utilizes an intermediate streptococcal
many of the genes encoding such traits have been host, a conjugative plasmid, and a vector molecule
cloned from both species, and their sequences deter- derived from a plasmid of mutans streptococcal
mined. However, assessments of the contributions of origin, to mobilize an E. coli/Streptococcus shuttle
these genes and their products to the cariogenicity vector into S. sobrinus. The second method involves
of the human mutans streptococci requires the ability the production of S. sobrinus cells competent for
to replace or complement the original wild-type transformation by electroporation. Both protocols
genes with genetically altered genes Whereas have been used for the construction of isogenic
numerous strains of S. mutans can achieve a state of mutants of S. sobrinus, and should be applicable to
natural competence for transformation, or can be other species and strains of streptococci not other-
transformed by electroporation, which renders them wise susceptible to genetic manipulation.
amenable to genetic manipulation, there are no
(Gas Pac H 2 C0 2 generator) for 4 h. Spread 0.1 colonies on bile esculin agar plates, whereas
ml of the undiluted transformation mixture, as S. gordonii recipient strain, and transconju-
well as of 10-fold dilutions up to 10-4, on BHI gants will not) and to check for the presence
agar plates containing 500 Ilg/ml of Km to of both plasmids by standard techniques.
select for transformants. If the vector has an B. Mobilization of plasmid ONA from S. gordonii to
insert with a selectable marker, e.g., the S. sobrinus strain 6715. The matings are con-
SpR (R = resistance, S = susceptible) gene from ducted in the same manner as in A. 3) above,
E. faecalis, spc [18], then the presence of this except that S. gordonii containing pAM~l plus
marker can either be selected for directly, or the mobilizable plasmid is used as the donor, and
subsequent to direct selection for KmR. Choose a strain of S. sobrinus resistant to Rf and Fa [So
a few colonies to be tested for the presence of sobrinus 6715RF; 2] serves as the recipient. BHIG
intact plasmid ONA by standard plasmid agar containing 25 Ilg/ml each of Rf and Fa are
isolation and restriction endonuclease diges- used to select for the recipient strain, and for
tion procedures. selection of specific transconjugants Km, Sp, or
3. Transfer of pAM~l to the Challis strain of S. both, are also included, as described below. Km
gordonii containing a mobilizable plasmid is used for selection of the mobilizable plasmid
Since pAM~lmay suffer deletion of a portion (or, e.g., Sp, if the mobilizable plasmid contains
of its transfer region upon prolonged storage a S. sobrinus gene that has been inactivated by
and/or growth of strain OLl harboring it [19], insertion of the E. faecalis spc gene). Only those
it is best to transfer this plasmid by conjuga- transconjugants of S. sobrinus that have received
tion (deletion also may occur as a consequence the mobilizable plasmid will be able to grow in
of transformation) to strains already harboring the presence of Km and/or Sp, or in which the
the mobilizable plasmid. Obtain overnight insertionally inactivated gene has integrated into
cultures of strains OL1 containing the mobi- the recipient chromosome via a single (resistant
lizable plasmid and E. faecalis strain JH201 to both Km and Sp) or double (resistant to Sp
(pAM~l) by transfer of 40 III of frozen only) crossover event.
glycerol stock to 4.0 ml of BHI without added C. Transformation of S. sobrinus 6715 by electro-
carbohydrate, but with appropriate antibiotic poration. Transfer 25 III of a frozen glycerol stock
for plasmid selection (Km for the mobilizable of S. sobrinus to 2.5 ml (1: 100 dilution) of
plasmid and 10 Ilg/ml Em for pAM~ 1) and THBYE, and incubate at 37 DC -6-8 h. Transfer
incubation at 37 DC. Transfer 0.25 to 0.50 ml 0.25 ml of the growing culture (even if only
of each overnight culture to 5 ml BHIG (no barely turbid) to 5 ml (1:20 dilution) THBYE +
antibiotic) in 13 mm x 100 mm screw cap 300 mM L-threonine and incubate overnight at
tubes. Incubate in a 37 DC water bath and 37 DC in the presence of 10% CO 2 • Transfer the
follow growth to 00 600 of 0.5. Chill the first overnight culture to 47.5 ml of pre-warmed
culture to reach an 00600 of 0.5 on ice until (37 DC) THBYE + 300 mM L-threonine in a 50
the second culture also reaches the same 00. ml capacity screw cap disposable conical cen-
Transfer 0.1 ml of donor culture and 0.1 ml trifuge tube. Mix and transfer 5 ml to a 13 mm x
of recipient culture to a 1.5 ml capacity 100 mm screw cap tube. Incubate both tubes in a
microfuge tube containing 0.8 ml of BHI. 37 DC water bath. Follow growth of the culture by
Controls contain 0.1 ml of donor culture (or measuring the 00 600 of the 5 ml culture at
recipient culture) plus 0.9 ml BHI. Vortex -30-60-min intervals. At an 00600 of 0.2 to 0.3,
mating and control cultures and centrifuge in transfer the 5 ml culture to the 45 ml culture and
a microfuge for 5 min to pellet cells. Remove chill in an ice-water bath for 10 min. Pellet the
the supernatant fluids with sterile tips for a cells in a table-top centrifuge at 4,000 rpm, for 15
1 ml capacity pipettor. Resuspend the cell min, at 4 DC. Pour off the supernatant fluid and
pellet in residual liquid (-10 Ill) and spot the immediately transfer the tube to an ice-water bath
entire suspension on a BHI agar plate. Incubate (if the pellet is not tight, centrifuge again, at a
4 h at 37 DC anaerobically (GasPak H 2C0 2 higher speed if necessary). Resuspend the pellet
generator). Scrape cells off the plate with a in 10 ml of chilled EPM, insuring that all clumps
sterile plastic disposable inoculating loop, are broken up. Add 15 ml of chilled EPM and mix
resuspend in 0.5 ml BHI, and vortex. Spread well. Centrifuge as above. Repeat resuspension of
0.1 ml aliquots onto BHI agar plates con- the cells in 10 + 15 ml of chilled EPM and
taining Em + Km (for presence of pAM~l in centrifugation one time, and then resuspend the
strain OLl containing mobilizable plasmid), pellet in 10 ml of chilled EPM (for a final wash)
and incubate anaerobically at 37 DC for 24 to and centrifuge as above. After discarding the final
48 h. Pick a few representative colonies to test supernatant fluid, add 0.5 ml of chilled EPM to
for inability to hydrolyze bile esculin (E. residual EPM in the tube and resuspend the cells
faecalis donor strain will produce black well. Chill in an ice-water bath for 45 min. Place
89
a pre-chilled electroporation cuvette (0.1 cm [16], the lacZ-mcs (0.9 kb) from pGEM7Zf(-) from
electrode gap) in ice and spot 1 to 2 III of trans- Promega, and intact pVA380-1 (4.2 kb) [13,17,21].
forming DNA at the top of the cuvette. Wash the The latter fragment was obtained by partial digestion
DNA down the side of the cuvette with 20 III of ofpVA380-1 with HaeIl, due to the presence oftwo
chilled cell suspension. Apply a pulse at 1.6 kV, sites for this enzyme in the pVA3 80-1 molecule (see
400 n, 25 IlFad. Record time constant (should be Figure 1). The 8.2 kb derivative, pDL289Ll202, was
-10 msec). Immediately after pulsing, return the derived from pDL289 (8.4 kb) by Exonuclease III
cuvette to ice. Transfer the cells from the cuvette, digestion of the latter, in both directions, after
with a sterile multi-flex microcapillary pipette tip, linearization by digestion with Stul, for which there
to a sterile 1.5 ml capacity microfuge tube con- was a single site located near the center of the -ori
taining 0.9 ml THBYE. Incubate in a 37 DC water of pVA3 80-1 [17]. Three of the sites in the mcs of
bath for 90 min. Centrifuge approximately 1 min pDL289 and pDL289Ll202, EcoRI, Clal, and HindIII,
to pellet the cells, pour off the medium, and allow also occur in the mob gene of the respective plasmids,
the tube to drain on its side. Resuspend the cells and cannot be used for cloning into either plasmid
in 100 III of THBYE and spread 50 III on THBYE without destroying the mobilizability of the vector.
agar containing the appropriate selective anti- When strains of S. gordonii Challis containing
biotic (Sp for pDL278, Km for pDL289 or pAMP1 plus either pDL289 or pDL289Ll202 were
pDL289Ll202, and/or antibiotic necessary to select used as donors in conjugation experiments with S.
for insertionally inactivated gene, see above). sobrinus 6715-RF, Km resistant transconjugants were
Incubate at 37 DC in the presence of 5-10% CO 2 • obtained at frequencies of 3 x 10-6 and 1 x 10-7 per
Transformant colonies should be visible after recipient CFU, respectively. Less than 3% and 12%
36 h, but may require up to 72 h of incubation. of the transconjugants containing pDL289 and
Always include a no DNA control treated in pDL289Ll202, respectively, contained pAMPI [2].
exactly the same manner as the transformation Results of subsequent experiments in which either
culture. vector containing cloned DNA fragments was
mobilized into S. sobrinus, revealed that the mobi-
lizing plasmid, pAMP 1, was present in anywhere
4. Results and discussion from 1 to 10% of transconjugants selected only for
the mobilized plasmid. The reasoning behind the
The ability to mobilize plasmid DNA into S. sobrinus construction of pDL289Ll202 was that interuptions of
was demonstrated with a derivative of pVA380-1 in the minus origin of the pVA380-1 portion of pDL289
which the kan gene of pDL413 [16] had been cloned would yield a much less stable plasmid in strepto-
into a restriction endonuclease site (Scal) located in coccal hosts, due to the accumulation of single-
a non-essential region of the plasmid. This deriva- stranded replicative intermediates [17]. It was
tive, pDL287, was transferred from S. gordonii reasoned that this would render the plasmid more
Challis (pAMP1 + pAM287) to S. sobrinus 6715-RF readily curable, and that the presence of an abun-
at frequencies in the range of 10-5 per recipient dance of single stranded replicative intermediates
colony forming units (CFU) [13]. Less than 2% of would facillitate recombination between the host
transconjugants selected for the presence of pDL287 chromosome and homologous DNA that had been
(resistant to Km) did not contain pAMP1 (susceptible cloned into the vector. A higher rate of curing of
to Em). Thus, transconjugants containing only the pDL289Ll202 over pDL289 has been demonstrated
mobilizable plasmid could be obtained quite readily, (3 to lO-fold), but the effects of the presence of
without the need to cure such transconjugants of the single-stranded DNA containing sequences with
mobilizing plasmid. The next step toward making S. homology to the host chromosome, on the frequency
sobrinus amenable to recombinant DNA technology of homologous recombination, have yet to be
was the construction of a suitable mobilizable E. assessed.
coli/Streptococcus shuttle vector. One example of the utility of pDL289Ll202 for the
The construction of pDL289, the E. coli/ construction of isogenic mutants of S. sobrinus is
Streptococcus shuttle vector mobilizable by pAMP1, described briefly here, and in greater detail in
has been described [1], as has the deletion deriva- Buckley et al. [2]. A spc gene [18] was inserted into
tive of it, pDL289Ll202 [2]. However, complete an EcoRV digested 2.7 kb HindIII fragment of S.
physical maps of these plasmids, that would permit sobrinus that contained the scrA gene encoding the
immediate assessment of useful restriction endonu- sucrose permease, EIl'llC [4]. This resulted in the
clease sites, were never published. Such a map of insertional inactivation of the scrA gene by replace-
pDL289Ll202 is presented in Figure 1. The original ment of the 226 bp EcoRV fragment internal to scrA
shuttle vector, pDL289, was constructed by the with the spc gene, as illustrated in Figure 2. The
sequential addition of HaeIl fragments containing the inactivated gene was subcloned into pDL289Ll202,
pSC101 origin of replication (1.9 kb) obtained from and the resultant recombinant plasmid was used to
pGB2 [6], a kan gene (1.4 kb) obtained from pDL413 transform S. gordonii Challis. After introduction of
90
II!pJH1kan
Hind III
~I- pOl289l1202JscrA IIIlC
11.8kb Hind III
2.7kb
3.6kb
pSC101 rep
serA
- - > II:
>
II: -'0
'2 8
w W8
.l:
..
J:
..
:I:
~ (,L:::.
serB serA
2.7kb
'0
.S; -g
I J:
/I I
oDL289A202
w/uu.al;:·:·:·:·:·:·:·:·:·:·:·: ,W'uhW/4¢vu4W/u/4
scrAlS/JC
I~
I
I
I 4.3kb
Figure 2. Diagrams of double and single crossover events between serA in the chromosome of S. sobrinus and serA
interupted by spe, present on pDL289Ll202. Top segment depicts a double crossover event between the S. sobrinus chro-
mosomal wild-type serA gene within a 2.7 kb HindIII fragment of DNA, and the plasmid-borne spe-inactivated serA gene
in a 4.3 kb HindIII fragment, resulting in replacement of the wild-type gene on the chromosome with the inactivated
gene in a 3.6 kb HindIII fragment. Lower segment depicts a single crossover event between the same chromosomal
wild-typegene and the spe-inactivated serA gene resulting in integration of the entire pDL289Ll202/serAspe plasmid,
and the presence of two copies of serA in the S. sobrinus host chromosome, a wild-type allele within a 2.7 kb HindIIl
fragment, and an inactivated (by spe insertion) allele within a 4.3 kb HindIII fragment.
91
pAM~l into a representative transformant, S. between the EcoRI site in the mcs and that in the mob
gordonii (pDL289LVscrAspc + pAM~1) was used as gene, would not hybridize to the probe. The second
a donor in matings with S. sobrinus 6515-RF as the possibility described above would yield a hybridizing
recipient. Transconjugants were obtained on media EcoRI fragment that was much larger than 10.6 kb,
containing Rf, Fa, and Sp, and then tested for resis- since the scrA gene of S. sobrinus 6715 is in an
tance to Km and to Em. Two different resistance EcoRI fragment of approximately 14 kb [4].
phenotypes were observed, SpRKms, and SpRKmR. Transconjugants containing both an integrated and a
None of the transconjugants tested were resistant to replicating plasmid would yield two hybridizing
Em. Total cell DNA was prepared from representa- EcoRI fragments, the 10.6 kb fragment, and the
tive transconjugant cultures and digested with larger one.
HindU!. After separation of the DNA fragments by The transformation system described under pro-
agarose gel electrophoresis, Southern blots were cedures has been used to transform S. sobrinus 6715
prepared and hybridized with a probe consisting of with the E. coli/Streptococcus shuttle vectors,
a DNA fragment internal to scrA, and including both pDL278 and pDL289, as well as with recombinant
sides of the two EcoRV sites. Two types of results DNA derivatives of pDL278 and pDL289~202.
were obtained, as predicted in Figure 2. Those Representative transformation frequencies obtained
transconjugants with the SpRKms phenotype yielded with each of these plasmids, all purified from E. coli
only a 3.6 kb HindIII fragment that hybridized to the DH5u, are shown in Table 1. At a concentration of
scrA-specific probe. In these transconjugants, the 2.7 0.5 Ilg/ml, pDL278 transformed S. sobrinus at a
kb HindIII fragment containing the wild-type scrA frequency of 1.6 11 x 10 3/llg. This same plasmid, and
gene was increased to 3.6 kb via a double crossover with the same batch of competent cells, when present
event in which the inactivated scrA gene replaced the at a concentration of 1.0 Ilg/ml yielded a frequency
wild-type gene (Figure 2, top segment). Greater than of 5.4 x 102/llg of DNA, suggesting that the trans-
50% of transconjugants selected for resistance to Sp forming DNA reached saturation between 0.5 and 1.0
only expressed the SpRKms phenotype, indicating Ilg/ml. A fragment of S. sobrinus 6715 genomic DNA
that double-crossover events occur at relatively high containing intact serB plus the region 5' of scrB
frequencies in S. sobrinus. Transconjugants with the encoding the putative promoter for scrA [5] (here
SpRKmR phenotype yielded two HindIII fragments designated scrAP), was cloned upstream of the pro-
that hybridized to the serA-specific specific probe, moterless cat gene of pCAT3-Basic (Promega). A
one of 2.7 kb and the other 4.3 kb in size. The size DNA fragment containing scrBscrAPcat was then
of the HindIII fragment on pDL289~202 containing subcloned onto pDL278, and the recombinant DNA
the inactivated scrA gene is 4.3 kb in size. Although molecule was used to transform S. sobrinus.
this latter group of transconjugants were not sub- Transformation frequencies obtained with pDL278
jected to further analysis, three possibilities would were on average only approximately four times
explain these results. The first is that the plasmid, higher than those obtained with the recombinant
pDL289~202/scrAspc is replicating in the transcon- pDL278 molecule (Table 1). Isolates of S. sobrinus}
jugant strictly as an extrachromosomal element, and containing pDL278/scrBscrAPcat are resistant to Cm.
the wild-type scrA gene has not been affected. The Whether or not the plasmid is functioning as an
second possibility is that the entire plasmid has independent replicon, or has integrated into the S.
integrated into the host chromosome via a single sobrinus chromosome via a single crossover event,
crossover event in which the wild-type and inter- or both, has yet to be determined. In general,
rupted scrA genes each consist of DNA derived from frequencies of transformation of S. sobrinus with
the incoming plasmid and a portion derived from the the mobilizable shuttle vector, pDL289, were at least
recipient chromosome, as depicted in the bottom 10-fold lower than the frequencies obtained with
segment of Figure 2, and the plasmid has been lost. pDL278. A fragment of S. sobrinus chromosomal
A third possibility is that integration of the plasmid DNA from which most of scrA and scrB was deleted
has occurred by a single crossover event, but that it and replaced by the spc gene was subcloned into
also continues to replicate in the host as an inde-
pendent replicon. The three possibilities could be Table 1. Frequencies of transformation of S. sobrinus
distinguished by conducting Southern hybridization 6715 with different plasmids
experiments similar to those described above, but
with DNA from the transconjugants with a SpRKmR Transforming plasmid Frequencies of transformation"
phenotype that has been digested with a different
pDL278 0.54-1.6 X 103
enzyme, e.g., EcoR!. In this case, transconjugants pDL278scrBscrAPcat 2.30-3.0 X 102
in which the plasmid is replicating solely as an pDL289 0.15-1.5 X 102
independent replicon would yield a fragment that pDL289scrABspc 0.05-0.9 X 102
hybridized with the scrA-specific probe of approxi-
mately 10.6 kb in size. A second EcoRI fragment, " Transformation frequencies expressed as transfor-
1.2 kb in size (see Figure 1), representing the DNA mants/Ilg DNA.
92
19. Macrina FL, Keeler CL Jr, Jones KR, Wood PH 23. Perry D, Kuramitsu HK (1981). Genetic transforma-
(1980). Molecular characterization of unique deletion tion of Streptococcus mutans. Infect Immun 32:
mutants of the streptococcal plasmid, pAM~ 1. 1295-1297.
Plasmid 4: 8-16. 24. Westergren G, Emilson CG (1983). Prevalence of
20. Macrina FL, Tobian JA, Jones KR, Evans RP, Clewell transformable Streptococcus mutans in human dental
DB (1982). A cloning vector able to replicate in plaque. Infect Immun 41: 1386-1388.
Escherichia coli and Streptococcus sanguis. Gene 19:
345-353.
2l. Macrina FL, Wood PH, Jones KR (1980). Genetic
transformation of Streptococcus sanguis (Challis) with Address for correspondence: Donald J. LeBlanc, Lilly
cryptic plasmids from Streptococcus ferus. Infect Research Laboratories, Eli Lilly and Company, Lilly
Immun 28: 692-699. Corporate Center, Drop Code 0438, Indianapolis, IN
22. Murchison HH, Barrett JF, Cardineau GA, Curtiss R 46285, USA
3rd (1986). Transformation of Streptococcus mutans Phone: (317)-433-3922; Fax: (317)-276-1743
with chromosomal and shuttle plasmid (p YA629) E-mail: leblanc_donaldj@lilly.com
DNAs. Infect Immun 54: 273-282.
Methods in Cell Science 20: 95-106 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. We have devised a procedure using cosmid clones were analyzed by restriction enzyme
immune sera to identify antigen-encoding genes of digestions and clones containing distinct inserts were
strains of Enterococcus faecalis. First, genomic chosen for subcloning. Sublibraries were screened
cosmid libraries containing large inserts were con- with one of the five sera, and immunopositive sub-
structed and screened with sera from patients with clones were subjected to DNA sequencing. BLASTX
enterococcal infectious endocarditis and with serum and BLASTN at NCBI were used to search for
from a rabbit immunized with surface proteins of an database similarities.
enterococcal endocarditis isolate. Immunopositive
1. Introduction 2. Materials
of the above diluted DNA to tube 1,20 DNA fragments of the desired size (35
~l to tubes 2 through 10. Keep all tubes to 50 kb for pBeloBACll, 30 to 40 kb
on ice. for pTEX5176, and 20 to 35 kb for
3) Add 2 units of Sau3AI to tube 1, mix pLAFRx). Dialyze against TE at 4 DC,
gently. Transfer 20 ~l from tube 1 to change buffer 3 to 4 times.
tube 2, mix gently. Transfer 20 ~l from 7) Transfer the DNA solutions to fresh
tube 2 to tube 3, mix. Continue till tube microfuge tubes and precipitate with
9. Discard 20 ~l from tube 9. Do not ethanol.
add anything to tube 10. All the tubes 8) Dissolve the DNA in TE to an approx-
should be kept on ice in this step. imate concentration of 400 ng/~l,
4) Incubate all tubes at 37 DC for exactly measure DNA concentration and check
1 hour. by electrophoresis through a 0.4%
5) Heat the tubes at 70 DC for 15 minutes agarose gel. Store at 4 DC.
to inactivate the enzyme. Spin briefly. 3. Preparation of vector DNA
Add 2.2 ~l lOx gel loading buffer, load a. Large-scale preparation of cosmid DNA
onto a 0.4% agarose gel. Run the gel uses the alkaline lysis procedure followed
slowly « IV/cm). Take pictures. by CsCI-ethidium bromide gradient cen-
b. Large-scale preparation of partially trifugation [22].
digested genomic DNA b. Digest 5 ~g clean DNA with 1 unit of
1) Choose a condition in the pilot experi- BamHI at 37 DC for 1 hour. Remove an
ment that gives the highest yield of aliquot and apply to a 0.7% agarose gel to
DNA fragments with sizes of 20-50 kb, examine if the digestion is complete. If it
in our case, it was about 0.7 units is not, add more enzyme and continue
Sau3AI/mg DNA at 37 DC for 1 hour. incubation for another hour.
Digest each 100 ~g of high-molecular- c. When digestion is complete, extract the
weight genomic DNA with 0.07 and sample with phenol:chloroform and pre-
0.14 units of Sau3AI, respectively, for cipitate with ethanol. Dissolve in 50 ~l TE.
1 hour at 37 DC. Remove 5 ~l DNA solution and save for
Since the conditions in the large scale later use.
digestion are not absolutely identical to d. Add 10 ~l lOx shrimp alkaline phosphatase
the conditions in the pilot, it is worth- (SAP) reaction buffer, 45 ~l water and 1
while to do an additional digestion unit SAP to the DNA solution, incubate at
using one of the neighboring conditions. 37 DC for 1 hour. Heat at 65 DC for 20 min
2) Add EDTA to a final concentration of to inactivate the enzyme.
12 mM at the end of the reactions (for e. Extract twice with phenol: chloroform,
a 200 ~l reaction, add 4.8 ~l of 0.5 M precipitate with ethanol. Dissolve in 50 ~l
EDTA, pH8.0). Load approximately 5 TE (approximately 100 ng/~l). Measure
~g of DNA from each digestion reaction DNA concentration.
onto a 0.4% agarose gel to examine the f. Test ligations of vector DNA
extent of the digestions. Pool the digests Set up six 5 ~lligation reactions containing
and store at 4 DC. the following:
3) Prepare a 38 ml 10-40% continuous 1) 100 ng SAP treated vector + 50 ng
sucrose density gradient in a Beckman BamHI digested pBluescript SK (-) (as
SW 28 ultracentrifuge tube (or equiva- control insert DNA) + 1.5 units of T4
lent). Adjust the volume of the pooled DNA ligase.
digests to 2 ml with TE, and carefully 2) 100 ng SAP treated vector + 50 ng
load onto the gradient. Centrifuge at Bam HI digested pBluescript SK (-), no
24,000 rpm for 20 hours at 20 DC. ligase.
4) Prepare a rack of microfuge tubes. A 40 3) 100 ng SAP treated vector +1.5 units T4
ml gradient will need about 80 tubes. DNA ligase.
Number all tubes and place them in 4) 100 ng SAP treated vector, no ligase.
appropriate racks in order. Collect 5) 100 ng BamHI digested, vector not
sucrose fractions, -0.5 ml/tube. treated with SAP + 1.5 units T4 DNA
5) Dilute 20 ~l of every fourth fraction ligase.
with 20 ~l water, add 4.4 ~l of lOx 6) 100 ng Bam HI digested, vector not
gel loading buffer. Analyze by elec- treated with SAP, no ligase. Add 0.5 ~l
trophoresis through a 0.4% agarose lOx ligation buffer, and water to each
gel. mixture. Incubate at room temperature
6) Pool the gradient fractions containing for 4 hours or at 14 DC overnight. Add
98
0.6 III gel loading buffer and load onto to calculate the efficiency as plaque
a 0.7% agarose gel to examine the forming units/Ilg DNA. Pick a number of
extent of ligations. colonies and inoculate LB broth with
4. Ligation of cosmid vector to partially digested appropriate antibiotics to prepare DNA
genomic DNA, in vitro packaging and storage (will be described below). Digest the DNA
a. Set up two ligation reactions both con- with restriction enzymes to determine the
taining 1 III vector DNA, 0.5 III lOx sizes of the inserts.
ligation buffer, 0.5 III T4 ligase, and 1 III e. When the test packaging produces good
water. Add 2 III partially digested genomic results, a normal scale packaging can be
DNA to the first tube, and 2 III water to performed following the instruction
the second. Incubate at room temperature manual. Briefly, the remains of the first
for 4 hours or 14°C overnight. ligation mixture is split into two and added
b. Preparation of host bacteria: inoculate 5 ml to two packaging extracts. After incubation
of LB with 0.2% (w/v) maltose-l0 mM at RT for 2 hours, add 500 III of SM buffer
MgS04 with a fresh single colony of E. coli followed by 25 III of chloroform to each
DH5a and the control host VCS257 reaction. A positive control with lambda
(included in the Gigapack III Gold DNA and VCS257 is also carried out.
Packaging Extract kit), respectively, shake Based on the colony numbers from the test
at 37°C for 4-6 hours (do not grow past packaging and plating, dilute the packaging
an 00 600 of 1.0). Harvest the bacteria by reaction to a level that will yield approxi-
centrifugation at 4000 rpm for 10 minutes. mately 100 to 200 colonies/plate. Use a
Gently resuspend the cells to an 00 600 of freshly prepared batch of cells for plating.
0.5 with sterile 10 mM MgS04 (should be f. Storage of the cosmid libraries: add 200 III
used immediately after this). LB broth with the appropriate antibiotics to
c. In vitro packaging and plating follow the the wells of 96-well micro titer plates. Pick
instructions from the Gigapack III Gold with sterile toothpicks single colonies from
Packaging Extract kit with slight modifi- the primary plating to each of the wells.
cations. A small-scale test packaging Incubate the micro titer plates at 37°C
should be carried out first. Quickly thaw a overnight with shaking. Add 30 III of sterile
packaging extract, carefully split into 4 glycerol to each well (final concentration
aliquots. To each aliquot, add 0.5 III of the 15 %), transfer 115 III culture from each
first ligation mixture (vector + insert), 0.5 well to a new microtiter plate as a back up.
III of the second ligation mixture (vector Store both at -80°C. Store the remains of
alone), 0.5 III of wild-type lambda DNA the packaging reaction mixture at 4 °C in
(included in the kit), and 0.5 III of water, the presence of chloroform in a clean glass
respectively. Mix well by stirring each tube tube.
with a pipet tip. Incubate the tubes at room C. Preparation of the antisera.
temperature for 2 hours (do not exceed 2 1. Source of antisera
hours). Add 125 III of SM buffer to each Four sera were collected from patients diag-
tube, add 5 III of chloroform and mix nosed by their physicians as having E. faecalis
gently. Store at 4 °C until ready for titering endocarditis. A rabbit serum was obtained
(1 to 2 days). from a rabbit immunized with surface proteins
d. Titering the test packaging reactions: of an E. faecalis endocarditis isolate [24].
prepare a 1: 10 and a 1:50 dilution of each 2. Testing the antisera against enterococcal
of the packaging reactions in SM buffer. strains and E. coli by Western blot analysis:
Mix 25 III of each dilution with 25 III of the a. Surface protein extracts were prepared
prepared host cells. For lambda DNA, using the detergent Zwittergent 3-12 [12].
VCS257 is used as plating cells, and for the Inoculate BHI broth with single colonies of
others, DH5a is used. After incubation of E. faecalis strains and LB broth with E.
the tubes at room temperature (RT) for 30 coli, and shake overnight at 37°C. Adjust
minutes, 200 III of LB broth is added to to a density of about 160 Klett units
each sample. Incubate the tubes at 37°C (measured in a Klett-Summerson photo-
for 1 hour with occasional shaking. Plate electric colorimeter) with BHI or LB broth.
the cells on LB agar plates with appropriate Take an equal volume from each adjusted
antibiotics. Incubate overnight at 37°C. culture, harvest the cells by centrifugation
Count the colonies and calculate the titer to at 4000 rpm for 10 minutes, and wash once
colony forming units (cfu)/Ilg DNA. with equal volume of phosphate buffered
Plating of the lambda control follows the saline (PBS). Resuspend in 1% of the
instruction manual and plaques are counted adjusted culture volume in PBS with 0.2%
99
Zwittergent 3-12. Place the suspension in prong Boekel replicator. Incubate at 37°C
a roller for 1 hour at RT, transfer to fresh overnight. Lift the filters from the plates, and
microfuge tube and centrifuge for 5 place in a chloroform chamber colony side up,
minutes at 10,000 g. Dialyze the super- for 15 minutes (this should be done in a
natant against 50 mm Tris-HCI, pH 7.5 chemical fume hood). Transfer the filters to
overnight at 4°C, and store at -20°C. lysis buffer I and shake at RT for 12-16 hours.
b. SDS-polyacrylamide gel electrophoresis Alternatively, transfer the filters to lysis buffer
(PAGE) is performed based on the Laemmli II which contains a higher concentration of
system, using a 4% stacking and 10% sep- lysozyme, incubate at RT for 1 hour, transfer
arating gel in a Tris-glycine buffer system to a fresh batch of lysis buffer II and incubate
[22]. Add an equal volume of 2x loading at RT for another hour. Transfer to TNT buffer,
buffer to each extract from about 2 ml of use a gloved finger to wipe off any residue of
the initial adjusted culture, boil for 5 the colonies from the filters, incubate for 10
minutes and subjects to SDS-PAGE. minutes at RT with shaking. Repeat twice. The
c. Transfer proteins from the gel to an filters are ready to use in immunoblotting, or
Immobilon-P transfer membrane using the can be wrapped in Saran Wrap and stored at
MilliBlot™-SDE System. Incubate the 4 °C for 1-2 days.
membrane in 3% skim milk blocking buffer 2. Blocking, incubation with antiserum and
for at least 1 hour at room temperature with protein A-peroxidase and color development
gentle shaking, and then incubate with the are the same as in Western blot.
primary antisera solution (1:500 dilution in 3. Re-screen. Immunopositive clones from the
1% skim milk) overnight at 4 °C with initial screening are streaked and picked to a
gentle shaking. Wash the membrane with new 96-well microtiter dish containing 200 ml
1% skim milk 3 times (5 minutes each of LB broth with appropriate antibiotics in
time), add the protein A-peroxidase each well, and incubated at 37°C overnight
solution, and incubate for at least I hour with shaking. Add glycerol to each well, and
at RT. Wash the membranes thoroughly 3 split the contents into two dishes as in the
times with 50 mm Tris, pH 7.4 (5 minutes storage of library clones. Inoculate these
each time), and then incubate with the color clones onto the filters and screen again as
development mix for 30 minutes, covered described above. All five sera are used in the
with a sheet of aluminum foil. Rinse the second screen.
membranes thoroughly with distilled water, E. Subsequent analysis of antigen-expressing cosmid
and air dry at RT. clones
3. Absorption of antisera with E. coli lysates. 1. DNA preparation and restriction enzyme
a. Preparation of lysates follows the method digestion (RED) analysis
described [22]. Briefly, grow the E. coli a. Grow up clones in 10 ml of LB broth with
host strain containing the cloning vector in appropriate antibiotics, and carry out the
100 ml of LB broth with appropriate antibi- standard SDS-alkaline lysis method [22].
otics. Harvest the cells by centrifugation at Resuspend the cell pellet in 150 III of
4000 rpm for 10 minutes. Resuspend the solution I, add 300 III of freshly made
cells in 3 ml of 50 mM Tris-HCI (pH 8.0) solution II, mix, incubate at RT for 5
and 10 mM EDTA (pH 8.0). Freeze and minutes, add 225 III of solution III, mix and
thaw for a few times, and then sonicate to chill on ice for 5 minutes. Centrifuge for
lyse the cells. Store the lysate at -20°C. 10 minutes in a desktop centrifuge (15,000
b. Absorption: dilute 100 III of the antiserum rpm), transfer the supernatants to clean
with 850 III of 1% skim milk, add 50 III of microfuge tubes. Extract with phenol:chlo-
the lysate, and incubate at RT for 4 hours roform, and precipitate with ethanol.
with gentle shaking. Store the absorbed Resuspend the DNA pellets in 50 III of TE
serum at -20°C. with RNase.
D. Immunoscreening of the cosmid libraries for b. Set up restriction digestions in 50 III
antigen-expressing cosmid clones reaction volumes. For pBeloBACll clones,
1. Preparation of filters. Label a number of sterile use 25 III of the miniprep DNA, and for
NitroPlus nitrocellulose transfer membrane pLAFRx and pTEX5176 clones, use 10 III
filters which have been cut to the size of a 96- of DNA. Add 5 III lOx reaction buffer, 1 III
well microtiter plate (or 137 mm circular of enzyme (EcoRI or HindIII), and water to
filters), and carefully place each on top of a bring the volume up to 50 Ill. Incubate at
LB agar plate with the appropriate antibiotics. 37°C for 2-3 hours. Add 5.5 III lOx gel
Inoculate library clones stored in 96-well loading buffer, apply to a thick 0.7%
microtiter plates onto the filters using a multi- agarose gel and carry out electrophoresis.
100
c. Compare RED patterns to find clones with the remaining 19 III DNase I treated cosmid
overlapping inserts. DNA, add 5 III lOx Klenow buffer, 1 III
2. Western blot analysis lOx BSA, 1 III dNTP mix (25 mM of each
a. Preparation of protein extracts from recom- dNTP), 21 III water and 3 III Klenow
binant clones involved the use of 3 fragment. Incubate at RT for 20 minutes,
methods. First, the detergent Zwittergent heat at 75°C for 15 minutes to inactivate
3-12 is used as in the preparation of surface Klenow fragment. Extract with
protein extracts from enterococcal strains. phenol:chloroform, precipitate with ethanol
Second, 0.5 ml of an overnight culture of and resuspend in 20 III of TE.
recombinant clones is centrifuged, resus- e. Plasmid pBluescript SK (-) is used to clone
pended in 40 III of Ix SDS-sample buffer, fragments from cosmid DNA. Preparation
boiled for 5 minutes and applied to SDS- of pBluescript SK (-) follows the procedure
PAGE. Third, 0.125 ml of ice-cold 100% used for cosmid vectors, except that EcoRV
trichloroacetic acid is added to the super- is used instead of BamHI. Test ligations are
natant of 1.2 ml of overnight culture, the carried out using 50 ng of vector and 25
tubes are chilled on ice for 5 minutes and ng of a blunt ended DNA fragment as the
centrifuged for 15 minutes at the highest control insert. Ligation mixtures are incu-
speed in a desktop centrifuge at 4 0c. After bated overnight at 14°C. At the end of the
the centrifugation, 1 ml of ice-cold acetone ligation, 1I25th of the ligation mix is used
was added to each pellet, the tubes were to transform DH5a competent cells (will be
vortexed briefly and centrifuged again for described below) to assess the quality of
5 minutes. The pellets are then air dried, the prepared vector.
resuspended in Ix SDS sample buffer, f. Ligation of DNase I treated cosmid to
boiled for 5 minutes and subjected to SDS- pBluescript SK (-) vector. Set up a 5 III
PAGE. ligation reaction with 1 III of SAP treated
b. SDS-PAGE and Western blot are as pBluescript SK (-),2.5 III insert DNA, 0.5
described above. III lOx ligation buffer, 0.5 III T4 DNA
3. Subcloning of antigen-encoding genes from ligase, and 0.5 III water. For ligation
immunopositive cosmid clones. control, use 0.5 III pBluescript SK (-)
a. Preparation of cosmid DNA. The same without SAP treatment, and 2.5 III water
method used to purify cosmid vectors instead of the insert DNA. Incubate
(alkaline lysis and CsCl-ethidium bromide overnight at 14°C. Dilute the reactions to
gradient centrifugation) is used. 50 III with TE, use 2 III to transform 100
b. Pilot experiments to determine the condi- III of DH5a competent cells.
tions for DNase I treatment of cosmid g. Preparation of competent cells and trans-
DNA. Dilute 5 Ilg of cosmid DNA in 52.5 formation are based on the one-step TSS
III Ix DNase I reaction buffer, take a 5 III method [4].
aliquot and keep on ice. Dilute DNase I to h. About 600 to 1000 transformants from each
0.01 units/ill in Ix reaction buffer, add 2.5 cosmid can be screened in a similar way
III of the diluted DNase I to the DNA as described for the immunoscreening of
solution, and incubate at RT. Take a 5 III the cosmid library. The differences are that
aliquot after every minute, add 1 III of (a) after the colonies grow up on the
0.1 M EDTA (pH 8.0) to each aliquot primary transformation plates, they are
immediately and keep on ice. After the last lifted onto NitroPlus nitrocellulose transfer
aliquot is done, add gel loading buffer and membrane filters directly; (b) immuno-
apply to a 0.7% agarose gel, followed by positive clones from the first screen are
electrophoresis. In our case, 4-5 minutes streaked and then spotted onto LB agar
of incubation at RT produced DNA frag- plates containing 100 Ilg of ampicillin and
ments largely concentrated at 0.5 to 2 kb. 0.5 mm IPTG; after overnight growth, the
c. Incubate 5 Ilg of cosmid DNA with 2.5 colonies are lifted and subjected to another
units of DNase I in 100 III reaction at RT round of screening; (c) tertiary screening is
for 4-5 minutes, stop the reaction by also carried out when necessary.
adding 12 III of 0.1 m EDTA (pH 8.0). 2. DNA sequencing of the immunopositive sub-
Extract twice with phenol:chloroform, pre- clones and sequence analysis
cipitate with ethanol, and resuspend in 20 a. Templates for sequencing are prepared
III of TE. Take 1 III for 0.7% agarose gel using the Qiagen plasmid mini kit, and
electrophoresis to check the extent of primers are for the T3 and T7 promoter
degradation. regions on pBluescript SK (-). DNA
d. Fill-in the ends with Klenow fragment: to sequencing reactions are performed using
101
the Taq dye-deoxy terminator kit and run seemed that enterococcal DNA in pTEX5176 was not
on either an ABI 373A or 377 DNA seque- very stable. Clones we generated in two other
nator. vectors, pBeioBACll (low copy number) and
b. Delete the pBluescript SK (-) vector region pLAFRx (medium copy number), did not show this
sequence from the DNA sequences using un stability. Cosmid pBeloBAC 11 is an expression
a sequence editor such as Seqed in the vector derived from the bacterial F factor, a low copy
GCG software package. The sequences are number plasmid [24]. Other F factor-based vectors
submitted through the Internet to the have been shown to maintain very large inserts in E.
BLAST (BLASTX and BLASTN) network coli [23]. Cosmid pLAFRx was derived from the
service at NCB I to look for homologous RK2 R-factor, has a copy number of about 10
sequences in the databases. copies/cell, and is not an expression vector (Table 1,
Figure 2). The majority of clones in libraries con-
structed with these two vectors seemed to have the
4. Results and discussion expected insert sizes (Table 2).
a The background is calculated as the percentage number of colonies on the vector only plate versus colonies on the
vector plus insert plate.
b Library pTEX5176-0GIRF was screened with a rabbit serum only, pTEX5I76-TX52 was not screened, the others were
screened with a patient serum first and the initial immunopositive clones were rescreened with five sera.
C Genome coverage is calculated as: the number of clones screened x average insert size/genome size.
d For pTEX5176-0GIRF, the number indicate clones reacted with the rabbit serum; for the others, the numbers indicate
1 2 3 4 5 6 7 8 9 10 11 12 13 14 KD
- 99
- 64
- 50
- 30
- 3J
Figure 3. Western blot analysis of E. faecalis strains with one of the patient sera. All samples were prepared using the
Zwittergent method. From lane 1 to lane 14 are: TXll, TX6S, TX 20, TXS2, TX44, TX12, TX17, TX1, TX4, TXS,
OG1RF, an E. coli recombinant clone, and DHSalpBe1oBACll, and the molecular weight standard (see Blue™ Pre-
stained Standards, Novex, San Diego, CA). The patient serum was diluted 1000 fold.
of Western blot analysis of immunopositive cosmid taining a putative polysaccharide biosynthesis gene
clones (data not shown): 1) there were differences cluster. We do not believe it would have been
in the antigen bands with protein samples prepared possible to isolate this clone with the standard small
by different methods, e.g., some antigen bands only insert expression library approach.
appeared in Western blots using the Zwittergent The possibility that a sequence of interest is
extract; 2) some cosmid clones were obviously present in a random library can be estimated using
positive in the immunoscreens, but did not show any the following equation based on Poisson distribution:
clear antigen bands in Western blots, possibly N = In(1 - P)/ln[l - (JIG)] where I is the average size
because the antigen co-migrated with a host band that of the inserts, G is the size of the genome, and N is
had cross reactivity with the antisera, or the antigen the number of independent clones that must be
was aggregated and did not dissolve easily in the gel screened to isolate a particular sequence with
loading buffer. In addition, one clone turned out to probability P (10). For a 99% probability of isolating
produce a polysaccharide antigen in E. coli, a sequence of interest, the equation predicts that the
explaining why it behaved differently than protein total number of base pairs screened (J x N) be 4.6-
antigens. fold in excess over the total number of base pairs in
The procedure of subcloning antigen-encoding the genome (G). According to this calculation, N
genes from immunopositive cosmid clones has been should be 380 for pBeloBACll clones (J = 34 kb),
applied successfully to at least 38 clones, and several and 530 for pLAFRx clones (J = 24.5 kb). However,
thousand transformants can be easily obtained from it is known that certain regions of a genome are more
each sublibrary. Twenty-five clones gave 53 difficult to clone, possibly due to unique DNA
immunopositive subclones while 13 did not produce structure or toxic functions encoded in the region,
any immunopositive subclones, possibly because the and consequently the library is not completely
antigen-encoding genes were unstable in a high-copy random. Therefore we screened a larger number of
number vector or the production of antigen required clones to increase the chance of isolating the antigen
more than one gene. genes. Critical evaluation as to whether we have
BLAST searches of the DNA sequences from the isolated all the antigen-encoding genes is difficult to
immunopositive subclones showed that a wide range make because: (1) it is not known how many antigens
of genes were cloned (Table 3), including those the enterococcal strains produce, and (2) it is not
encoding transporters, potential virulence factors, cell clear how many antibodies against enterococci are
surface proteins, metabolic enzymes and hypothet- present in each serum at high enough titer for immun-
ical proteins. The majority of these were predicted to odetection. However, a comparison of the results
be surface or membrane proteins, consistent with the obtained by Western blot analysis to those obtained
hypothesis that these proteins could be seen by the by immunoscreening, assuming that each antigen
immune system in infection. In addition, we also band on the Western blot represents a different
identified a pBeloBACll clone (45 kb insert) con- antigen, can provide an estimate. There were 10 to
104
all 5 sera
subclone
screen
53 immunopositive
clones
DNA sequencing
/ sequence analysis
39 subclones
Figure 4. A flow chart of the immunoscreening, cloning and DNA sequencing results.
20 antigen bands in most of the strains tested (Figure 3. Calbiochem Co., La Jolla, CA 92037, USA
3), and at least 18 unique putative antigen-encoding 4. Cambridge Technology, 23 Elm Street, Watertown,
genes that showed sequence similarity to genes in the MA 02172, USA
database (Table 3) were found by immunoscreening. 5. Corning Glass Works, Corning, New York 14831,
Taking into consideration the 14 sequences from USA
6. Difco Laboratories Inc., Detroit, MI 48232-7058, USA
immunopositive subclones that did not have any
7. Fisher Scientific, Fair Lawn, New Jersey 07410, USA
database similarity (Figure 4), the number of poten- 8. Klett MFG. CO. INC., New York, USA
tial antigen-encoding genes isolated by immuno- 9. Micro Separations Inc., Westboro, MA 01581, USA
screening is of the same order as the number of 10. Millipore Co., Bedford, MA 01730, USA
antigen bands observed in the Western blots. Two 11. New Englsnd Biolabs Inc., 32 Tozer Road, Beverly,
previously identified E. faecalis genes, one of which MA 01915-5599, USA
encodes an antigen associated with endocarditis 12. Novex, 11040 Roselle St., San Diego, CA 92121,
infections (EfaA) [17], and the other encoding a USA
surface protein (autolysin) [2], were detected by our 13. Qiagen Inc., 9600 De Soto Avenue, Chatsworth, CA
immunoscreening. These results suggest that our 91311, USA
libraries are representative, and that immuno- 14. Sigma, PO Box 14508, St. Louis, MO 63178, USA
15. Stratagene, 11011 North Torrey Pines Road, La Jolla,
screening is reasonably thorough. However, we note
CA 92037, USA
that each library only identified a few antigen- 16. United States Biochemical Co., PO Box 22400,
encoding genes per genome equivalent cloned (Table Cleveland, Ohio 44122, USA
2), clearly lower than the abundance of antigen-
encoding genes. This is likely due in part to the non-
randomness of libraries, discussed above, but may References
also relate to the low copy number of the vectors.
Since inserts are large, expression in E. coli relies 1. Allen AG, Perham RN (1991). Two lipoyl domains
on endogenous signals, and at low copy number, this in the dihydrolipoamide acetyltransferase chain of the
may be inefficient. It is possible that expression is pyruvate dehydrogenase multienzyme complex of
affected by the location of the gene in the insert, i.e., Streptococcus faecalis. FEBS Lett 287: 206-210.
inserts in pBeloBACll with an antigen encoding 2. Beliveau C, Potvin C, Trudel J, Asselin A, Bellemare
gene near the lac promoter are more likely to be G (1991). Cloning, sequencing, and expression in
immunopositive. Thus, high coverage is important. Escherichia coli of a Streptococcus faecalis autolysin.
J Bacterioll73: 5619-5623.
It is likely that libraries made in high copy number
3. Chen J-D, Morrison DA (1988). Construction and
expression vectors, such as pBluescript SK (-), may
properties of a new insertion vector, pJDC9, that is
facilitate detection of antigens with lower titer anti- protected by transcriptional terminators and useful for
bodies in serum. However, antigens dependent on cloning of DNA from Streptococcus pneumoniae.
large inserts, such as polysaccharides, would be Gene 64: 155-164.
missed. 4. Chung CT, Niemela SZ, Miller RH (1989). One-step
preparation of competent Escherichia coli: Trans-
formation and storage of bacterial cells in the same
Acknowledgment solution. Proc Nat! Acad Sci USA 86: 2172-2175.
5. Clarke L, Carbon J (1976). A colony bank containing
synthetic ColEl hybrids representative of the entire E.
This work was supported by grant AI 33516 from the
coli genome. Cell 9: 91-99.
PHS. 6. Dean CR, Poole K (1993). Expression of the ferric
We are very grateful to Ronald Christopher enterobactin receptor (PfeA) of Pseudomonas aerug-
Mackenzie for his technical support in constructing inosa: Involvement of a two-component regulatory
the genomic and DNase I random libraries, per- system. Mol Microbiol 8: 1095-1103.
forming DNA sequencing and advice on sequence 7. Ebbole DJ, Zalkin H (1987). Cloning and characteri-
analysis, Kavindra V. Singh and Monjula zation of a 12-gene cluster from Bacillus subtilis
Chidambaram for general technical support, H. encoding nine enzymes for de novo purine nucleotide
Shizuya and M. Simon at the California Institute of synthesis. J BioI Chern 262: 8274-8287.
Technology, Pasadena, CA, for providing us with the 8. Ebel-Tsipis J, Botstein D, Fox MS (1972).
cosmid vector pBeloBACll, Laura Bankey and Beto Generalized transduction by phage P22 in Salmonella
typhimurium. I. Molecular origin of transducing DNA.
Zuniga for technical support.
J Mol BioI 71: 433-448.
9. Friedman AM, Long SR, Brown SE, Buikema WJ,
Ausubel FM (1982). Construction of a broad host
Notes on suppliers range cosmid cloning vector and its use in the genetic
analysis of Rhizobium mutants. Gene 18: 289-296.
1. AMRESCO, Solon, Ohio 44139, USA 10. Furst P, Mosch H-U, Solioz M (1989). A protein of
2. Bio-Rad Laboratories, 2000 Alfred Nobel Dr., unusual composition from Enterococcus faecium.
Hercules, CA 94547, USA Nucleic Acids Res 17: 6724.
106
11. Ganeshkumar N, Hannam PM, Kolenbrander PE, 18. Murray BE (1990). The life and times of the entero-
McBride BC (1991). Nucleotide sequence of a gene coccus. Clin Microbiol Rev 3: 46-65.
coding for a saliva-binding protein (SsaB) from 19. Murray BE, Singh KV, Ross RP, Heath JD, Dunny
Streptococcus sanguis 12 and possible role of the GM, Weinstock GM (1993). Generation of restriction
protein in coaggregation with actinomyces. Infect map of Enterococcus faecalis OGI and investigation
Immun 59: 1093-1099. of growth requirements and regions encoding biosyn-
12. Kessler RE, Yagi Y (1983). Identification and partial thetic function. J Bacteriol 175: 5216-5223.
characterization of a pheromone-induced adhesive 20. Mytelka DS, Chamberlin MJ (1996). Escherichia coli
surface antigen of Streptococcus faecalis. J Bacteriol jliAZYoperon. J Bacteriol 178: 24-34.
155: 714-721. 21. Rodriguez F, Grandi G (1995). An operon encoding
13. Khandke KM, Fairwell T, Acharya AS, Trus BL, a novel ABC-type transport system in Bacillus
Manjula BN (1988). Complete amino acid sequence subtilis. Microbiology 141: 1781-1784.
of streptococcal PepM49 protein, a nephritis-associ- 22. Sambrook J, Fritsch EF, Maniatis T (1989). Molecular
ated serotype. Conserved conformational design cloning, a laboratory manual. Cold Spring Harbor,
among sequentially distinct m protein serotypes. J BioI NY: Cold Spring Harbor Laboratory Press.
Chern 263: 5075-5082. 23. Shizuya H, Birren B, Kim U-J, Mancino V, Slepak
14. Klein JR, Henrich B, Plapp R (1991). Molecular T, Tachiiri Y, Simon M (1992). Cloning and stable
analysis and nucleotide sequence of the envCD operon maintenance of 300-kilobase-pair fragments of human
of Escherichia coli. Mol Gen Genet 230: 230-240. DNA in Escherichia coli using an F-factor-based
15. Klose KE, Mekalanos JJ (1997). Simultaneous pre- vector. Proc Natl Acad Sci USA 89: 8794-8797.
vention of glutamine synthesis and high-affinity trans- 24. Xu Y, Jiang L, Murray BE, Weinstock GM (1997).
port attenuates Salmonella typhimurium virulence. Enterococcus faecalis antigens in human infections.
Infect Immun 65: 587-596. Infect Immun 65: 4207-4215.
16. Lansing M, Lellig S, Mansolf A, Martini I, Crescenzi
F, O'Regan M, Prehm P (1993). Hyaluronate
synthase: Cloning and sequencing of the gene from Address for correspondence: George M. Weinstock,
Streptococcus sp. Biochem J 289: 179-184. Department of Microbiology and Molecular Genetics,
17. Lowe AM, Lambert PA, Smith AW (1995). Cloning University of Texas Medical School, 6431 Fannin St.,
of an Enterococcus faecalis endocarditis antigen: Houston, TX 77030, USA
Homology with adhesins from some oral streptococci. Phone: (713)-500-6083; Fax: (713)-500-0652
Infect Immun 63: 703-706. E-mail: georgew@utmmg.med.uth.tmc.edu
Methods in Cell Science 20: 107-111 (1998)
© 1998 Kluwer Academic Publishers.
Molecular Microbiology & Immunology, University of Missouri-Columbia Medical School, Columbia, Missouri, USA;
3 Department of Medicine, Veteran Affairs Medical Center and University of California - San Francisco, San
Francisco, California, USA; 4 Division of Infectious Diseases, Children's Hospital & Medical Center, University of
Washington, Seattle, Washington, USA
Abstract. Many bacteriologic studies, including cumbersome and severely limit the number of
cellular invasion and adherence assays, require organisms or conditions which can be tested. As a
enumeration of viable organisms or colony forming potential alternative, we describe a simple, rapid
units. When attempting to screen large numbers of and inexpensive soft agar-based technique for semi-
clinical or environmental isolates or laboratory- quantitative determination of bacterial colony counts
derived mutants for differences in invasion or adher- directly within the wells of a 96-well microtiter
ence phenotype, standard plating methods can be plate.
Key words: Bacterial adherence, Bacterial invasion, Bacteriological techniques, Invasion assay, Microbial
colony count, Soft agar
modified our protocol to eliminate the labor-inten- cases, a replica plate with 200 IJ,1 of fresh
sive plating steps, and employed soft agar to enu- media per well may be inoculated with 10 IJ,1
merate the bacteria directly within wells of a 96- of the overnight cultures and growth monitored
well micro titer plate. We subsequently adapted by optical density measurement using a
this methodology to develop a screening assay for microplate reader.
streptococcal adherence to human platelets. 2. Inoculation of cell monolayers
Immediately prior to the invasion assay, each
plate is vortexed gently to resuspend settled
2. Materials bacteria. Optical density measurement can be
obtained to identify any strains or mutants
1. 96 well tissue culture plates, flat bottom, sterile which grew poorly, such that any spuriously
with lid (Costar #3598). low input inoculum can be considered in inter-
2. Todd Hewitt Broth (Difco #DF0492-17-6). pretation of final invasion assay results. Using
3. Bacteriologic grade agar (Difco DFOI40-01-0). an 8- or 12-channel pipettor, 10 IJ,1 of each bac-
4. A549 human lung carcinoma cell line (ATCC terial culture is transferred as an inoculum to
#CCL-185). a corresponding well in a 96-well plate con-
5. RPMI 1640 tissue culture media, with HEPES taining 200 IJ,1 antibiotic-free tissue culture
(Sigma #R 6504). media and a confluent A549 cell monolayer.
6. Fetal calf serum, heat inactivated (Sigma Alternatively, the plates may be inoculated
#F 4135). using a Boekel 99-pin stainless steel replicator
7. 12-channel pipettors (Labsystems #4510040 and which transfers a standard volume of the liquid
4510050). culture by capillary action.
8. Boekel 96-pin stainless steel replicator (Fisher 3. Antibiotic protection method for determination
#05-450-9). of cellular invasion
9. Reagent reservoir (Sigma #R 1936). Following inoculation, the 96-well tissue
10. Bent stainless steel manifold (Drummond #3-00- culture plates are centrifuged at 800 Xg for 10
094). min to place GBS at the surface of the cell
11. Gentamicin sulfate (Sigma #G 1264). monolayer, then incubated for 2 h at 37 DC
12. Penicillin G (Sigma #P 7794). with 5% CO 2 to allow cellular invasion by the
13. 0.25% TrypsinlEDTA solution (Sigma #T 4049). GBS. After the incubation, medium is removed
14. Triton X-I00 (Sigma #X-I00). from the monolayers by gentle aspiration of
15. Poly-L-Lysine (Sigma #P 4832). the wells using a bent-hub 8-channel stainless
16. Brain-Heart Infusion (BHI) Broth (Difco steel manifold attached to a vacuum source.
DF0037-07-0). The subsequent wash and treatment steps
17. Tyrode's salt solution (Sigma #T 2397). all involve liquids dispensed from a sterile
reagent reservoir. Monolayers are washed x3
by adding 200 IJ,1 of phosphate buffered saline
3. Procedures (PBS) via a multichannel pipettor, followed by
gentle aspiration of the wash buffer with the
A. Cellular Invasion Assay vacuum manifold. Alternatively, removal of
1. Preparation of bacteria and tissue culture cells medium or wash buffer can be accomplished
A549 lung epithelial cells are seeded and by rapid inversion and brief shaking of the
grown in tissue culture media (RPMI media + plate over a sink or disposal container. After
10% fetal calf serum) in 96-well tissue culture the primary wash steps, 200 IJ,1 of tissue culture
plates until confluent monolayers are formed. medium containing 100 IJ,g/ml gentamicin and
The day before the screening assay, single 5 mg/ml penicillin G is added to each well,
colonies of GBS mutants are picked with a and the plates incubated for 2 h at 37 DC with
sterile toothpick and used to inoculate 200 IJ,1 5% CO 2 to kill extracellular and surface-
of Todd Hewitt broth (THB) in individual adherent bacteria. The monolayers are once
wells of 96-well microtiter plates. Control again washed x3 with PBS. Finally, 50 IJ,1 of
wells on each plate are inoculated with either a 1:4 mixture of 0.25% trypsin/EDTA solution
the wild-type GBS strain or a noninvasive bac- and 0.025% Triton X-100 is added to each
terial isolate (e.g., Escherichia coli strain DH5 well, the plates incubated for 10 min at 37 DC
or Streptococcus gordonii Challis). The plates in order to disrupt the epithelial cell mono-
are incubated overnight at 37 DC to allow the layers and liberate intracellular bacteria.
bacteria in each well to reach stationary 4. Semiquantitative determination of bacterial
growth phase. Cellular invasion by certain colony counts using soft agar
bacterial species may require factors expressed Todd Hewitt soft agar (THSA) medium is
only during exponential growth phase. In such prepared in advance by using 0.7% Bacto
109
agar (Difco) rather than our standard 1.5%, forming units represent possible low-platelet-
maintained in liquid form by incubation in a binding mutants, whose phenotype is subse-
45-50 DC water bath, and dispensed into a quently confirmed by a quantitative platelet
reagent reservoir just prior to use. Addition of binding assay [11].
150 ~l of THSA to each well using the multi-
channel pipettor results in uniform dispersion
of the 50 ~l lysate and solidification within 4. Results and discussion
a few minutes at room temperature. The
microtiter plates are incubated overnight at Figure 1 demonstrates GBS recovered within micro-
37 DC, and the next day bacterial colonies titer wells by the soft agar technique following
are observed growing within the THSA. invasion of lung epithelial cell monolayers. Serial
Identification of isolates or mutants that are twofold dilutions of the organism were used as
invasive (some colonies present), hyperinva- initial inoculums over a range at which invasion is
sive (many colonies present) or noninvasive approximately linear, in order to demonstrate the
(no colonies present) is easy and reproducible. observed colonial morphology and reproducibility of
Mutants with suspected alterations in invasion the assay. Each dilution was performed in triplicate,
phenotype are subsequently confirmed by and three replica wells were assayed by spread
means of a standard 24-well plate quantitative plating on Todd-Hewitt agar in standard petri dishes.
cellular invasion assay [9]. After overnight incubation, high colony counts
B. Platelet adherence assay imparted a fine granular appearance to the solidified
1. Preparation of bacteria and platelet mono- soft agar, whereas at low colony counts, larger
layers discrete colonies were evident. Below approximately
To identify transposon mutants of Streptococ- 1,000 colonies per well, even two-fold differences
cus sanguis deficient in binding to human in colony number were easily discernable. For quan-
platelets, we have performed a similar 96- titative comparison, the colony forming units counted
well microtiter plate screening assay. Briefly, on the spread plates for each dilution were as follows
washed human platelets are obtained from (+ st dev): 1260 + 80, 635 + 32, 301 + 34, 164 + 20,
fresh blood donated by healthy volunteers, 79 + 4, 42 + 6, 19 + 6, 12 + 8, and 4 + 2.
isolated by centrifugation (100 xg, 15 min), Figure 2 illustrates how a low platelet binding
followed by washing, fixation in 0.8% mutant of S. sanguis can be readily identified by
formaldehyde (30 min at 37 DC), additional visual inspection following the semiquantitative soft
washing, and suspension in Tyrode's solution. agar-based screening platelet adherence assay. A
Platelet monolayers are prepared by placing similar screening assay allowed identification of low
107 fixed platelets into each well of 96-well platelet binding variants of Staphylococcus aureus
tissue culture plates pretreated with poly-L- [11]. In additional experiments using appropriate soft
lysine (0.01 % solution). agar media, we have successfully adapted our method
2. Semiquantitative determination of platelet to assay the invasion potential of Escherichia coli,
binding using soft agar Burkholderia cepacia and Haemophilus inJluenzae.
Overnight cultures of S. sanguis mutants are The soft agar microtiter plate technique should be
grown in BHI broth, washed and resuspended generalizable to a host of cellular adherence and
in Tyrode's salt solution. One hundred ~l of invasion assays, microbicidal assays and numerous
each bacterial suspension is transferred (_107 other bacteriologic experiments which can be per-
cfu) onto the immobilized platelets in a corre- formed in a micro titer well and require only semi-
sponding microtiter well and rocked gently quantitative interpretation. Once a particular assay
at 4 DC for 1 h. The unbound organisms is developed, 'standard curves' from known serial
are then removed by washing three times dilutions of the organism grown in soft agar media
with Tyrode's solution. The wells are treated can be constructed and used to assign approximate
with trypsin (l mg/ml, 10 min) to release the numerical values to matching experimental wells.
adherent bacteria. Brain-Heart Infusion soft We have found several advantages to the use of
agar (BHISA) medium is prepared in advance the soft agar microtiter well assay as compared to
by using 0.7% Bacto agar (Difco), maintained standard bacteriologic plating methods. First, the
in liquid form by incubation in a 45-50 DC entire well is assayed for colony forming units,
water bath, and dispensed into a reagent reser- eliminating both sampling error in obtaining a small
voir just prior to use. Using a multichannel aliquot and plating error associated with 'hockey-
pipettor, 175 ~l of BHISA is added to each sticking' or other dispersion techniques. Second, the
well. After overnight incubation at 37 DC, assay is extremely rapid, requiring but seconds for
the number of organisms per well is assessed delivery of the soft agar with a multichannel pipettor.
qualitatively by visual inspection. Those wells Severalfold more samples can be screened efficiently
with markedly decreased quantities of colony in a given experiment, and inters ample variation,
110
Figure 1. Semi-quantitative invasion assay in which serial dilutions of group B streptococci are added to lung epithe-
lial cell monolayers in microtiter wells, extracellular bacteria eliminated by washing and gentamicin treatment, and
intracellular bacteria recovered by addition of soft agar and overnight incubation at 37 DC. The experiment was performed
in triplicate and colony counts confirmed by standard plating methods.
Figure 2. Use of soft agar to identify low platelet binding variants of Streptococcus sanguis M99. One of the center
wells is much clearer, indicating greatly decreased colony forming units associated with low binding to platelets immo-
bilized on the microtiter well surface.
due to time elapsed between individual platings, is in a single well; alternatively, appropriate dilution
avoided. Third, the assay is easy to interpret, since into a replica microtiter plate can be performed prior
positive and negative controls can be placed on the to addition of the soft agar. We recognize that a soft
same plate for direct visual comparison with sample agar microtiter well assay may not be applicable to
wells. Lastly, the assay requires almost no additional organisms requiring high oxygen tensions and those
expense, in contrast to the considerable cost and/or acutely sensitive to the initial temperature elevation
labor involved in preparing large numbers of standard required to maintain the agar in liquid form.
agar petri dishes. Stocks of soft agar medium can Formation of large or confluent surface colonies
be made in advance, allowed to solidify, and the would also tend to obscure organisms embedded
required amount melted using a microwave on the within the agar. Nevertheless, we encourage other
day of the assay. investigators interested in large scale screening of
All soft agar assays should be calibrated such that microorganisms for viable colony counts to consider
< 1,000 colony forming units are typically recovered this semi-quantitative technique. By delivering the
111
agar to the organism (rather than the other way 3. Francisco DE, Mah RA, Rabin AC (1973). Acridine
around), substantial laboratory time, effort and orange epifluorescent technique for counting bacteria
expense may be saved. in natural waters. Trans Am Microsc Soc 92: 416-
421.
4. Lundgren B (1981). Fluorescein diacetate as a stain
of metabolically active bacteria in soil. Oikos 36:
Acknowledgments 17-22.
5. Noble PA, Ashton E, Davidson CA, et al. (1991).
This work was supported in part by grants HD 07233 Heterotrophic plate counts of surface water samples
and AI 01451 (VN), AI 29549 (ALS), AI 32506 by using impedance methods. Appl Environ Microbiol
(PMS) and AI 30068 (CER) from the National 57: 3287-3291.
Institutes of Health. 6. Norris KP, Powell EO (1961). Improvements in deter-
mining total counts of bacteria. J R Micros Soc 80:
107-119.
Notes on suppliers 7. Peck R (1985). A one-plate assay for macrophage bac-
tericidal activity. J Immunol Methods 82: 131-140.
1. Corning Costar Corporation, One Alewife Center, 8. Pinder AC, Purdy PW, Poulter SA, et al. (1990).
Cambridge, MA 02140, USA Validation of flow cytometry for rapid enumeration of
2. Difco Laborotories LTD, P.O. Box 14B, Central bacterial concentrations in pure cultures. J Appl
Avenue, West Molesey Surrey, England, KT8 2SE Bacteriol 69: 92-100.
3. Drummond Scientific Co., 500 Parkway Blvd., 9. Rubens CE, Smith S, Hulse M, et al. (1992).
Broomal, PA 19008, USA Respiratory epithelial cell invasion by group B strep-
4. Fisher Scientific, 711 Forbes Avenue, Pittsburgh, PA tococci. Infect Immun 60: 5157-5163.
15219, USA 10. Selan LE, Berlutti FN, Passariello CE, Thaller MC,
5. Labsystems Oy, P.O. Box 8, FIN-00881 Helsinki, Renzini G (1992). Reliability of a bioluminiscence
Finland ATP assay for detection of bacteria. J Clin Microbiol
6. American Type Culture Collection (ATTC), 12301 30: 1739-1742.
Parklawn Drive, Rockville, MD 20852, USA 11. Sullam PM, Bayer AS, Foss WM, et al. (1996).
7. Sigma, 3050 Spruce Street, St. Louis, MO 63103, USA Diminished platelet binding in vitro by Staphylococ-
cus aureus is associated with reduced virulence in a
rabbit model of infective endocarditis. Infect Immun
64: 4915-4921.
References
Abstract. Two pieces of data are needed to fully map probing unambiguously reveals the order of those
the location of a transposon inserted in a plasmid, the restriction sites. End-probing is similar to end-
site of insertion and the transposon's orientation. labeling, except that the uncertainties inherent in the
Both of these parameters can be determined from a radiolabeling reaction (and the problems of working
map of restriction sites, which can be derived by with radionucleotides) are avoided. In this paper
end-probing. Like restriction mapping, end-probing we discuss the use of end-probing as a means to
reveals the distance between restriction sites on a map insertions of transposon Tn917 into the
plasmid. In contrast to restriction mapping, end- Streptococcus agalactiae plasmid pGB354.
1. Introduction 2. Materials
11. Prehybridization buffer (in TBS) 3. Inoculate a single colony into 3 ml THB-1O
- 1% Casein. f..1g/ml cm and 10 f..1g/ml em.
- 0.05% Tween-20. 4. Incubate overnight at 37 De.
- Dissolve the casein by heating gently 5. Pellet culture in a benchtop centrifuge at
(below boiling) with stirring for 2 hours. 13,600 g.
12. Hybridization buffer (in dH 20) 6. Resuspend the pellet in 1.5 ml PBS, repellet
- Ix Denhardt's solution. as before.
- 45% deionized formamide. 7. Resuspend the pellet in 40 f..11 protoplast
- 0.1% SDS. buffer.
- 5x SSe. 8. Add 2 f..11 of 10 U/f..11 mutanolysin, incubate
- 20 mM sodium phosphate buffer pH 7.0. at 37 DC for 1 hour.
- 10% dextran sulfate. 9. Pellet protoplasts as before, resuspend pellet
13. Prewash Buffer (in dH 20) in 250 f..11 of buffer PI from Qiaprep Spin Kit.
- 2x SSC. Process the plasmids, thereafter, exactly
- 1% SDS. according to the manufacturor's instructions.
14. Wash buffer (in dH 20) Note that, after lysis, the solution should
- 0.16x SSe. clear. If the culture remains turbid it is very
- 0.1% SDS. unlikely that the cells lysed sufficiently.
15. Buffer 1 (in dH 20) 10. Elute the plasmid DNA off of the spin column
- 2 mM MgCI 2 • in 50 f..11 10 mM Tris pH 8.5.
- 0.1 M Trizma pH 7.5. 11. Estimate the plasmid concentration assuming
- 0.1 M NaCl. an absorbance of 1.0 equates to a 50 f..1g/ml
- 0.05% TritonX-100. DNA concentration (given a 1 cm light path)
16. Blocking buffer (in PBS) [11].
- 1% Casein. D. PCR amplification of biotinylated end-probe
- 1% Tween-20. 1. Dilute pMHL201 to 1 ng/f..11 and dilute
- Dissolve the casein by heating gently primers oMLl 04 and oMLl 05 to 5 f..1M in
(below boiling) with stirring for 2 hours. 10 mM Trizma pH 8.0. (The primers were
17. Binding buffer (in PBS) designed amplify a 487 bp fragment adjacent
- 5% Tween-20. to the unique Pvull site in pMHL201, see
18. Detection buffer I (in PBS) Figure 1.)
- 1% Tween-20. 2. Amplify the biotinylated probe using PCR
19. Detection buffer II (in dH 20) Non-radioactive Labeling System, according
- 0.1 M Trizma pH 9.5. to the manufacturer's recommendations. Set
- 0.1 M NaCl. up four control reactions that omit one of the
- Autoclave. DNA components or MgCl z. Set up trial
- 50 mM MgCl 2 (added fresh with each use reactions having 1,2,4, or 6 mM MgCl z. (A
from a 1.0 M stock). faint 560 bp contaminating band is seen with
20. SSPE (in dH 2 0) 2-6 mM MgCl z.)
- 175.3 g NaCl. 3. Separate the fragments from the biotinylated
- 27.6 g NaH 2P0 4 ·H20. cytosine with a Centri-sep column, according
- 7.4 g EDTA. to the manufacturer's instructions.
- Raise to 800 ml with dHzO and pH to 7.4 E. Test the labeling reaction
with 10 M NaOH. - Dilute the probe through 8 twofold dilutions in
- QS to 1 L and autoclave. 10 mM Tris pH 8.0.
B. Primers - Spot 1 f..11 of each dilution onto a nylon
1. oMLl04 membrane.
AAT ATC CCT TTT GTT GTA GAA AC - Crosslink with UV at 120 mJ/cmz.
2. oMLI05 - Block the membrane by incubating with block-
CAG ATT TAA ATT CTG ATT TTG AAG ing buffer for 2 hours at room temperature.
3. Tn917 L - Wash three times in binding buffer for 3
AGA GAG ATG TCA CCG TCA AG minutes at room temperature.
4. Tn917 R-ext - Incubate the membrane with streptavidin-
TAG GCC TTG AAA CAT TGG TT peroxidase diluted 5000-fold in binding buffer
C. Plasmid preparation for 1 hour at room temperature.
1. Streak COHI [pMHL201] onto THA plate - Wash the membrane three times in blocking
supplemented with cm to 10 f..1g/ml and em buffer for 15 minutes.
to 10 f..1g/ml. - Wash the membrane twice in detection buffer
2. Incubate overnight at 37 De. I for 5 minutes.
116
- Dry the membrane on Whatman paper. - Incubate in blocking buffer for 1 hour at room
- Detect the probe using the Supersignal substrate temperature, exchange for fresh buffer, and
according to the manufacturer's instructions. incubate 30 minutes further.
F. Restriction digests - Wash twice for 5 minutes in binding buffer.
- Set up 4 digests of pMHL201 using 250 to 500 - Cover the membrane in binding buffer with a
ng of plasmid per digest in Ix NEB 2 buffer. 5000: 1 dilution of streptavidin-peroxidase (it
- Add 10 units of Pvull and digest for 1 hour at helps to have a small, flat-bottomed container
37°C. to minimize the volume of peroxidase required,
- Dilute Bell in Ix NEB 2 buffer to produce 10, we found the tops of pipette tip racks to work
3.33, 1, and 0.4 units/~1. well) and incubate for 1 hour at room
- When the Pvull digest is complete, add 1 ~l temperature.
from one of the Bell dilutions to each of the - Wash the membrane three times for 5 minutes
complete Pvull digests, and digest for 5 at room temperature in blocking buffer.
minutes at 50°C. - Wash three more times in detection buffer II
- Add 4 ~l of 6x sample buffer to each 20 ~1 for 5 minutes at room temperature.
digest, and resolve the fragments in a 0.7% - Perform chemilumenescent detection using
agarose gel in Ix TBE at 2-3 V/cm. Supersignal in accordance with the manufac-
- Stain the digested fragments for 10 minutes in turer's instructions. Exposure times vary
TBE/ethidium bromide. between 2 and 20 minutes.
- Destain in TBE for 10 minutes, and photograph H. Sequencing
with UV transillumination. - Sequence from the left and right terminal
G. Southern blotting. Blot the DNA onto nylon repeats of Tn917 with primers Tn917-L and
membrane using standard Southern blotting Tn917-R-ext (respectively), using the ABI
methods [12], with the following modifications Prism Dye Terminator Cycle Sequencing Core
- Soak the agarose gel in lOx SSC for 30 Kit.
minutes. - Desalt with Centri-sep columns.
- Depurinate in 0.25M HCI for 30 minutes. - Dry samples in a concentrator.
- Denature the DNA with two 30 minute - Fluorescent sequencing was performed at the
incubations in denaturing buffer. Fred Hutchinson Cancer Research Center
- Prepare the gel for transfer with two 30 minute facility (FHCRC).22
incubations in neutralization buffer.
- Use overnight capillary transfer to move the
DNA onto the membrane. 4. Results and conclusion
- After transfer, wash the membrane in 5x SSPE
for 5 minutes at 60°C. Dry the membrane on The approach is diagrammed in Figure la. First the
the bench for 30 minutes (it should remain plasmid was digested to completion at the unique
slightly damp) and UV crosslink the DNA to Pvull site. The linearized plasmid was then partially
the filter at 120 mJ/cm. digested at the 5 Bell sites. This produces a compli-
- Incubate the membrane for 1 hour at 42°C in cated mixture of fragments, which would ordinarily
prewash buffer using a rotating hybridization. be difficult to interpret. However, there is an infor-
- Block in prehybridization buffer for 30-40 mative subset of fragments (thin gray lines), all of
minutes at room temperature. which have one end at the Pvull site and run clock-
- Convert the probe to single stranded form by wise to one of the Bell sites. Resolving the fragments
adding an equal volume (usually 50 ~l) of on a gel and hybridizing a probe (thick gray line) to
0.2 M NaOH and incubate the probe at 37°C a Southern blot identified this subset. The probe was
for 30 minutes. generated by PCR amplification of sequences
- Remove the prehybridization buffer and add 10 adjacent to the Pvull site.
ml hybridization buffer with the denatured Figure Ib shows such a blot. Plasmid pMHL201
probe, then incubate overnight at 42°C. was digested to completion with PvuII, then the
- Wash the membrane twice in wash buffer (to linear fragment was digested for 5 minutes with
cover the membrane) for 3 minutes at room various amounts of Bell (varying Bell concentrations
temperature on an orbital shaker at room altered the relative abundance of short vs. long DNA
temperature. fragments). The blotted fragments were probed with
- Wash twice more at 65°C. the biotinylated PCR fragment and the bands visual-
- Rinse the membrane twice in 2x SSC for 3 ized using a streptavidin-HRP conjugate in the
minutes at room temperature in the hybridiza- presence of a chemilumenescent substrate.
tion oven. Six bands hybridized with the probe; the 12 kb
- Block the membrane in Buffer 1 for 1 minute band was produced by Pvull cleavage (no Bell
at 65°C. cleavage) and the 5 smaller bands were each
117
a b
(
- 12.0 kb
pMHL201
4.43
3.75
3.67
Figure 1. End-probing schematic and results. (a) Schematic of end-probing method. Plasmid pMHL201, composed of
pGB354 with a Tn917 insertion, was digested to completion with Pvull and then partially digested with Bell (which
cleaves 4 times in Tn917 and once in pGB354). The fragments are separated on an agarose gel, blotted, and hybridized
with biotin-labeled probe (thick gray line). The probe hybridizes to all fragments that extend clockwise from the Pvull
restriction site (thin gray lines). (b) Results of end-probing. Bell concentrations used were 10,3.3, 1.1, and 0.4 units in
lanes 1 to 4, respectively . Biotinylated lambda/BstEIl fragments were used as size markers in lane 5. Bands were detected
using a streptavidin-HRP conjugate and a chemilumenescent substrate for HRP.
produced by Pvull cleavage at one end and Bell during the short incubations used in these experi-
cleavage at the other. The size of these bands, there- ments. A complete digest is a useful control in
fore , maps the distance from Pvull (clockwise) to a determining the number of expected fragments.
Bell site. Reading from the bottom to the top, there End-probing proved to be an efficient means of
are BelI sites at 3.67 kb, 3.75 kb, 4.43 kb, 6.43 kb, mapping a large number of transposon insertions. The
and 9.93 kb from the Pvull site. The pattern of Bell problematic aspects of end-labeling were avoided,
restriction sites showed that Tn917 is inserted with while the traditional advantage of end-labeling (the
its left terminal repeat (LTR) closest to the probe. unambiguous ordering of restriction sites on the
Within Tn917, the Bell site nearest the LTR is 1.83 plasmid) was maintained. End-probing is based on
kb from the LTR end, so the insertion site was at robust methods, and investigators with PCR and
1.84 kb (3.67-1.83) in pGB354. Southern blotting experience can expect to obtain
How accurate is end-probing? The transposon ' s interpretable results immediately. End probing is time
orientation and its exact insertion site could be consuming compared to a simple restriction digest
identified by sequencing. Twelve Tn917 insertions (probe production and verification, as well as the
were first mapped by end-probing and then washing and hybridizing steps, make it a two-day
sequenced. All twelve had been assigned the correct process at a minimum). Consequently, it may be best
orientation. The insertion site assignments made by exploited in projects where a single probe can be
end-probing were misplaced, on average, by 173 base used to analyze many plasmids simultaneously, e.g.,
pairs. The size of the average error was greatly to identify overlapping DNA fragments in a physical-
increased by a small number of assays in which the mapping project.
smallest bands were quite large (-6 kb). We note that
smaller and more accurately sized bands could have
been derived by stripping the probe off of the blot Acknowledgments
and reprobing with sequences from the opposite flank
of the Pvull site. This work was supported by NIH Grant AI25152 The
Problems with end-probing can occur when the Group B Streptococcus Initiative. Thanks to Paul
enzyme used for the partial digest cleaves different Framson and Harry Yim for help in creating
locations at significantly different rates (e.g. , [13]). pMHL201 and to Amanda Jones for a critical reading
If a given site is cleaved too slowly then the expected of this manuscript.
fragment may not accumulate to detectable levels
118
(*Current Address: University of California, San Diego, Department of Biology, 0116, 9500 Gilman Drive, La lolla,
California, USA)
Abstract. We have recently developed a novel sion of the phage lytic cycle. This method has
bacteriophage-protection system for Lactococcus been used successfully in our laboratory to map the
lactis, based on a two-component genetic 'trap'. An general location of a number of inducible promoters
inducible promoter from a lytic bacteriophage is used on the genomes of bacteriophages attacking lactic
to activate a lethal gene after infection, killing the acid bacteria. Once identified and cloned, small frag-
host cell and halting phage proliferation. To expand ments encoding inducible promoters can be partially
the potential use of this novel defense strategy, pro- sequenced and primer extension reactions carried out
moters specific to any particular phage of interest on phage RNA, isolated over the course of an infec-
must be available for fusion to a universal death tion, to pinpoint the precise location of the promoter.
gene. A method to localize regulated promoters In this study, we illustrate the use of the capping
within the context of the total phage genome was method to map the phage-inducible promoter on the
evaluated. The 'capping' activity of the vaccinia genome of the lactococcal bacteriophage skI. This
virus guanylyltransferase was exploited to label approach provides a rapid and efficient means to
newly synthesized mRNA extracted from infected identify promoter regions on the genomes of rela-
cells at sequential time points over the course of a tively uncharacterized phages. These promoters can
phage infection. The labeled mRNAs were then used then be used in a variety of applications, including
as probes in Southern hybridization reactions to phage-triggered defenses and inducible gene expres-
identify restriction fragments in the phage genome sion systems.
where new transcripts were initiated during progres-
~;~;;;:ln I
injected phage genome ~
signal
loJ
X
~
TGATGAGATATGTGACGGCTTCTGGTAATTTGTCACCTTTTGGGCGAAAACTGACAAGATAAAT
t
GTT~GTATCATCAAATAAAACAAATAAAGCCAGCGG~TATA]rCTGTTGGCTTTTTGT
t
Fspl l~~=:-::r!"~
GTGGAGAAAGTGAGGTGACCTCCCATAGCATTACGTGCTGACCGTACTGGTG~
- ~~~~
from partial or complete sequencing of phage accurate method is needed which could be univer-
genomes [2, 21, 28]. Neither sequencing nor shot- sally applied to different bacteriophages to identify
gun cloning promise to be effective methods to detect and characterize inducible promoters. Ideally, the
and isolate phage-specific promoters from lytic approach would not require detailed genetic infor-
phages that appear and destroy starter cultures mation about the phage or its genomic sequences.
in industrial fermentations. A rapid, efficient, and Transcription initiation sites used by the
121
30°C until OD600 reached 0.4 (approx. 3-4 RNA samples (total volume per sample
hours). 140 ~l).
B. Bacteriophage infection and phage DNA isola- 5. To generate short end-labeled RNA species,
tion hydrolyze the purified RNA samples with
1. Conduct bacteriophage infection as described 0.2 N NaOH (30 min, on ice), and neutralize
by Terzaghi and Sandine [27]. with 0.5 vol 1M Hepes acid as described pre-
2. Isolate phage DNA using the large scale viously by Haynes and Rothman-Denes [10].
protocol described by Raya et al. [23]. 6. Use short RNA species as hybridization probes
C. RNA Isolation to locate phage skI fragments which encode
1. All solutions and glass/plasticware must be phage-inducible transcription signals.
RNAse free. Plastic/glassware was soaked in E. RNAIDNA hybridization (all solutions and glassl
3% hydrogen peroxide, several hours or plasticware must be RNAse free)
overnight, rinsed well with picopure water 1. Digest phage skI genomic DNA (3-5 ~g) with
(double deionized water), and autoclaved, dry restriction enzymes EcoRV, HindIII, Sau3AI,
cycle, 40 minutes. Water (picopure) was auto- and SspI using the standard methods [24].
claved first in RNAse-free glassware and then 2. Add 10 units of RNAse inhibitor per sample
used to make desired solutions. Measuring and incubate the tubes at 37°C for 30 minutes.
spatulas were soaked in 3% peroxide and auto- 3. Heat the samples for 30 minutes at 65 °C (to
claved. open phage cohesive ends) and place in an ice
2. Incubate L. lactis MG 1363 cells at 30°C until bath until loading on a 1.2% SeaKem GTG
OD 600 = 0.4 is reached and then infect with agarose gel.
phage skI at multiplicity of infection (MOl) 4. Gel-separate restriction fragments and transfer
of 5 (five ml of phage stock @ 1 x 1010 pfu/ml to MSI Magnacharge nylon membranes as
was added to the 50 ml culture). described by Southern [25]. Perform transfer
3. At different time points over the course of overnight at room temperature. Use 20x SSC
the infection, remove 9 ml of cells from the buffer.
culture, centrifuge, and freeze the cell pellets 5. When transfer is completed, cross-link the
in an ethanol:dry ice bath. Isolate RNA from DNA to the membrane in UV-Stratalinker.
the frozen pellets using 1 ml of TRIzol™ 6. Pre-wet the filters in 5x SSPE and pre-
reagent according to the manufacturers instruc- hybridize for 1 hour at 42°C in prehybridiza-
tions. Add glass beads and homogenize the tion buffer, 30 ml (0.2 mllcm 2 of membrane
cells for 90 seconds in a Mini Bead Beater. 7 150 cm2) containing 50% formamide [8].
Determine RNA concentrations spectrophoto- 20 ml of hyb. buffer (approx. 0.13 mllcm 2 of
metrically, 1 OD 260 = 40 mcg/ml RNA. For membrane). Hybridize at high-stringency (at
pure RNA the OD26ofOD280 ration should be 2 42°C, over-night in 20 ml of hybridization
(> 1.8 is acceptable). buffer, identical to prehybridization buffer) to
D. 5'-end labeling of RNA samples (all solutions and 5'-end labeled RNA probes. The labeled RNA
glass/plasticware must be RNAse free) probe (100 mcg) was in a total volume of 213
1. Use the procedure originally described by microliters (213 mcl. = 140 mcl. (see step D4)
Haynes and Rothman-Denes [10] with the fol- + 70 mcl. of Hepes + 2.8 mcl of 10 N NaOH
lowing modifications: Incubate RNA (100 ~g) (see step D5). The probe was boiled for 5 min,
for 30 minutes at 37°C in a reaction buffer mixed with 0.5 ml of hybridization buffer
containing 50 mM Tris-HCI (pH 7.9), 1.2 mM and added to a bottle containing 19.5 ml of
MgCI 2, 6 mM KCI, and 2.5 mM DTT, with 10 the same buffer (0.13 mllcm 2 membrane).
units of guanylyltransferase and 3.5 nmol Hybridization was performed in roller oven.
a32p[GTP] (3000 Ci/mmol), in a total reaction 7. Perform post-hybridization washes at room
volume of 50 ~l. temperature:
2. Terminate the reaction by adding 1 mM EDTA - Wash filters once for 30 sec in 30 ml of 2x
(pH 8.0) and 0.2% SDS. Five microliters of SSPE, 0.1 % SDS to remove unbound probe
lOx stock solution (10 mM EDTA, 2% SDS, - Wash filters twice, for 15 min each time,
pH = 8) per 50 microliters reaction (see item in 200 ml 2x SSPE, 0.1 % SDS
Dl). - Wash filters twice, for 15 min each time,
3. Add 20 ~l of Ix STE buffer and separate the in 200 ml O.lx SSPE, 0.1 % SDS
RNA from the unincorporated nucleotides 8. Rinse filters in 2x SSPE and blot excess buffer
using NucTrapR probe purification columns by placing membrane on a piece of Whatman
according to the manufacturers instructions. paper.
4. When the purification is completed, wash the 9. Wrap the filters in plastic wrap and set up
columns with 70 ~l of Ix STE buffer and autoradiography. Use KODAK BIOMAX MS
combine eluates with the originally eluted film, with adequate intensifying screen.
123
('cap') from GTP to any RNA transcript possessing as a probe for Southern hybridization [25] to
a di- or tri-phosphate 5' termini, the newly initiated restriction fragments of phage genomic DNA immo-
prokaryotic transcripts, which have tri-phosphate bilized on the nylon membrane (Figure 3D). Strong
groups at their 5'-termini, are 'capped' in vitro. hybridization signals were obtained only with the
The capping method was used to identify precisely phage skI genomic fragments which also hybridized
the skI genomic regions where phage-specific tran- to transcripts generated during the middle-late stage
scription is initiated during the timely-regulated of lytic development (Figure 3C). Therefore, the
expression of phage genes. L. lactis MG1363 cells HindlII fragment 'D' and the Sspl fragment'!',
were infected with phage skI at MOl of 5 and total mapped previously with guanylyltransferase, were
RNA isolated from lactococcal cells at 3, 5, 8, and found to encode a single phage-inducible transcrip-
124
A
I
Sau3A1
EcoRV
~S""3A' ,Hlndlll
'Sspl
I Ii 1~~3AI
I Sspl tspl
, II
,
, Ss
III I I i
' SsPI
Sau3A1 I 'I I.ss
I
Sspl I
ISm<3AI
1 1 ~~:1
1 " coRY
.EcoRV
I ' ., . . I (fmdlll
EcoRV
I ~. li! l' I ! I Ii Ii ..Sspl
! I Ssp)
I I il.ECORV
I I ,"Sspl
cos cos
Hindlll "D"
SspJ "I"
5000
! ,
10000
" ,
'5000, 20000
1 ,25000
B J min, X min. D
2 J ~ 5 6 7 8 9 IU
-
/1;",1111 "IJ"
- 1Ii",1I 1I "I)'"
-
/li,,~1I 1 -11-
-
S"I'I -'"
- S"I'I •• , "
-
SSI" "I"
Figure 3. Location of phage skI genomic fragments encoding new transcripts initiated after infection of Lactococcus
lactis MG 1363. (A) Restriction map of bacteriophage skI adapted from [1]. Only relevant restriction sites are shown.
Overlapping DNA fragments HindIII 'D' and SspI 'I' are indicated. (B) Restriction enzyme patterns of phage skI restricted
with EcoRV (lanes 2, 6), HindIII (lanes 3, 7), Sau3AI (lanes 4, 8), and SspI (lanes 5, 9). The molecular weight standard
was I-kb ladder (GIBCO-BRL, lanes 1, 10). The HindIII fragment 'D' and SspI fragment 'I' are indicated by arrows.
(C) Hybridization profiles of Southern transfer containing phage skI genomic fragments restricted with EcoRV (lanes
2, 6), HindIII (lanes 3, 7), Sau3AI (lanes 4, 8), and SspI (lanes 5, 9) probed with 5' -end labeled RNAs isolated at 3
minutes and 8 minutes after the infection. Hybridization signals indicated by arrows represent newly initiated transcripts
corresponding to the DNA fragments HindIII 'D' and SspI '1'. (D) Hybridization profiles of Southern transfer con-
taining phage skI genomic fragments restricted with EcoRV (lane 2), HindIII (lane 3), Sau3AI (lane 4), and Ssp I
(lane 5) probed with the a32p[dCTP]-labeled skI DNA fragment encoding the phage-specific promoter. Hybridization
signals corresponding to the HindIII fragment 'D' and the SspI fragment 'I' are indicated by arrows.
tion signal. Only one such promoter was found to be was determined previously [1]. Northern analysis
encoded within the entire phage skI genome. In fact, revealed that early transcription of the skI genome
phage-specific promoters are not abundant in the was initiated within 5 minutes after infection,
genomes of lactococcal bacteriophages; <1>31 [20], followed by middle (7 to 10 minutes), and late (15
bIL4l [21], and c2 [28] each encode only a single minutes) transcription. The positions and lengths of
phage-inducible promoter. Therefore, use of the early and middle phage transcripts were determined
'capping' method to localize these rare genetic by Northern hybridization. The transcriptional starts
elements in the future will have an important advan- and exact lengths of late transcripts could not be
tage over shot-gun cloning or sequencing of phage determined due to RNA processing or degradation.
genomes, two labor intensive approaches used pre- The authors speculated that late skI transcripts could
viously to identify fragments encoding phage-specific be generated from either a late promoter located
promoters, Once identified, these DNA fragments within the middle region of the phage genome, or
can be cloned and sequenced. The precise location of by anti termination or processing of transcripts initi-
a phage-inducible promoter within a small DNA ated from a middle phage-specific promoter. Since
fragment could be identified using primer extension processed transcripts are not labeled by the capping
reactions, carried out on RNA isolated over the method, the late skI transcripts detected by Chandry
course of a phage infection. et al. [1] were most likely generated from the
A temporal transcription map of phage skI sole middle phage-specific promoter. Therefore, one
125
tion. In: RNA methodologies, pp 191-203. Academic arranged in an operon with llaIM, on the conjugative
Press, Inc. Lactococcus plasmid pTR2030. J Bacteriol 177:
9. Gasson MJ (1983). Plasmid complements of 134-143.
Streptococcus lactis NCD0712 and other lactic strep- 20. O'Sullivan DJ, Walker SA, West SG, Klaenhammer
tococci after protoplast-induced curing. J Bacteriol TR (1996). Development of an expression strategy
154: 1-9. using a lytic phage to trigger explosive plasmid ampli-
lO. Haynes LL, Rothman-Denes LB (1985). N4 virion fication and gene expression. Bio/Technology 14:
RNA polymerase sites of transcription initiation. Cell 82-87.
41: 597-605. 21. Parreira R, Valyasevi R, Lerayer ALS, Ehrlich SD,
11. Hill C, Pierce K, Klaenhammer TR (1989). The con- Chopin M-C (1996). Gene organization and tran-
jugative plasmid pTR2030 encodes two bacteriophage scription of a late-expressed region of Lactococcus
defense mechanisms in lactococci, restriction modifi- lac tis phage. J Bacteriol 178: 6158-6165.
cation (R+/M+) and abortive infection (Hsp+). Appl 22. Powell IB, Arnold PM, Hillier AJ, Davidson BE
Environ Microbiol 55: 2416-2419. (1989). Molecular comparison of prolate- and iso-
12. Itoh N, Mizumoto K, Kaziro Y (1984). Messenger metric-headed bacteriophages of lactococci. Can J
RNA guanylyltransferase from Saccharomyces cere- Microbiol 35: 860-866.
visiae. J BioI Chern 259: 13923-13929. 23. Raya RR, Kleeman EG, Luchansky JB, Klaenhammer,
13. Mizumoto K, Lipman F (1979). Transmethylation and TR (1989). Characterization of the temperate
transguanylylation in 5' -RNA capping system isolated bacteriophage <1>adh and plasmid transduction in
from rat liver nuclei. Proc Natl Acad Sci USA 76: Lactobacillus acidophilus ADH. Appl Environ
4961-4965. Microbiol 55: 2206-2213.
14. Monroy G, Spencer E, Hurwitz J (1978a). Purifica- 24. Sambrook J, Fritsch EF, Maniatis, T (1989).
tion of mRNA guanylyltransferase from vaccinia Molecular cloning: a laboratory manual, Second
virions. J BioI Chern 253: 4481-4489. edition. Cold Spring Harbor, NY: Cold Spring Harbor
15. Monroy G, Spencer E, Hurwitz J (1978b). Char- Laboratory.
acteristics of reactions catalyzed by purified 25. Southern EM (1975). Detection of specific sequences
guanylyltransferase from vaccinia virus. J BioI among DNA fragments separated by gel elec-
Chern 253: 4490-4498. trophoresis. J Mol BioI 98: 503-517.
16. Monsalve M, Mencia M, Rojo F, Salas M (1995). 26. Stoddard SF, Howe MM (1989). Localization and
Transcription regulation in Bacillus subtilis phage regulation of bacteriophage Mu promoters. J. Bacteriol
<1>29: expression of the viral promoters throughout the 171: 3440-3448.
infection cycle. Virology 207: 23-31. 27. Terzaghi BE, Sandine WE (1975). Improved medium
17. Nuez B, Salas M (1993). Bacteriophage Nf DNA for lactic streptococci and their bacteriophages. Appl
region controlling late transcription: structural and Microbiol 29: 807-813.
functional homology with bacteriophage <1>29. Nucleic 28. Waterfield NR, Lubbers MW, Polzin KM, Le Page
Acid Res 21: 2861-2865. RWF, Jarvis AW (1996). An origin of DNA replica-
18. O'Sullivan DJ, Klaenhammer TR (1995). C LtaI is a tion from Lactococcus lac tis bacteriophage c2. Appl
bifunctional regulatory protein of the llaI restriction Environ Microbiol 62: 1452-1453.
modification operon from Lactococcus lactis. In:
Feretti JJ, Gilmore MS, Klaenhammer TR, Brown F
(eds), Genetics of the streptococci, enterococci, and Address for correspondence: T. R. Klaenhammer, Depart-
lactococci, vol 85: Dev BioI Stand, pp 591-595. ment of Food Science, North Carolina State University,
Basel: Karger. Raleigh, NC 27695-7624, USA
19. O'Sullivan DJ, Zagula K, Klaenhammer TR (1995). Phone: (919) 515-2971; Fax (919) 515-7124
In vivo restriction by LlaI is encoded by three genes, E-mail: klaenhammer@ncsu.edu
Methods in Cell Science 20: 127-136 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. The secretion of heterologous proteins isolated from Bacillus circulans T3040 into S.
by Streptococcus gordonii from genes present on gordonii. A portion of the CITase gene devoid of its
plasmids or on the chromosome has been achieved signal sequence was fused inframe to the signal
with a novel recombination system. We have con- sequence of the Streptococcus sobrinus gtfI gene.
structed an integration-mediated transformation The CITase fused gene was then introduced into
system in oral streptococci to clone foreign DNA into either a resident plasmid or the S. gordonii chromo-
either resident plasmids or the host chromosome. In some. The presence of the enzymatically active
this system, Escherichia coli plasmid pACYC184 CITase in the culture fluids from plasmid-borne
was extensively modified and utilized to construct transformants was confirmed. These results indicate
anchor, integration, and heterodimer plasmids for that the integration-mediated transformation system
introduction of the foreign genes. In order to evaluate described is an effective means of engineering
this system, we cloned the gene for cycloiso- recombinant streptococci capable of secreting hetero-
maltooligosaccharide glucanotransferase (CITase) logous proteins.
glucan synthesis. The expression of these molecules gene. To fuse the gtfI secretion domain encoding
has been recognized as an important virulence factor 38 amino acids and the CITase structural gene,
in dental decay. One possible approach for reducing PCR amplification was carried out using approx-
cariogenicity would be to construct recombinant oral imately 200 ng of pNSOI (see Figure 4D) as a
commensal bacteria which produce enzymes leading template with 10 pmoles of each primer in a final
to inhibition of GTF activity. One of the objectives volume of 25 ilL in a GeneAmp PCR System
of the present study was to construct S. gordonii 9600 thermocycler! under the following condi-
strains able to secrete the enzyme cycloisomal- tions: (i) initial denaturation, 94 DC for 3 min; (ii)
tooligosaccharide glucanotransferase (CITase) which 30 cycles of amplification, denaturation at 94 DC
has been demonstrated to inhibit glucan formation by for one min, primer annealing at 66 DC for 2 min,
mutans streptococci [4]. We have now utilized the and extension at 72 DC for 3 min with AmpliTaq
integration strategy to secrete CITase from S. DNA polymerase!; and (iii) final extension, 72 DC
gordonii. for 10 min. The PCR product was blunt-ended
Another objective of the present study was to with T4 DNA polymerase2 in the presence of
expand the versatility of the previous system. In the dNTP, and pNS03 (see Figure 4E) was isolated
newer version, the anchor plasmid (containing target following standard kinase 2 treatment, ligation, and
DNA sequences for integration of heterologous transformation [1].
DNA) was introduced into either a resident plasmid C. Transformation of S. gordonii
or the host chromosome and acts as the common Transformation of S. gordonii Challis can be
integration site. This strategy allowed us to integrate carried out as previously described [13] with
heterologous genes originally present on a single E. slight modifications. Add the host bacterium (1:20
coli plasmid into either multi-copy plasmids or the dilution) to 1.0 ml of brain heart infusion 3 (BHI)
single copy chromosome. broth containing 10% heat -inacti vated horse
serum4 (HIHS-BHI) and maintain the culture at
37 DC. When bacteria harboring plasmids are
2. Materials transformed, no antibiotics need to be added to
the media for positive selective pressure. After
A. Equipment 16 h incubation, transfer a 50 III aliquot of this
1. PCR amplifier.! overnight culture into 1.0 ml of fresh HIHS-BHI
B. Chemicals and reagents and incubate at 37 DC for 30 min. Transfer the
2. T4 DNA polymerase. 2 competent cells (100 Ill) into fresh tubes con-
3. Brain heart infusion broth. 3 taining 100-200 ng of appropriate DNA and
4. Erythromycin. 4 incubate at 37 DC. After 30 min, add 100 III of
5. Spectinomycin. 4 fresh HIHS-BHI and continue the incubation for
6. Blue dextran. 5 an additional 2 h. Select transformants on BHI
7. Q-Sepharose. 5 agar plates supplemented with either erythro-
8. Horse serum. 4 mycin 4 (Em, 10 Ilg/ml), or spectinomycin4 (Sp,
9. Agarose, SeaKem. 6 200 Ilg/ml), following 2 days of incubation at
37 DC under anaerobic conditions.
D. Enzyme assays
3. Procedures Since the CITase exhibited dextranase activity [9],
blue dextran was used to monitor CITase activity
A. Recombinant DNA procedures in this study. To screen for S. gordonii CITase
The CITase gene (GenBank accession number positive transformants, use BHI plates containing
D61382) isolated from Bacillus circulans T3040 0.5% blue dextran 5 and examine each for clearing
was originally cloned into the pUC derivative, zones around the colonies after 48 h of anaerobic
pCI429 [9]. DNA purification, endonuclease growth at 37 DC. To detect CITase activity in cell
restriction, ligation, and E. coli transformation samples, zymography was also carried out.
were carried out under standard conditions as Dialyze culture fluids (50 ml) from CITase
previously described [1]. Nucleotide sequencing positive S. gordonii cells against 10 mM Tris-HCl
was carried out as outlined earlier [15]. buffer (pH 8.0) and apply onto a Q-Sepharose 5
B. PCR amplification column (0.6 x 15 cm) at 4 DC. Elute the proteins
Since the signal peptides of both the CITase and with the same buffer containing sodium chloride
the GTF-I enzymes consist of the first 38 amino (0 to 0.5 M linear gradient) at approximately
acids [3, 9], a set of two PCR primers comple- 0.1 ml per min and subject the eluates to 7.5%
mentary to the respective regions were designed: polyacrylamide gel electrophoresis under non-
5'-CGCACTAGCTACTGAAGCACC-3' for the denaturing conditions [13]. After electrophoresis,
gtfI (GenBank D90213) and 5'-TCAGGCTCTG- overlay the gel with 0.7% agarose6 containing
GCGGCATCGAG-3' for the CITase structural 1.0% blue dextran and incubate at 37 DC. After
129
--
Asc
Integration System Integration System
chromosome
/ chromosome
-i.'.
Asc ABc
j.
S. gordonii wHd
EooRV
(J
~sc
t?
reS
ReSident
o Plasmid Not
.I
o 0
Emr
Chimeric
Integration
Plasmid
linearized DNA
linearized DNA
reS Not
foreign DNA
ABc
I---L\
~ I \ ~ .. m ______ •
ABc
\
linearized DNA MCS
, 1---LI I i ~ __ m m . . _ .
~"'tm~...",,_________Ch,"~\
Double crossing-over
Asc Asc
1--1-\ 1
~M M~ ~
Int~rant Plasmid Chromosome
1- _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ,
Figure 1. Overview of the SCIS and MPIS strategies. See text for details.
130
pACYC184 [2], and its replication ongm, p15A, the two regions, p15A and Tc s , present within the
were used throughout this study. Initially, pACYC184 acceptor DNA and linearized integration plasmid are
was extensively modified and a portion of the Tc f identical, double cross-over integration resulted in
gene was replaced with either an Emf or Spf gene (in replacement of the Emf gene with the Spf gene as well
this example, only the Emf version is depicted). In as the heterologous DNA fragment.
the anchor plasmid, the remaining 3'-portion of the
Tc gene was designated Tc s • Using this anchor Construction of resident, anchor, heterodimer, and
plasmid, both resident and heterodimer plasmids integration plasmids
were constructed. The former plasmid was trans-
formed into S. gordonii cells to establish the Multi- To construct resident, anchor, heterodimer, and
copy Plasmid Integration System (MPIS), while integration plasmids, E. coli plasmid pACYC 184
the latter plasmid was integrated into S. gordonii was extensively modified (Figure 2). Both BstBI
chromosome as a basis for the Single-copy (nucleotide position 3716) and Bell (3541) sites were
Chromosome Integration System (SCIS). Next, converted to AscI and a 170-bp DNA fragment was
foreign DNA was cloned into the integration plasmid removed. This manipulation resulted in the rendering
(Spf version) which was a derivative of the anchor of a unique BstBI site (1318). Next, the Sall site
plasmid. Following the linearization of the chimeric (2145) positioned in the middle of the Tc f gene was
integration plasmid with the restriction enzyme AscI, converted into a Not! site following linker ligation
competent S. gordonii cells were transformed. Since (A). This modified pACYC plasmid and pResEmNot
o E
Nco Pst EcoAV Pst Asc
I I ,I I, I
MO rep
pVA380-1 basic replicon (2.5Kb Asct fragment)
"S. Asci pMD749E-Tc «
>
U1 LO
» ~ EcoRV
7.0Kb C.
BslSl
C Eco
Sal
Asc
+ Tcs
..
Not
Resident Plasmid
,
Ba
BsSI. No~
>
BslSl
BsSI. No~ digestion ligation
point pOint
t
Pst Asc
G
IF
Not
Anchor Plasmid
BslSl p15A
pResEmNot
1.6Kb
Not
>
Asci
.. BslSl
pTF11
6.5Kb
MCS Pst
digestion...... Pst
point
Asc
ligation/
Pst point
Heterodimer Plasmid
BsISI
Kmr
Integration Plasmid
Figure 2. Construction of resident, anchor, heterodimer, and integration plasmids. For details, see the text. In heterodimer
plasmid (0), both ligation and digestion sites are such that the orientation of the two coupler fragments is the same as
is present on the chromosome. The structure of the MCS of the integration plasmid pMDINlO-Tc (H) is: NotI-BglII-
EcoRI-BamHI-HindIII-BclI-NotI in the clockwise orientation. The MCS structure between EcoRI and HindIII is the
same as that of pUC vectors. The MCS structure of the other integration plasmid pMDINll-Tc is the same except that
it is in the opposite orientation relative to the plasmid.
131
(B) were digested with BstBI and NotI and the (B), Sp' versions of resident (pMD749S-Tc), anchor
800-bp BstBI-NotI fragment corresponding to the 5' (pMDI84S), and integration plasmids (pMDINlOS-
end of the Tc' gene in the modified pACYC was Tc and pMDINllS-Tc) were also constructed (data
replaced with the gel-purified Em' gene containing a not shown).
portion of the p15A ori to yield the anchor plasmid
pMD 184E (C). To the unique AscI site of the anchor Construction of pTS204 and related plasmids;
plasmid, the pVA380-1 [14] replicon (D) or pTS204 introduction of 'coupler fragments'
(F, for construction of this plasmid, see below) was
introduced and the resident plasmid pMD749E-Tc (E) In order to develop a SCIS not only for S. gordonii
and heterodimer plasmid pTFll (G) were isolated. but also for use in other transformable bacteria, we
The anchor plasmid was also further modified introduced 'coupler fragments' originating from the
and the integration plasmids, pMDINlOE-Tc and S. mutans GS-5 chromosome as outlined in Figure
pM DIN llE-Tc (H), were constructed. In Figure 2, 3. The S. mutans GS-5 gtfB and gtfC genes are
only the Em' versions are depicted. Using arranged tandemly on the chromosome (A). The 250
pResSpNot, a counterpart plasmid of pResEmNot bp PstI-BglII fragments flanking the gtfB and gtfC
0
A I
2
I
4
I
6
I
8
I
10
I
12
I
14Kb
I
PBgI H EB HH B PES HH B Sst Sph Bgi BgIP
II III
I II I I I In III
I I I I I I I II
5' - S. mutans gtfB -gtfC 3'
0 2 4 10Kb
B I I I
6
I
8
I I
H H H H
I 121
I I I
- S. gordonii gttG
Pst
Pst
0 Pst
BsSl
>
PSt!
BgAI
... en
'Q,
Hind Pst
Bam Kpn Bam G
I I I I
xo
Sp/' Pst
E Kmr en
'0..,
pTS222S
5.2Kb
~ Hind F
BsIBI p15A
Hind
pResKmHind
2.0Kb
PSt!
...
>
Him II I
Xho
HSca H
II I
Figure 3. Construction of pTS204 and related plasmids as well as the strategy for introduction of the coupler fragments.
132
genes, referred to as 5' and 3', respectively, were suggested the possibility that the secretion domain
cloned into the PstI site of the plasmid pResAmpPst originating from B. circulans was inactive in strep-
(not shown), and pTS204 (C) was constructed. Into tococci. A similar observation was also made for the
the unique BglII site of the plasmid was ligated the secretion of a S. mutans glue an-binding domain from
Spr gene and pTS212S (D) was isolated. To introduce S. gordonii [14]. Therefore, we initially isolated a
the anchor plasmid into the S. gordonii chromosome, portion of the CITase gene devoid of the 700 bp 5'-
the 1.5 kb HindIII fragment present within the S. region (429~Pst~Eco) and then fused the remainder
gordonii gtfG gene (B) was cloned into the interme- ofthe gene inframe with the 5' -end of the S. sobrinus
diate plasmid, pResKmHind (E), and pTS221 (F) was gtfI gene coding for the signal sequence.
constructed. The unique Seal site found in the 1.5 To accomplish this, the 429~Pst~Eco fragment
kb fragment was next converted into a PstI site using was cloned into the integration plasmid pMDIN10S-
linker DNA. Following PstI digestion and ligation of Tc and pTFOl (C) was isolated. To construct the
these two p1asmids, pTS222S (G) was obtained. secretion domain, the 1.1 kb BamHI -SalI fragment
of the CITase gene and the 1.2 kb DraI-BclI fragment
Cloning of a portion of the CITase gene and from the S. sobrinus gtfI gene (B) were cloned into
replacment of its secretion domain with that from pResEmNotMCS10, a derivative of pResEmNot
the S. sobrinus gtfl gene (see Figure 2B), and pNSOl (D) was constructed.
Two PCR primers were designed to anneal with the
The gene coding for the CITase was first isolated on 3' -end of the signal peptide coding region of the gtfI
a 5 kb Sau3AI fragment and the non-essential 3'- gene and 5' -end of the structural gene of the CITase
region (1.2 kb) was removed by PstI digestion to gene. Following PCR amplification, self-ligation, and
produce 429~Pst (Figure 4A, 3.8-kb BamHI-PstI transformation, pNS03 was isolated (E). As shown
fragment). Our preliminary experiments indicated in Figure 5, DNA sequencing of the junction region
that although this gene was expressed in E. coli, S. of pNS03 confirmed that the secretion domain of the
gordonii harboring this gene secreted only very GTF from S. sobrinus was fused with the CITase
small amounts of CITase into the culture fluids. This structural gene in the correct reading frame.
0 2 3 4 5Kb
AI I I I I
Bam Him Eco Sal Sal Sal Him Sal Sal Sal Pst
I I I I I I I II I I I
B. circulans CITase gene
Eco Sal Sal Sal Him Sal Sal Sal Pst
I I I I I II I I I
429APsL1Eco
Bam Him Eco Sal
II I I
B Cia HimBgI Kpn Sac Him
• I II I I I
S. sobrinus gtfl gene
Ora Bel
I • I
c Eco
D E
pTF01 Eco
ind
7.4Kb
Figure 4. Cloning of a portion of the CITase gene and replacement of its secretion domain with that from the S.
sobrinus gtfI gene. The hatched region at the 5'-end of the gtfI open reading frame (B) corresponds to the signal sequence
coding region. Small inverted arrows in (D) denote the direction of PCR amplification.
133
Sequential integration of the CITase gene using the linearized pTFOl (bottom, line F) and
pSN03 (bottom, line H), integrants II and III were
The strategy for integrating the CITase gene into the isolated and the CITase gene was successfully inte-
resident plasmid (upper) or host chromosome (lower) grated into the chromosome of integrant III (bottom,
is depicted in Figure 6. This strategy involved line I).
initially integrating the CITase gene without a func-
tional signal sequence into the target region via the Secretion of CITase from s. gordonii
acceptor sequences. Subsequently, the GTF signal
sequence was fused to the CITase gene following Figure 7 shows the expression and secretion of
homologous recombination. S. gordonii harboring the CITase from plasmid-borne S. gordonii transfor-
resident plasmid pMD749E-Tc (Emr-version, top, line mants. When S. gordonii harboring pTF02-Tc and
A) was made competent and AscI-digested pTFOl pNS04-Tc were spread on BHI agar plates containing
(top, line B) was transformed into these cells. The 0.5% blue dextran, only the latter transformant exhib-
expected genotype of the initial plasmid-integrants ited clear halos around the colonies suggesting that
would be EmS and Spr. However, most of the trans- enzymatically active CITase proteins were secreted
formants were resistant to both Em and Sp. S. (Figure 7A, right). Plasmids were isolated from these
gordonii secondary transformants generated using the cells and subjected to agarose gel electrophoresis.
plasmids isolated from the initial transformation Figure 7B clearly indicated that following the
introduced into parental S. gordonii did exhibit the sequential integration of linearized DNA, the fused
EmS and Spr phenotypes and transformants harboring CITase gene was successfully introduced into the
pTF02-Tc were isolated (top, line C). These results resident plasmid. The presence of the active CITase
suggested that integration of the linearized plasmid enzyme in the culture fluids was also confirmed by
occurred in only a portion of the multi-copy plasmids zymography (Figure 7C).
in each cell in the initial transformation. Therefore, On the other hand, the single copy chromosome
each cell would contain a mixture of Emr and EmS integrant did not exhibit halos around the colonies on
plasmids. Next, pNS03 was linearized by digesting blue dextran plates. Therefore, chromosomal DNA
with AscI (top, line D) and S. gordonii harboring was isolated from integrants II and III, and the
pTF02-Tc was transformed. Transformants were presence of the CITase gene in the chromosome was
screened as described above and S. gordonii har- examined by PCR amplification (Figure 7D). Using
boring pNS04-Tc was isolated (top, line E). a set of primers corresponding to the p15A and
The target site for chromosomal integration used CITase sequences (see Figure 6), 1.8 kb and 2.4 kb
was within the S. gordonii gtfG gene (1.5 kb HindIII bands were obtained from integrants II and III (lanes
fragment, bottom, line A). pTS222S was digested 1 and 2, respectively). After Notl digestion of these
with AscI (bottom, line B) and transformed into S. two fragments, the former band yielded l.4-kb and
gordonii. An EmS and Spr integrant designated O.4-kb bands, while the latter fragment produced 1.3-
dBCSpr was isolated (lower line C) and 'coupler kb and 1.l-kb bands (lanes 3 and 4, respectively).
fragments' were successfully introduced into the host Since the 1.4-kb and O.4-kb bands corresponded to
chromosome. This strain was further transformed the Spr gene sequences containing a portion of p15A
with PstI-digested heterodimer plasmid, pTFll, ori and with the 5 -end ofthe 429LlPstLlEco while the
f
(bottom, line D) and integrant I was constructed latter 1.3-kb and 1.1-kb bands coincided with the Emr
(bottom, line E). Following sequential integration gene containing a portion of p15A ori and the 5 -endf
32 34 36 38. 40 42 44
TCC CCC CCG CAA GCG CTA GCA TCA GGC TCT GGC GGC ATC GAG
B. circulans CITase ~~~~~~~~-=~~~~==
Ser Pro Pro Gln Ala Leu Ala Ser Gly Ser Gly Gly Ile Asp
A
GGT GCT TCA GTA GCT AGT GCG GAT ACA GAC ACT GCT AGT GAT
s. sobrinus gtfl B
Gly Ala Ser Val Ala Ser Ala Asp Thr Asp Thr Ala Ser Asp
GGT GCT TCA GTA GCT AGT GCG TCA GGC TCT GGC GGC ATC GAG
pNS03
Gly Ala Ser Val Ala Ser Ala Ser Gly Ser Gly Gly Ile Asp C
Figure 5. Nucleotide and corresponding amino acid sequences around the signal peptide cleavage sites. The amino acid
sequence of the first 32 to 45 residues from the initiator Met of the B. circulans CITase (A), S. sobrinus GTF-I (B) and
pNS03 fusion protein (C) as well as the corresponding nucleotide sequences are shown. The nucleotide sequences of the
two PCR primers utilized to fuse the gifI secretion domain and the CITase structural genes are underlined. The arrow
denotes the signal peptide processing site of both the CITase and the GTF-I proteins.
134
Asc NotH E S
pNS03 I......!I. .11 I I
0
p1SA Em'
0 2 4 6 8 10Kb
I I I I I I
HSca H
wild
II I A
HP P H
pTS222S II I I1 I B
S' SpI' 3'
HP P H
dBCSp"
I 11
5' SpI'
1I
3'
I c
PAS<; Not As<; P
pTF11 I I· . --.11 t----- U 0
s· Cmf p1S" Em' Tc8 3-
As<; NotH E S
pNS03 I......!I• .11. I I
H
p1S" Emf
integrant III
1 I I. . ~I· .11 ~ I t----- II I
S' Cm' p1S" Emf CITase Tc8 3'
Figure 6. Sequential integration of the CITase gene. All constructs depicted were made in S. gordonii. For details, see
the text. The inverted arrows in S. gordonii integrants II and III (bottom, lines G and H) denote the primer annealing
sites and direction of PCR amplification for detection of the CITase gene integrated into the chromosome.
of the fused CITase gene, respectively, it was con- modified E. coli plasmid pACYC184 as the anchor
firmed that the CITase gene was successfully inte- plasmid. In this system, several DNA fragments
grated into the S. gordonii chromosome. Therefore, including anchor plasmids, coupler fragments, inte-
it is likely that the single copy of the CITase gene gration plasmids, and cloning plasmids were prepared
on the chromosome secreted only low levels of the and these DNA fragments were systematically
enzyme which could not be readily detected with the manipulated. To introduce the CITase gene into S.
blue dextran assay systems. gordonii, each of two 2S0-bp coupler fragments
originating from the S. mutans GS-S chromosome
were initially integrated into the host chromosome.
5. Discussion To subsequently integrate an anchor plasmid, a het-
erodimer plasmid was constructed since this con-
We have described an integration-mediated transfor- struct could readily provide the linearized DNA
mation system in S. gordonii using an extensively fragment in which the orientation of the two terminal
135
A B c o
M 1 2 34M 234 M 1 2 3 4
Figure 7. Secretion of CITase from S. gordonii. A, BHI agar plates containing 0.5% blue dextran spread with S. gordonii
transformants harboring pTF02-Tc (left) and pSN04-Tc(B). B, agarose gel electrophoresis of plasmids isolated from S.
gordonii transformants. Lanes 1 and 2, integration plasmids pMDINlOE-Tc; lanes 3 and 4, pTF02-Tc and pSN04-Tc. M
represents the molecular size marker ("-phage HindIII digest). C, zymogram of CITase activity. Samples in lanes 1 and
4 are the same as those in panel (B). For preparation of samples, see Materials and Procedures. D, agarose gel elec-
trophoresis of PCR products to detect the integrated CITase gene. Lanes 1 and 3, PCR product from integrant II; lanes
2 and 4, integrant III. Lanes 1 and 2, intact products; lanes 3 and 4, Notl digests.
regions (coupler fragments) are exactly the same as into a E. coli-streptococcal shuttle plasmid [5, 13].
that present in the host chromosome. The integra- However, we have demonstrated that such an
tion of the coupler fragments into any host chromo- approach does not appear to be suitable for all hetero-
some prior to the introduction of the anchor plasmid logous genes. For example, it was not possible to
would allow for the construction of a similar system stably clone a fusion gene composed of the S. mutans
in bacteria other than S. gordonii. This is an improve- gtfB secretory domain with the glucan-binding
ment of the original system developed in our domain DNA fragment from the S. mutans gtfD gene
laboratory [14] and the utility of coupler fragments into the shuttle plasmid pTS749 in E. coli [14]. For
as universal integration anchor sites for many this reason an earlier version of the MPIS and SCIS
bacteria is suggested. Indeed, these coupler fragments was developed, without the coupling fragment
were recently integrated into the S. milleri chromo- strategy, which was successful in secreting the glucan
some and the S. sobrinus gtfI gene was inserted into binding domain from S. gordonii [14]. The present
the chromosome and secreted GTF activity (data not strategy expands the potential usefulness of this
shown). Therefore, once DNA fragments of interest system to other transformable streptococci and poten-
are cloned into an E. coli integration plasmid, it is tially other gram-positive organisms as well. Whether
possible to introduce heterologous genes into either or not this strategy could also prove useful for strains
plasmids or chromosomes in S. gordonii, S. milleri transformable only by electroporation awaits further
and possibly other gram-positive transformable evaluation.
bacteria In attempting to secrete heterologous proteins
Although the CITase gene was integrated into the from streptococci, it would be practical to first utilize
S. gordonii chromosome, detection of CITase activity the simplest strategy for constructing a secretable
in the culture fluids was unsuccessful. It is possible derivative of the heterologous gene directly in a
that the promoter activity of the S. sobrinus gtfI gene shuttle plasmid in E. coli followed by transformation
may not be sufficient to secrete detectable amounts into the gram-positive bacteria. If difficulty in iso-
of enzyme protein. Additional approaches will be lating such a stable chimeric shuttle plasmid is
required to determine why the fusion protein was not encountered, either the MPIS or SCIS could prove
highly expressed from the S. gordonii chromosome. to be viable alternatives. Since the MPIS expresses
In the SCIS, utilization of the secretion domain from higher levels of the heterologous proteins, this system
the S. mutans GS-5 gtfB gene or the corresponding would likely be preferable for most purposes.
regions from other secreted proteins might be nec- However, maintenance of the relevant plasmids
essary to accomplish efficient secretion of heterolo- would likely require positive selection with anti-
gous proteins. biotics. For utilization in in vivo systems, this may
At first glance, the strategy depicted here would prove to be impractical. Nevertheless, non-antibiotic
appear to be excessively laborious since multiple selection strategies are available for plasmid main-
genetic engineering steps are required. Indeed, it has tenance [8].
been possible to construct secretion systems in S. An alternate strategy for secreting heterologous
gordonii using a simpler approach by cloning a proteins and peptides from S. gordonii has recently
fusion gene composed of a heterologous gene fused been developed [12]. This procedure utilizes a vari-
inframe to a streptococcal secretion domain directly ation of the surface expression system based on the
136
localization of the M-protein on the cell surface. By 2. Chang ACYC, Cohen SN (1978). Construction and
genetically engineering a stop codon between the characterization of amplifiable multicopy DNA
chimeric M-protein-heterologous protein sequences cloning vehicles derived from the p15A cryptic mini-
and the cell surface anchor sequences it has plasmid. J Bacteriol 134: 1141-1156.
3. Ferretti II, Gilpin ML, Russell RRB (1987).
been possible to secrete peptides comprising the
Nucleotide sequence of a glucosyltransferase gene
Porphyromonas gingivalis FimA fimbrillin subunit
from Streptococcus sobrinus MFe28. J Bacteriol169:
protein from S. gordonii. However, since only rela- 4271-4278.
tively small peptides have been secreted in this 4. Kobayashi M, Funane K, Oguma T (1995). Inhibition
system, it is not known if larger proteins can also be of dextran and mutan synthesis by cycloisomal-
secreted by this strategy. In this regard, a 98 kDa tooligosaccharides. Biosci Biotechnol Biochem 59:
protein has been secreted using the S. mutans gtfB 1861-1865.
secretory domain on a shuttle plasmid [13]. 5. Kubo S, Kubota H, Ohnishi Y, et al. (1993).
The ability to genetically engineer the oral com- Expression and secretion of an Arthrobacter dex-
mensal S. gordonii to secrete heterologous peptides tranase in the oral bacterium Streptococcus gordonii.
or proteins may have potential therapeutic value. Infect Imun 61: 4375-4381.
6. Loach DM, Jenkinson HF, Tannock GW (1994).
These organisms have been demonstrated to colonize
Colonization of the murine oral cavity by
the oral cavity of rodents [6] and may therefore be
Streptococcus gordonii. Infect Immun 62: 2129-2131.
implantable into the human oral cavity. Strains which 7. Loesche WJ (1986). Role of Streptococcus mutans in
stably secrete peptide antigens in the oral cavity may human dental decay. Microbiol Rev 50: 353-380.
elicit mucosal immunity against bacterial, fungal, or 8. Nakayama K, Kelly SM, Curtiss III R (1988).
viral pathogens. In addition, the secretion of peptides Construction of an asd+ expression cloning vector:
which interfere with the colonization of the patho- Stable maintenance and high level expression of
gens might also prove to be beneficial. Likewise, the cloned genes in a Salmonella vaccine strain.
secretion of bacteriocins or enzymes which interfere Biotechnology 6: 693-697.
with the growth or colonization of oral pathogens 9. Oguma T, Kurokawa T, Toke K, et aI. (1995). Cloning
might also prove useful. Therefore, it will be of and sequence analysis of the cycloisomaltooligosac-
charide glucanotransferase gene from Bacillus circu-
interest to pursue these possibilities not only for oral
lans T-3040 and expression in Escherichia coli. Appl
streptococci but for other organisms which could be Carb Sci 42: 415-419.
engineered with the MPIS or SCIS to antagonize 10. Pozzi G, Contomi M, Oggioni MR, et al. (1992).
microbial virulence in other areas of the human host Delivery and expression of heterologous antigens
(skin, reproductive tract, throat). Other genes besides on the surface of streptococci. Infect Immun 60;
the gtfG gene of S. gordonii or alternate promoter 1902-1907.
fragments may prove to be more suitable for such 11. Savijoki K, Kahala M, PaIva A (1997). High level
constructs. heterologous protein production in Lactococcus and
Lactobacillus using a new secretion system, based on
the Lactobacillus brevis S-layer signals. Gene 186:
Acknowledgments 255-262.
12. Sharma A, Sojar HT, Hruby DE, et al. (1997).
This study was supported in part by a Grant-in-Aid Secretion of Porphyromonas gingivalis fimbrillin
for General Individual Research Grants of the Suzuki polypeptides by recombinant Streptococcus gordonii.
Biochem. Biophys Res Commun (in press).
Memorial Grant of Nihon University School of
13. Shiroza T, Kuramitsu HK (1993). Construction of a
Dentistry at Matsudo (TS) and by grant DE03258 model secretion system for oral streptococci. Infect
from the National Institutes of Health (HK). Immun 61: 3745-3755.
14. Shiroza T, Kuramitsu HK (1995). Development of a
heterodimer plasmid system for introduction of het-
Notes on suppliers erologous genes into streptococci. Plasmid 34: 85-95.
15. Shiroza T, Ueda S, Kuramitsu HK (1987). Sequence
1. Perkin-Elmer Corp., Norwalk, CT, USA analysis of the gtfB gene from Streptococcus mutans.
2. Takara Syuzo, Kyoto, Japan J Bacteriol 169: 4263-4270.
3. Difco Laboratories, Detroit, MI, USA 16. Udaka S, Yamagata H (1993). High level secretion
4. Sigma Chemical Co., St. Louis, MO, USA of heterologous proteins by Bacillus brevis. Methods
5. Pharmacia Biotech., Piscataway, NJ, USA Enzymol217: 23-33.
6. BioProducts, Rockland, ME, USA
w. Todd Grey!, Joshua D. Lasker!, Roy Curtiss 1112 & Michael C. Hudson!
1 The University of North Carolina at Charlotte, Department of Biology, Charlotte, North Carolina, USA;
2 Washington University, Department of Biology, St. Louis, Missouri, USA
Abstract. Laboratory studies of bacterial gene lence-associated bacterial genes in vivo. This report
expression have not allowed the investigation of in describes a system, previously tested utilizing the
vivo factors crucial to pathogenesis. Recently, genetic fructosyltransferase (itf) gene of Streptococcus
tools have been used to identify in vivo-activated mutans, that allows the measurement of activity of
genes. These tools, however, have not allowed the specific virulence-associated streptococcal genes
measurement of specific expression levels of viru- while bacteria are living in the animal environment.
11.5 kb 23 kb
Figure 1. Region of the genome of S. mutans SMSI01 (ftf-cat) [6]. ORF, open reading frame; rbs, cat gene ribosome
binding site; CAT, promoterless chloramphenicol acetyltransferase gene; EM', erythromycin resistance gene; ftf,
fructosyltransferase gene; ., ftf promoter (duplicated); kb, kilobase pairs. The arrowheads depict the direction of tran-
scription.
138
It! gene. Compared to fructosyltransferase, the intra- - Sprague-Dawley rats (Charles River
cellular CAT molecule is relatively easy to easy for, Laboratories). 7
making the enzyme an extremely effective tool for - Standard rodent chow (Laboratory Rodent Diet
the study of factors influencing transcription of #5001, Purina Mills).8
bacterial genes. Understanding the role of various - Feeding bowls (5 cm deep with a 4 cm
environmental factors in influencing the expression opening, custom made).
of bacterial virulence-associated genes is extremely - Pipet tips (Fisher #21-244-1).
important in the study of the pathogenesis of any - Cotton-tipped applicators (Fisher #14-959-90).
bacterium. Because the mammalian environment is - Trizma base (Sigma #T6791).
so complex, it cannot be successfully duplicated in - Scalpel (Fisher #08-920A).
vitro. With regard to the oral streptococci, host- - Forceps (Fisher #13-812-41).
dependent factors such as carbohydrate consumption - Microcentrifuge tubes (Fisher #05-407-10).
amounts and animal age, for example, could influ- - 0.1 mm zirconium beads (Biospec).
ence bacteria present in the animal environment. - Chloramphenicol (Sigma #C0378).
Because the complex mammalian environment may - i4C-Acetyl CoA solution (ICN Pharma-
influence streptococcal gene expression in ways that ceuticals, Inc. #13070L).9
cannot be observed in the laboratory, we developed - Ethyl acetate (Fisher #BP1125-1).
a method where the expression of cat, controlled by - Polyethylene scintillation vials (Fisher #03-
the It! regulatory region, was used as a reflection of 337-11B).
It! expression while S. mutans was present in the - Bio-Safe II scintillation cocktail (Research
mammalian oral cavity. Products International Corp. #111195).10
It has been demonstrated that members of the rat - 100% ethanol (McCormick Distilling Co.
normal oral flora are sensitive to the antibiotic Inc.)Y
streptomycin [1, 8,9, 13]. Therefore, a stable, spon- C. Animal diets
taneous streptomycin-resistant (strf) version of S. - Dextrin (Sigma #D2006).
mutans fusion strain SMS 101 [6], designated - Polypeptone (Difco #DFOI23-17-3).
NCHI06, was isolated to facilitate strain enumeration. - L-methionine (Sigma #M6039).
- Calcium phosphate (dibasic, Sigma #C7263).
- Sodium chloride (Sigma #S7653).
2. Materials - D-Sucrose (Sigma #S7903).
- D-Glucose (Sigma #G7528).
A. Equipment - D-Fructose (Sigma #F2543).
- High speed centrifuge (RC5C, Sorvall - Vitamin Bi (thiamine, Sigma #T4625).
Instruments).i - Vitamin B6 (pyridoxine, Sigma #P9755).
Micropipetter (Oxford 40-200 III range, Fisher - Vitamin C (ascorbic acid, Sigma #A5960).
Scientific #2100231)? - Vitamin B3 (niacinamide, Sigma #N3376).
Vortex mixer (Vortex Genie 2, Fisher #12-812). - Vitamin B5 (calcium pantothenate, Sigma
Microscope (Micromaster HL, Fisher #12-576- #P57 10).
2S). - Vitamin B2 (riboflavin, Sigma #R4500).
- 37°C incubator (Isotemp, Fisher #11-690- - Vitamin E (tocopherol acetate, Sigma #T3001).
650D). - Vitamin D2 (ergocalciferol, Sigma #E5750).
- Mini Beadbeater apparatus (Biospec - Vitamin A (retinol acetate, Sigma #R4632).
Products).3 - Vegetable oil (Proctor and Gamble)Y
- Microcentrifuge (235C, Fisher). - Whole wheat flour (The Pillsbury CO.).13
- Water bath (Isotemp, Fisher #15-461-2).
- Liquid scintillation spectrophotometer (LS
6000 SC, Beckman Instruments, Inc.).4 3. Procedures
B. Supplies
- Brain Heart Infusion (BHl) broth and agar A. Str bacterial strain
(Difco Laboratories #DF0037-17-8, DF0418- Transfer an isolated colony of the streptococcal
17_7).5 cat fusion strain from a brain heart infusion (BHI)
- Todd Hewitt Broth (THB) (Difco #DF0492- agar plate into 45 ml of BHI broth (Difco) in a
17-9). 50 ml polypropylene tube. Grow anaerobically
- 50 ml polypropylene tubes (Fisher #05-538- (GasPak Plus Anaerobic System) at 37°C for
55). 3 days, standing, to achieve a dense culture
- GasPak anaerobic culture system (Fisher (approximately 10 10 colony forming units
#11-814-21). (cfu)/ml). Harvest bacteria by centrifugation at
- Streptomycin sulfate (Sigma Chemical Co. 4300 xg for 10 minutes at 4 °c to concentrate
#S6501).6 cells. Spread plate approximately lOll cfu on BHI
139
agar containing 200 Ilg/ml streptomycin sulfate any remaining diet with fresh food once each
(Sigma) and grow anaerobically at 37 DC for day.
4 days. Streak resistant clones for isolation on Transfer an isolated colony of the streptococcal
fresh media containing streptomycin. Uniform str cat fusion strain from a BHI agar plate con-
colony size will be indicative of stability of the taining streptomycin into Todd Hewitt broth
mutation responsible for resistance. Determine the (THB) (Difco) containing 1% sucrose and grow
maximal level of expressed streptomycin resis- anaerobically at 37 DC overnight. Concentrate
tance by streaking isolates on BHI agar containing bacteria by centrifugation at 4300 xg and suspend
increasing amounts of streptomycin. This will in fresh THB containing 1% sucrose to a density
dictate the amount of streptomycin to be used to of 1 x 1010 cfu/ml. Inoculate cell suspensions into
select for cat fusion strains once removed from the rat oral cavities on days 2 and 3 of the 4 day
the animal environment. inoculation period. Inoculation of animals is best
B. Animal inflections performed by 2 individuals. To accomplish animal
Maintain 18- to 40-day-old Sprague-Dawley rats inoculations on days 2 and 3 of the 4 day high
(Charles River) on indirect bedding in groups of sucrose diet, remove animals from their cages
4 throughout the experiment. Feed the animals singly. Cradle the animal, using both hands, while
standard rodent chow (Laboratory Rodent Diet not applying any pressure. Hold hands with
#5001, Purina) for at least 4 days prior to the thumbs up, knuckles forward, and I set of fingers
inoculation and experimental periods (allowing enclosed by the other so that a small hole will be
time for acclimation to the animal facility), and created that the rat can sit in with head protruding.
supply them with distilled water lacking fluoride. Mix the bacterial inoculum by pipetting in and out
To initiate the experiments, feed the rats soft several times with a micropipetter followed by
defined diet M2000 (containing 56% sucrose) [8] withdrawal of 100 III of suspension into the
(Table 1) for 4 days (inoculation period). The soft pipette tip. Gently steady the animal's head and
diet will facilitate retention of bacteria on the insert the pipet tip into 1 side of the rat's mouth.
teeth once infection has occurred. Feeding dishes Expel the solution slowly and observe that the
will either need to be purchased or made that will animal is drinking. If the animal is hesitant to
allow the rats to feed on the soft sticky diet drink the suspension, gently touch the pipet tip
without getting the food on their bodies and in to the tongue or mouth roof of the animal. This
particular on their feet (animals actually will usually will cause the animal to begin drinking.
become stuck to the bottom of their cages). We Administer the bacterial suspension until the pipet
had pottery made that were 5 cm deep with a tip is empty. Reinoculate the animal if less than
4 cm opening for this purpose. Add soft diet 75% of the suspension is retained. Do not provide
anytime animals ingest all provided and replace antibiotics to animals so that streptococcal fusion
strains can interact with members of the rat
Table 1. Diet M2000 normal oral flora.
C. Experimental treatment
Component Amount Following the 4 day inoculation period, place
animals on diet 2000 (which lacks sucrose) [8, 10]
Dextrin 115g (Table 2) containing 5% glucose for I to 2 days.
Polypeptone 270 g After this 1 to 2 day period, remove diet from the
L-methionine 10 g animals so that they fast overnight. Once fasted,
Calcium phosphate 8g separate animals into individual cages and feed
Sodium chloride 20 g
them diet 2000 containing a particular carbohy-
Sucrose 560 g
Vitamin BJ (thiamine) 25 Ilg drate at a 5% final concentration for 30 minutes,
Vitamin B6 (pyridoxine) 25 Ilg and during this time confirm that the animals
Vitamin C (ascorbic acid) 750 Ilg actually ingest the provided diet (confirmation
Vitamin B3 (niacinamide) 250 Ilg facilitated by use of individual cages). The
Vitamin Bs approach utilizing feeding following fasting will
(calcium pantothenate) 100 Ilg allow for precise control of exposure of strepto-
Vitamin B2 (riboflavin) 25 Ilg in 2.5 ml 0.1 N coccal cells to experimental carbohydrates.
NaOH D. Collection of bacteria and preparation of bacterial
Vitamin E lysates
(tocopherol acetate) 1 ml Following the experimental treatment of animals,
Vitamin D2 (ergocalciferol) 0.063 Ilg in 2 III ethanol
collect bacteria from the animals' teeth by
Vitamin A (retinol acetate,
2.9 x 106 U/g) 8.62 Ilg in 148 III ethanol
swabbing the tooth surfaces with sterile cotton-
Vegetable oil 6 ml tipped applicators soaked in 0.05 M Tris-HCI
Distilled water 400 ml buffer (pH 7.8). Collection of bacteria is best
performed by 2 individuals. Place the animal into
140
10. Keyes, PH, Jordan HV (1964). Periodontal lesions in chloramphenicol acetyltransferase specified by R
the Syrian hamster-III Findings related to an infec- plasmids. Nature (London) 282: 872-872.
tious and transmissible component. Archs Oral BioI 16. Sleigh, MJ (1986). A nonchromatographic assay for
9: 377-400. expression of the chloramphenicol acetyltransferase
11. Kiska, DL, Macrina FL (1994). Genetic regulation of gene in eucaryotic cells. Anal Biochem 156: 251-256.
fructosyltransferase in Streptococcus Mutans. Infect 17. Wexler DL, Hudson MC, Burne RA (1993).
Immun 62: 1241-1251. Streptococcus mutans fructosyltransferase (jtf) and
12. Mahan, MJ, Tobias JW, Slauch JM, Hanna PC, Collier glucosyltransferase (gtfBC) operon fusion strains in
RJ, Mekalanos JJ (1995). Antibiotic-based selection continuous culture. Infect Immun 61: 1259-1267.
for bacterial genes that are specifically induced during 18. Weyrauch CD, Burgess 10, Reagan SE (1995). Tooth
infection of a host. Proc Natl Acad Sci USA 92: loss during compulsory dental care. Military Med 160:
669-673. 194-197.
13. Ooshima T, Sumi N, Izumitani A, Sobue S (1988).
Effect of inoculum size and frequency on the estab-
lishment of Streptococcus mutans in the oral cavities
of experimental animals. J Dent Res 67: 964-968. Address for correspondence: Michael C. Hudson, Ph.D.,
14. Shaw WV (1979). Chloramphenicol acetyltransferase Department of Biology, The University of North Carolina
from chloramphenicol-resistant bacteria. Methods at Charlotte, 9201 University City Boulevard, Charlotte,
Enzymol 43: 737-755. NC 28223, USA
15. Shaw WV, Packman LC, Burleigh BD, Dell A, Morris Phone: 704/547-4048; Fax: 704/547-3457
HR, Hartley BS (1979). Primary structure of a E-mail: mchudson@email.uncc.edu
Methods in Cell Science 20: 143-151 (1998)
© 1998 Kluwer Academic Publishers.
Abstract. The objective of the current research was straining pattern of 23S and 16S rRNAs, either
to define a consistent and reliable method for the denaturing (formaldehyde/agarose) or nondenaturing
preparation of high quality, intact total RNA from (TAE/agarose) gel electrophoresis could be used to
Gram-positive oral bacteria. The method described, determine the general integrity of the RNA prepara-
adapted from previously published protocols [1, 8, tion. Analysis by either method revealed sharp rRNA
17], employs mechanical means to disrupt cells bands indicative of high quality RNA. Expression
and release RNA. Bacterial cells are subjected to of adherence-associated transcripts could be detected
vigorous vortexing in the presence of high density using formaldehyde/agarose gel electrophoresis and
glass beads and the denaturing agents, phenol and northern hybridization analysis. This method of RNA
sodium dodecyl sulfate. Under these conditions, dis- preparation proved to be useful for isolation of intact
ruption of the bacterial cell walls is rapid enough to RNA from the recalcitrant bacterium, Streptococcus
expose intracellular contents to the denaturing agents parasanguis, and may prove useful for other such
before endogenous ribonucleases have a chance to oral bacteria.
hydrolyze RNA. Based on the ethidium bromide
Key words: Adherence, Gene expression, RNA isolation, Streptococci, Transcriptional regulation
Abbreviations: cpm =counts per minute; DEPC =diethyl pyrocarbonate; EDTA =ethylenediaminetetraacetic
acid, EtBr =ethidium bromide; kb = kilobases; mRNA = messenger RNA; MOPS = 3-(N-morpholino)propane-
sulfonic acid; RNA = ribonucleic acid; RNase =ribonuclease; rRNA =ribosomal RNA; SDS = sodium dodecyl
sulfate; THB = Todd Hewitt Broth; Tris = Tris[hydroxymethyl]aminomethane
1. Introduction the oral cavity [11, 12, 15]. Adherence of oral strep-
tococci to this wide range of substrates is facilitated
The sanguis streptococci (including Streptococcus by multiple adhesins exposed on the surface of the
parasanguis, S. sanguis, S. gordonii and S. oralis) bacterial cell [12]. These adhesins are considered to
are the primary colonizers of the tooth surface and be important factors in determining the success of
are found abundantly in human dental plaque [6], oral streptococcal colonization and survival within
the forerunner of dental caries and inflammatory the human host.
periodontal disease. In addition, when the oral Given the constantly changing environmental con-
streptococci gain access to the bloodstream, they ditions micro-organisms face when attempting to
can successfully colonize heart tissue, becoming a colonize the human host, it is not surprising that they
major cause of subacute bacterial endocarditis [21]. have evolved the ability to regulate the expression of
Successful colonization and the persistence of these adherence- and other virulence-associated genes. The
organisms within the constantly changing conditions differential expression of these genes enables the
of the oral cavity or within heart tissue implies that bacteria to selectively produce factors necessary for
they have evolved mechanisms adaptive to their successful adaptation to changing host conditions.
survival in these unique ecological niches. A critical For example, the types of adhesins expressed by the
first step in the colonization process is the ability of oral streptococci at the mucosal surfaces of the oral
the bacterium to attach firmly to the host surface. cavity may be very different from those required for
Adherence is particularly important in areas such as adherence to heart tissue. The regulation of bacte-
the oral cavity, where mucosal surfaces are constantly rial virulence factors is often associated with envi-
being washed by host fluids. Numerous studies ronmental cues such as growth phase, carbon source,
suggest that oral streptococci bind to a variety of pH, temperature, osmolarity, iron or oxygen levels
substrates in the mammalian host, including salivary and several of these changes in gene expression
components, extracellular matrix or serum compo- appear to be regulated at the transcriptional level
nents, host cells and other microbial cells present in [2, 5, 9, 10, 14, 16, 22].
144
anaerobic environmental conditions. Include LiCl and 2 volumes of 100% ethanol. Mix well
this medium in the growth chambers so that and place at -20 DC for at least 30 min.
the medium has equilibrated with the envi- 5. Centrifuge 10K for 15 min. Pour off super-
ronmental conditions of each growth chambers natant and wash pellet gently with ice cold
prior to use. 70% ethanol. Centrifuge for 5 min. Remove as
2. After overnight incubation, the cultures will much of the ethanol as possible and then allow
be in stationary phase (Do not incubate the the pellet to air dry by inverting the tube onto
cultures longer than 18 h. After this point cells a clean KimWipe for 5 min at room tempera-
enter a logarithmic death phase and will go ture. Resuspend the pellet in 200 III DEPC-
through a long lag phase when resuspended in treated water. Freeze RNA in aliquots at
fresh medium). Add two mls of the aerobic -70 De.
culture to each of the two flasks containing D. Analysis of RNA integrity
125 ml of THB equilibrated to aerobic condi- Before proceeding to northern hybridization
tions. Incubate at 37 DC, 5% CO 2 until mid-log analysis or to other applications, it is necessary
growth phase (about 3.5 h). Do the same to first assess the integrity of the isolated RNA.
for the culture growing under anaerobic con- Most often, RNA is analyzed by electrophoresis
ditions. through a denaturing agarose gel in the presence
3. Pour the cultures from each growth conditions of ethidium bromide. Several denaturing agents,
into five 50 ml disposable polypropylene cen- such as formaldehyde, glyoxal and dimethyl sul-
trifuge tubes and incubate on ice for 10 min. foxide (DMSO) or methyl mercuric hydroxide,
e. Preparation of total RNA can be used [1]. However, formaldehyde is used
For preparation of total RNA, perform all steps most frequently because it is less toxic than
at 4 DC and, with the exception of 10% SDS, keep mercuric hydroxide and formaldehyde gels are
all solutions and tubes on ice. Chill the glass less difficult to run than those containing
beads on ice before use. Centrifuge larger glyoxallDMSO [1]. Integrity of RNA can also be
volumes in a refrigerated Sorvall RC5B cen- assessed rapidly by electrophoresis on a nonde-
trifuge using a pre-chilled SS34 rotor. The 50 ml naturing agarose gel. This latter method elimi-
and 15 ml polypropylene tubes will fit into a nates problems associated with toxic chemicals.
SS34 rotor if a nut, rather than the rotor cover, Both methods are presented below. The protocol
is used to secure the rotor. Alternatively, other for analysis of RNA using a denaturing agarose
types of refrigerated centrifuges can be used. For gel is adapted from Ausubel et al. [1]. Both
smaller volumes use a refrigerated microcen- methods, as well as northern hybridization
trifuge or a microcentrifuge kept in a cold room. analysis, are performed on 7 x 10 cm horizontal
The procedure below is for 250 ml of culture submarine gels. Longer gel rigs can be used if
grown under one environmental condition. More additional resolution is required. Use distilled
than one culture can be processed simultaneously. water and sterile Pipetman tips and microfuge
1. Centrifuge tubes for 10 min, 6K, 4 DC. tubes that have been reserved for use with RNA
Aspirate or carefully pour off supernatant and applications.
resuspend bacteria in a total volume of 4.0 ml 1. Analysis of RNA using a denaturing agarose
of ice cold TE, pH 8.0. Transfer the bacteria gel
to a 15 ml disposable polypropylene centrifuge a) Preparation of lOX MOPS buffer: lOX
tube. MOPS buffer is 0.2 M 3-(N-morpho-
2. Add 3.5 g chilled acid-washed glass beads, lino)propanesulfonic acid (MOPS), 50 mM
3 ml of 100:24: 1 (v/v/v) acid-saturated sodium acetate, 10 mM EDTA, pH 8.0. To
phenol/chloroform/isoamyl alcohol and 0.25 prepare one 1, add 41.9 g of MOPS and
ml 10 % SDS to the 15 ml tube and mix well 4.1 g of sodium acetate to 800 ml of water
to ensure that beads are suspended. Vortex and adjust the pH to 7.0. Add 20 ml of a
immediately for a total of 3 min at highest 0.5 M stock solution of Na2EDTA, pH 8.0
speed. After each min, place on ice for 1 min. and adjust the volume to 1 1 with water.
Repeat two more times. Store at 4 DC, protected from light.
3. Centrifuge for 1 min at 6K. Transfer top b) Preparation of a 0.44 M formaldehyde
aqueous phase to a clean 15 ml polypropylene denaturing gel: To make 50 ml of a 1%
centrifuge tube, making sure not to take any agarose/formaldehyde gel, melt 0.5 g of
of the interface. Add an equal volume of agarose in 43.2 ml of water and cool to
100:24: 1 (v/v/v) phenollchloroform/isoamyl about 65 DC. Add 1.8 ml of formaldehyde
alcohol and vortex at top speed for 10 sec. and 5 ml of lOx MOPS buffer to the
Centrifuge 1 min at 6K. Repeat extraction 3 melted agarose, swirl to mix and pour into
more times. gel tray. Caution: Formaldehyde vapors
4. To precipitate RNA, add 0.1 volume of 10 M are toxic. Prepare solutions containing
147
stranded DNA probes labeled by nick transla- first investigated the utility of a RNA isolation kit for
tion to a specific activity of at least 1 x 108 yeast and Gram-positive bacteria commercially avail-
cpm//-lg using [a- 32P]dCTP using the able from Gentra Systems, Inc. (15200 25th Ave. N.,
GibcoBRL Nick Translation System according Suite 104, Minneapolis, MN 55447), for the isolation
to the manufacturer's directions. Unincor- of RNA from S. parasanguis. This kit (PUREscript,
porated label is removed from the probe using Cat. No. R-6000) employs a proprietary 'Lytic
TE SELECT-D, G25 spin columns according Enzyme Solution' to generate spheroplasts. Using
to the manufacturer's directions. Add the dena- this kit, RNA was isolated from S. parasanguis, fol-
tured probe directly into the prehybridization lowing the manufacturer's protocol, and then RNA
solution (about 500,000 cpm of the probe/ml integrity was evaluated using native and denaturing
hybridization solution). agarose gel electrophoresis and northern hybridiza-
6. Following hybridization, wash the filter for tion analysis. Some degradation of the RNA was
10 min at room temperature in 2x SSC, evident and the quality of the Northern blot, using
0.5% (w/v) SDS. Repeat. Wash at 65°C for this RNA, was not satisfactory (data not shown).
15 min in Ix SSC, 0.1 % SDS. Repeat. Wash Since all of the solutions in the kit were RNase free
15 min at 65°C in 0.2x SSC, 0.1 % (w/v) SDS. and were handled carefully, the most likely explana-
Repeat. tion for degradation of the RNA was that, for S.
7. Expose the filter to Kodak film at -70°C with parasanguis cells, the enzymatic disruption was too
an intensifying screen. Develop the film after slow and inefficient. Good lysis of S. parasanguis
24 h. cells was not observed following incubation with the
'Lytic enzyme solution'. We concluded that a strong
physical force may be necessary to disrupt the cell
4. Results and discussion walls of the oral streptococci.
A number of reports have indicated that RNA can
Our interest in studying the expression of specific be released from cells that are difficult to disrupt by
streptococcal adherence-associated genes by northern vigorously vortexing cells in the presence of glass
hybridization has led us to explore alternative beads and denaturing agents, phenol and SDS [1,
methods for the isolation of high quality, intact RNA 8,17]. We adapted this method for use with S.
from S. parasanguis. Although useful information parasanguis and found that the streptococcal cells
about the steady-state levels of RNA produced from could be disrupted quickly and effectively by this
a specific gene can be garnered with lower quality mechanical means to yield high quality RNA. The
RNA preparations using slot or dot hybridization of procedure requires few manipulations and is done in
RNA [18], coupling gel electrophoresis with northern the cold room, or on ice, with prechilled reagents to
hybridization offers several advantages. First of all, minimize RNase activity. Another advantage of the
transcript size can be determined using gel elec- method is that it prevents RNA degradation by
trophoresis. More importantly, however, it is possible RNases during lysis since samples are always in the
to detect the presence of multiple transcripts presence of the denaturing agents. The use of phenol
produced from a single gene or operon or to detect in methods for RNA preparations was first described
changes in the relative size of a transcript produced by Kirby [13] and phenol extraction is an effective
from a single gene or operon. This could become means of inactivating and removing RNases. In the
extremely important when comparing gene expres- past, however, the preparation of phenol for use in
sion under different environmental conditions when, molecular biology experiments was difficult and
not only message abundance but also message size often hazardous, with the phenol being subject to
or number, may vary in response to environmental varying impurities. Now, the use of phenol is safer
signals. In addition, gel electrophoresis allows for the and more convenient due to the introduction of
direct comparison of relative message abundance and prepared high quality, RNase-free, saturated liquid
transcript sizes between samples on the same blot. phenols by numerous biotechnology companies. In
The blot can be stripped and rehybridized several addition, the method does not require hot phenol for
times with probes to other virulence-associated genes the extraction of RNA as does our previous method,
or to other genes of interest, greatly facilitating thus safety risks associated with the handling of hot
analysis of gene expression. phenol are minimized.
This approach, however, requires high quality, The general integrity of the RNA released from
intact RNA. The objective of the current research was cells was assessed based on the typical ethidium
to improve upon methods developed in our labora- bromide staining pattern of total RNA after elec-
tory for the preparation of RNA [4] by defining a trophoresis on both a simple native agarose gel
protocol that would consistently and reliably yield (Figure 1A) or on a denaturing formaldehyde/agarose
high quality RNA from S. parasanguis. Although gel (Figure lB). Total RNA preparations contain pri-
methods based on enzymatic digestion of cells walls marily rRNA. If the two bands, representing the 23S
have not been optimal with the oral streptococci, we and 16S, in either lane were not sharp, it would be
149
indicative of RNA degradation during sample prepa- avoided and gels can be run on the laboratory bench
ration. This was not the case. Two sharp bands appear rather than in a fume hood.
in each lane on both gels, indicating high quality, To analyze the integrity of the total RNA prepa-
intact RNA had been isolated from both sample rations further, we employed northern hybridization
preparations. Chromosomal DNA is also apparent in analysis using an adherence-associated gene, limA,
the RNA preparations and is particularly evident from S. parasanguis FW213 [3,4,6] as a hybridiza-
when the RNA is analyzed on native agarose gels tion probe. RNA was denatured with formaldehyde/
(Figure lA). The method releases both RNA and formamide and separated on a 0.44 M formalde-
DNA, so an A z60 reading will be a measure of total hyde/1 % agarose gel. This procedure gave good
nucleic acid. Lithium chloride precipitation is incor- separation of RNAs over a range of molecular
porated into the protocol as this salt preferentially weights as indicated by the distinct RNA markers
precipitates RNA. While, the RNA preparation still ranging from 461 to 4061 bases (Figure 2A).
contains some contaminating DNA, this usually will Addition of EtBr directly to RNA samples prior to
not interfere with most studies, including S I, primer loading on the formaldehyde/agarose gel gave excel-
extension and northern hybridization analysis. If lent visualization of RNA with ultraviolet light
further purification of the RNA is required, DNase immediately after electrophoresis (Figure 2A). It was
treatment can also be used [1, 18]. Expected yields also advantageous to be able to view the RNA at any
from a 250 ml mid-log culture of S. parasanguis are point during electrophoresis. If the RNA has not been
approximately 500 )lg of RNA. separated sufficiently, electrophoresis could be con-
Both native and denaturing agarose gels gave com- tinued. After transfer to a nitrocellulose membrane
parable results (Figure IA and IB), indicating that by capillary action, the gel retained undetectable
either type of gel could be used to assess RNA amounts of RNA indicating that the stained RNA had
quality. Native gels offer an advantage over dena- been transferred efficiently (Figure 2B).
turing gels in that sample and gel preparation is easier
and therefore somewhat quicker. Toxic chemicals are
M I 2 M I 2 2
2 2
+- DNA
... 3.3 kb
DNA .....
+- 23S
+- 16S
23S .....
16S .....
A B c
Figure 2. Northern hybridization analysis of RNA isolated
from S. parasanguis FW213. RNA samples were prepared
and separated on a 1% agarose/formaldehyde gel as
described in Procedures. (A) Electrophoretic pattern of
EtBr-stained RNA before transfer to nitrocellulose. Lane
A B M, RNA molecular weight markers (2 (J.g) are, from top
to bottom, 4061,2193, 1701, 1280,754,461 bp; lanes 1
Figure 1. Electrophoretic pattern of RNA isolated from and 2, RNA samples (10 (J.g) prepared from cultures grown
S. parasanguis FW213 using vigorous vortexing of cells anaerobically or aerobically in 5% CO 2 , respectively. (B)
in the presence of glass beads and denaturing agents. Gel depicted in (A) after transfer of RNA to nitrocellu-
EtBr staining of rRNA reveals RNA integrity. Samples lose by capillary action as described in Procedures. (C)
were prepared and gels electrophoresed as described in Northern blot of RNA samples transferred to nitrocellu-
Procedures. (A) Native 1% agarose gel in TAE buffer. lose and analyzed for the presence of FimA mRNA using
Lanes 1 and 2 are RNA samples prepared from cultures a nick-translated vector probe containing internal fimA
of S. parasanguis FW213 grown anaerobically or aerobi- sequences as described in Procedures. Lanes 1 and 2, total
cally in 5% CO 2, respectively. (B) Denaturing 1% agarose RNA prepared from cultures grown anaerobically or aer-
gel in formaldehyde/MOPS buffer with the same samples. obically in 5% CO 2, respectively.
150
Our laboratory has shown previously that the limA Notes on suppliers
operon produces a 3.3 kb transcript when grown aer-
obically in THB with 5% CO 2 [4]. The results of 1. VWR Scientific Products Corp., 501 Heron Dr.,
northern blotting of total RNA isolated from S. Bridgeport, NJ 08014, USA
parasanguis FW213 grown either anaerobically or 2. Forma Scientific, Box 649, Marietta Ohio 45750-0649
aerobically in 5% CO 2 are shown in Figure 2C. The USA
3. DuPont, 31 Pecks Lane, P.O. Box 5509, Newtown, CT
appearance of nondegraded FimA mRNA (Figure 2C,
06470-5509 USA
Lane 2) further demonstrates that the described 4. Bio-Rad Laboratories, 2000 Alfred Nobel Drive,
method for preparing total RNA from S. parasanguis Hercules, CA 94547 USA
yields high quality, intact RNA. The ability to isolate 5. Sigma Chemical Company, P.O. Box 14508, St. Louis,
high quality RNA is the first and often most critical MO 63178 USA
step in performing many fundamental molecular 6. Gibco BRL, P.O. Box 68, Grand Island, NY 14072-
biology experiments and it is essential to analyzing 0068 USA
gene expression. For our work, it has made it possible 7. New England Bio1abs, 32 Tozer Road, Beverly, MA
to begin to analyze the regulation of adherence-asso- 01915-5599 USA
ciated genes in S. parasanguis FW213. Northern 8. 5 Prime-3 Prime, Inc., 5603 Arapahoe Road, Boulder,
hybridization analysis suggets that expression of CO 80303 USA
9. NEN Life Science Products, 549 Albany Street, Boston,
FimA is stimulated at the transcriptional level during
MA 02118-2521 USA
aerobic growth (Figure 2C, Lane 2) but not during
anaerobic growth (Figure 2C, Lane 1). Further
analysis of environmental signaling may reveal other References
pathways that may be involved in the regulation of
adherence-associated gene expression. 1. Ausubel FM, Brent R, Kingston RE, Moore DD,
In summary, we describe here a simple, reliable Seidman JG, Smith JA, Struh1 K, eds (1989). Current
and relatively rapid method for preparing high protocols in molecular biology. New York: John
quality, intact total RNA from the Gram-positive Wiley and Sons.
bacteria, S. parasanguis. This procedure may have 2. Caparon MG, Geist RT, Perez-Casal J, Scott JR
general applicability for use with oral bacteria or (1992). Environmental regulation of virulence in
other cells with tough walls that are difficult to group A streptococci: transcription of the gene
disrupt. The use of vigorous vortexing with glass encoding M protein is stimulated by carbon dioxide.
J Bacteriol 174: 5693-5701.
beads greatly assists the initial disruption of cells and,
3. Fenno JC, LeBlanc DJ, Fives-Taylor PM (1989).
because the sample is always in the presence of Nucleotide sequence of a type 1 fimbrial gene of
phenol/SDS, this method prevents RNA degradation Streptococcus sanguis FW213. Infect Immun 57:
during lysis. The resulting RNA preparation is useful 3527-3533.
for routine Northern hybridization analyses and can 4. Fenno JC, Shaikh A, Spatafora G, Fives-Taylor P
also be used for primer extension and S 1 analyses. (1995). ThefimA locus of Streptococcus parasanguis
Because of the relative ease with which this proce- encodes an ATP-binding membrane transport system.
dure can be performed, it is possible to process Mol Microbiol 15: 849-863.
several samples at the same time. This feature is par- 5. Finlay BB, Falkow S (1997). Common themes in
ticularly important when comparing the steady-state microbial pathogenicity revisited. Microbiol Mol BioI
level of a message under various growth conditions Rev 61: 136-169.
6. Fives-Taylor PM, Macrina FL, Pritchard TJ, Peene SS
or over time. This method of RNA preparation will
(1987). Expression of Streptococcus sanguis antigens
facilitate analyzes of the expression of virulence- in Escherichia coli: cloning of a structural gene for
associated genes in S. parasanguis and in other oral adhesion fimbriae. Infect Immun 55: 123-128.
streptococci. 7. Frandsen EVG, Pedrazzoli V, Kilian M (1991).
Ecology of viridans streptococci in the oral cavity and
pharynx. Oral Microbiol Immunol 6: 129-133.
Acknowledgments 8. Gagnon G, Vadeboncoeur C, Gauthier L, Frenette M
(1995). Regulation of ptsH and pts/ gene expression
We are grateful for very helpful experimental in Streptococcus salivarius ATCC 25975. Mol
suggestions from Dr Michel Frenette (University Microbiol16: 1111-1121.
of Laval, Quebec City, Quebec, Canada), Dr 9. Gibson CM, Caparon MG (1996). Insertional inacti-
vation of Streptococcus pyogenes sod suggests that
Dominique Galli (Indiana University, Indianapolis,
prtF is regulated in response to a superoxide signal.
IN) and Dr Mary Tierney (University of Vermont,
J Bacteriol 178: 4688-4695.
Burlington, VT). We would also like to thank Dr 10. Hudson MC, Curtiss R III (1990). Regulation of
Tierney for a critical review of this manuscript. expression of Streptococcus mutans genes important
This research is support by Public Health Service to virulence. Infect Immun 58: 464-470.
Grant R37-DEllOOO from the National Institutes of 11. Jenkinson HF (1995). Genetic analysis of adherence
Health. by oral streptococci. J Ind Microbiol 15: 186-192
151
12. Jenkinson HF, Lamont RJ (1997). Streptococcal requirements of Streptococcus mutans and other oral
adhesion and colonization. Crit Rev Oral BioI Med streptococci. Infect Immun 11: 656-664.
8: 175-200. 20. Terleckyj B, Willett NP, Shockman GD (1975).
13. Kirby KS (1968). Isolation of nucleic acids with Growth of several cariogenic strains of oral strepto-
phenolic solvents. Meth Enzymol 12B: 87-99. cocci in a chemically defined medium. Infect Immun
14. Kiska DL, Macrina FL (1994). Genetic regulation of 11: 649-655.
fructosyltransferase in Streptococcus mutans. Infect 21. Weinberger I, Rotenberg Z, Zacharovitch D, Fuchs J,
Immun 62: 1241-1251. Davidson E, Agmon J (1990). Native valve infective
15. Kolenbrander PE, London J (1993). Adhere today, endocarditis in the 1970's versus the 1980's: under-
here tomorrow: oral bacterial adherence. J Bacteriol lying cardiac lesions and infecting organisms. Clin
175: 3247-3252. Cardiol 13: 94-98.
16. Mekalanos JJ (1992). Environmental signals control- 22. Wexler DL, Hudson MC, Burne RA (1993).
ling expression off virulence gene determinants in Streptococcus mutans fructosyltransferase lftf) and
bacteria. J Bacteriol 174: 1-7. glucosyltransferase (gtjBC) operon fusion strains in
17. Putzer H, Gendron N, Grunberg-Manago M (1992). continuous culture. Infect Immun 61: 1259-1267.
Co-ordinate expression of the two threonyl-tRNA
synthetase genes in Bacillus subtilis: control by
transcriptional antitermination involving a conserved Address for correspondence: Dr Paula Fives-Taylor,
regulatory sequence. EMBO J 11: 3117-3127. University of Vermont, Department of Microbiology &
18. Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Molecular Genetics, Stafford Hall, Burlington, VT 05405-
cloning: a laboratory manual 7.0-7.87. Cold Spring 0084, USA
Harbor: Cold Spring Harbor Laboratory Press. Phone: (802) 656-1121; Fax: (802) 656-8749
19. Terleckyj B, Shockman GD (1975). Amino acid E-mail: pfivesta@zoo.uvm.edu
Methods in Cell Science 20: 153-163 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. A new integrational vector, pFP1, con- tested on agar containing different sugars, some 19
taining a promoterless lacZ gene has been con- distinct clones with sucrose- and/or glucose-respon-
structed for use with Streptococcus mutans. The sive promoters were isolated. Sequence analysis
vector can be grown in Escherichia coli, but cannot indicated that the cloned DNA encoded all or part
replicate in S. mutans. pFPl can be transformed into of 29 putative proteins with 52% to 100% similarity
S. mutans with selection for kanamycin resistance. to known proteins. When assayed for ~-galactosidase
It integrates into the chromosome when homologous activity in BTR medium containing 2% sucrose, glu-
DNA is present in the vector. pFPl provides a way cose or maltose, several genes showed some evidence
for cloning and identifying new genes of S. mutans. of sugar regulation, including gtfBK, ftf, scrA, and
When a number of S. mutans transformants were most dramatically for sucrose regulation, fruA.
EcoRI,1
EcoRI Sacl,?
Sstl Ndel,13
Kpnl Sall,19
Smal Pstl,25
BamHI Notl,31
Xbal Spel,39
Sail BamHI,46
Pstl
Sphl
Pstl
lacZ
ori pPP207
7500 bps
II Ball
I
V
Pstl,1
Notl,6
Spel,14
BamHI,'21
MOB . . . ~ LACZ
pFP1
6900 bps
lacZ
Figure 1. Plasmid pFPl was constructed in two steps. The integrational vector pSF151 (kindly provided by Dr L. Tao,
Oklahoma City, Oka.) was digested with PstI and PvuII, and ligated to the promoterless lacZ fragment (PstI-BalI) from
pPP207 [49]. The smaller arrows flanking the multi cloning site in pFPl indicate the direction of the two oligonucleotides
(MOB and LACZ) used both for PCR and sequencing. Abbreviations: Km, kanamycin resistance; Ori, E. coli origin of
replication.
are designed on the sequence published by Sato et al. analysis, cells of GS-5 or of its L 7 derivative are
[38], and thefifprimers are designed on the sequence grown in BTR containing 2% glucose. The next day
published by Shiroza et al. [40]. The SpeI restriction the cells are washed once with 1 volume of BTR
site is added to the 5' of the sequence of the primers containing no sugars and then diluted 1110 in the
SCRA1 and FTF1, while the BamHI restriction site same medium. The cells are grown in BTR con-
is added to the 5' of SCRA2 and FTF2 primers. These taining 2% sucrose, 2% glucose, or 2% maltose. One
restriction sites are used to clone the serA and the fif ml of the culture is harvested at an OD at 675 nm of
genes upstream of the laeZ promoterless fragment -0.8. (or at various other OD when specified) and
in pFPl. used for the ONPG analysis. Assays for ~-galactosi
For o-nitrophenyl-~-D-galactopyranoside (ONPG) dase activities are performed on toluene-permeabi-
156
lized cells, and ONPG is used as the substrate as prepared from E. coli and also polymerase chain
described by Nicholson and Setlow [28]. One ml of reaction (PCR) products obtained from the plasmids.
the culture is transferred to a 1.5-ml Eppendorf tube Appropriate oligonucleotides were synthesized and
and centrifuged for 5 min at 4,000 rpm. To the pellet used to sequence regions of interest (based on
are added sequentially 1 ml of Z buffer (60 mM suggested open reading frames [ORFs]) in the
Na 2HP0 4 , 40 mM NaH 2P0 4 , 1 mM MgS0 4 , 10 mM opposite direction. Potential ORFs were analyzed
KC1, 50 mM ~-mercaptoethanol) and one drop using the BLAST program for proteins. The results
(about 10 Ill) of toluene. The tubes are then vortexed of this analysis are shown in Table 1. All plasmids
for 15 s and are left 10 min at room temperature. The yielded ORFs showing similarities to proteins or
tubes are then opened and transferred to a 28 DC ORFs in GenBank. In most plasmids analyzed, the
waterbath. After 10 min 200 III of the ONPG solution cloned inserts included portions of single genes. In
(4 mg/ml ONPG in Z buffer) is added to the tubes. four instances, the amino terminus of one putative
The time is recorded at this point. When the solution gene product or an entire gene and the carboxy
has become clearly yellow, the reaction is stopped by terminus of another (dagK and sgp, tuf and tpi, acmA
adding 300 III of 1 M Na 2C0 3 , and the time is and dac, and glnB and serC) were present in the
recorded again. The cells are spun down at 13,000 cloned DNA. In one instance the insert contained the
rpm for 4 min, and the supernatant is transferred to amino domains of divergently transcribed genes
a cuvette and the OD 42o measured with a spec- (ORFI and pepX). In each case, ~-galactosidase
trophotometer. The ~-galactosidase activity is expression may indicate expression of the gene closer
calculated according to the formula: Miller units = to, and in the same direction as, lacZ; there are no
A420 x 1000/reaction time (min) x OD 67s ' data to indicate if the more distal gene is coregu-
lated with the more proximal gene. Two clones were
recovered coding for different portions of the same
4. Results gene, mfd.
Integration of the plasmids into the S. mutans
S. mutans GS-5 was transformed with 94 distinct chromosome is presumed to be by a Campbell-like
pooled-plasmid preparations (12 plasmids per pool), mechanism. This presumption was confirmed in the
with selection for Kmr on TH agar containing 2% case of transformants obtained with the plasmids
sucrose. Transformants were screened for ~-galac pFP11 and pFPI9, which contained, respectively, a
tosidase activity. Of the 94 plasmid pools tested, 64 portion of gtjBIC and a portion of fruA (see Table
gave some transformants with detectable ~-galac 1). gtjBIC analysis by Southern blots of appropriately
tosidase activity. All these transformants were then restricted DNA indicated that the plasmid pFP11 had
patched onto three TH-X-Gal agars containing, indeed integrated into the region of homology on the
respectively, 2% sucrose, 2% glucose, and no added chromosome by a single cross-over (Campbell-like
sugar. From 23 pools, transformants were obtained recombination) (data not shown). In the case offruA,
that exhibited a potentially sugar-regulated pheno- two primers were designed based on the sequence
type, displaying substantially higher ~-galactosidase of Burne et al. [7]: FRUA1, 5'-CACTGGAATAAC-
activity on TH agar with no added sugar than on TH CAATGGT; and FRUA2, 5'-GACTTGTCCTAA-
agar with 2% sucrose or 2% glucose. Individual CAAGACT, both external to the region included in
plasmid preparations of each of the 12 members of the plasmid. Analysis by PCR using two sets of
the 23 pools were then used to transform S. mutans, primers, FRUAI-LACZ and FRUA2-MOB, con-
and transformants were again selected on TH -Km firmed the integration of the plasmid into the region
plates containing X-/Gal and 2% sucrose. Seventy- of homology on the chromosome.
seven of the 276 plasmids were found to give trans- In 13 of the 26 clones the insert was entirely
formants that exhibited a possible sugar-regulated internal to the putative gene. In these cases the gene
phenotype. Thus, several plasmid pools contained would be insertionally inactivated by Campbell-like
more than one plasmid that led to a possible sucrose- integration. As a result, it can be inferred that the
regulated phenotype. In a first analysis, 26 of 77 were following genes are not essential: adhE, bglH, fruA,
chosen for sequencing. The inserts analyzed ranged gcp, gtjBIC, gtfD, leuD, ilvG, mfd, sapR, uvrA, and
from 112 to 1600 base pairs. yieG; it is already known thatfruA, gtjBIC, and gtfD
To begin characterization of the inserts fused are not essential genes for S. mutans.
to the lacZ reporter gene, an oligonucleotide Data obtained from plates containing TH and 2%
was used to obtain single-stranded sequence, using sucrose or 2% glucose were difficult to interpret,
a primer that hybridized to lacZ, 5'- presumably due to the presence of 0.2% glucose
GTTGGGTAACGCCAGGG. In addition, single- added to the TH medium. Consequently, we assayed
stranded sequence was obtained for the other end of the transformants on BTR X-gal plates containing 2%
the insert, using a primer that hybridized to mob, glucose or 2% sucrose [39]. The results of this
5'-CACGACCAGTCTTTCTGCA (Figure 1, arrows). screening were easier to interpret. Specifically, one
Sequences were performed using the entire plasmids purB transformant gave white colonies on glucose-
157
pFPl-l U75476 purB; adenylosuccinate lyase (Bacillus sub til is) [10, 11, 25] 75/86 53 b
pFPI-2 U75471 livG; liv-J from Eo coli [2, 43] 54/68 90 c
(high similarity also with the livG gene product from Salmonella
typhimurium, with the IktB gene product of Pasteurella
with the gltL gene product of Eo coli, and with the rtx gene
product of Actinobacillus pleuropneumoniae)
pFPI-3 U75473 mfd; transcription repair coupling factor (Bo sub til is) [30] 64/83 98 c
pFPI-4 U75474 mfd; transcription repair coupling factor (Bo subtilis) [30] 73/87 72 c
pFPI-5 U75472 bglH; ~-glucosidase (Bo subtilis) (Do Le Coq et aI., direct 58176 ++c
submission to the EMBL/GenBanklDDBJ data banks, 1994)
pFPI-6 d U78605 d ORFl; hypot. ORFI in pepX 5' region (Lactococcus lactis) 74/86 78 b
[24, 25]
pFPI-6 e U78605 pepX; X-prolyl-dipeptidyl-peptidase (L. lactis, L. lac tis subspo 55170 127 b
cremoris) [26, 27]
pFPI-7 e U78599 acmA, N-acetylmuramidase (Lactococcus lactis) [5] 34/60 164b
(high similarity also with the autolysin of Enterococcus
faecalis and Enterococcus hirae)
pFPI-7 d U78599 d dac; D-D-carboxypeptidase (Eo faecalis) (So Evers & 38/65 136 f
Po Courvalin, unpublished)
pFPI-8 U78602 yca]; hypot. Protein HI1590 (Haemophilus inJluenzae) [12] 41/64 211b
(Wo Oswald, Thesis, Justus Liebig Universitatet, Frankfurt,
Germany 1994)
pFPI-9 U75478 adhE; alcohol dehydrogenase (Eo coli) [16] 44173 52e
(high similarity also with adhE gene product of
Entoemeba histolytica, Clostridium acetobutylicum, Citrobacter
freundii, Saccharomyces cerevisiae, Zymomonas mobilis,
Schizosaccharomyces pombe, and Bacillus methanolicus)
pFPI-lO U75477 oppF; oligopeptide transport ATP-binding protein (Bo subtilis) 53171 109b
[33, 37]
pFPl-11 M17361 gtfBIC; glucosyltransferase Band C (So mutans) [41, 47] 100/100 46 c
pFPI-12 U75475 lauD; hypothetical ABA transporter in leuD 3' region 65/82 58 c
(L. lactis) [15]
pFPI-13 U78604 Membrane protein; (Streptococcus pneumoniae) (Jo Krauss & 34/62 129c
R. Hakenbeck, unpublished sequence in GenBank)
pFPI-14 e dagK; diacylglycerol kinase (So mutans) [48] 99/100 137g
pFPI-14 L03428
e sgp; GTP-binding protein ERA homolog (So mutans) [48} 100/100 128b
pFPI-15 U78601 gcp; sialoglycoprotease (Po haemolitica) [1] (Mo Do Potter & 47173 157c
R. Yo Co Lo, unpublished)
pFPI-16 M29296 gtjD; glucosyltransferase D (So mutans) [19] 100/100 146c
pFPI-17 3 U78606 glnB; nitrogen regulatory protein (Azospirillum brasilense) [9] 63177 57 f
pFPI-17 e U78606 d serC; phosphoserine aminotransferase (Yersinia enterocolitica) 57175 84 b
[29]
pFPI-18 U75479 uvrA; excinuclease ABC subunit A (Mycoplasma genitalium) [13] 69/81 134c
(high similarity also with the uvrA gene product of Mycoplasma
capricolum, Micrococcus luteus, Neisseria gonorrhoeae, Eo Coli,
So typhimurium, H. inJluenzae, and Pseudomonas aeruginosa)
pFPI-19 L03358 fruA; exo-~-D-fructosidase (So mutans) [7] 99/99 79 c
158
Table 1. (Continued)
and sucrose-containing BTR. Transformants from grew each transformant of S. mutans GS-5 L7 in BTR
pFP-2 to pFP-20 had blue colonies on sucrose, but medium to an OD 675 of 0.8. BTR medium con-
white colonies on glucose-containing X-gal plates. taining either 2% or 0.2% sucrose, or 2.0 or 0.2%
Lastly, six transformants had blue colonies on glucose, and the specific activity of ~-galactosidase
glucose- and on sucrose-containing X-gal plates. was determined and is expressed as Miller units.
Among the colonies that were blue on sucrose- but Table 2 provides the results obtained using the L 7
white on glucose-containing X-gal plates were derivative of S. mutans GS-5. However, we found
transformants with a lacZ insert in the gtfB/C and in no difference qualitatively between results obtained
the fruA genes (pFP-ll and pFP-19 in Table 1). with the GS-5 parent, or with the L7 derivative, and
As an additional control, we cloned the ft! gene media containing 2.0% or 0.2% sugar.
and the serA gene in the pFPl vector and transformed The purB strain which had white colonies on X-
S. mutans, selecting for Campbell-like insertions. gal plates supplemented with sucrose or with glucose
Both transformants gave blue colonies in sucrose- had low levels of ~-galactosidase (Table 2). The six
and white colonies in glucose-containing media. A transformants (numbers 21-26) that gave blue
transformant containing the region downstream of the colonies on both glucose and on sucrose had specific
3' terminus of the serA gene gave white colonies in activities of ~-galactosidase that ranged from less
both sucrose- and glucose-containing media (data not than 16 to 524 Miller units in both sugars (Table 2).
shown). However, the most interesting results were obtained
In order to analyze our results in greater detail, we from transformants that had blue colonies in sucrose
159
Table 2. ~-galactosidase activity of S. mutans transfor- in sucrose than in glucose, and glucose does not
mants increase the specific activity of the enzyme. In each
case shown in Figure 2, the specific activity of lacZ
GS-517::pFPl ~-galactosidase specific activity is affected by the time of the analysis.
derivatives (in Miller units)
A) gtfB/C::lacZ B) ftf::lacZ
40-r---------------------------, 40
0 0
30
.!1 30 .!1
'c:J 'c:J
~ a.... ~ t- a
~ ~
~ C)
g
.S: 20 g .S: 20
.?;- ci .?;- ci
·s ·s -1
~
-1
0 131\1 0
131\1
+--+--;
U U
1;= 10 1;= 10
'0 '0
Ql
Ql
a. a.
CI) CI)
0 -2 0 ·2
0 2 4 6 8 0 4 6 8
C) scrA::lacZ D) fruA::lacZ
40 160
140
0 0
30 VI 120
.!1 ~
'c:J :J
~ '0 ~
100
a....
~ cii ~ C)
20 g .S: 80 g
.S:
.?;- ci .?;-
·s ci
·s -1
0 60
-1
0
131\1 131\1
U U
1;= 10 1;= 40
'0 '0
Ql
Ql
a. a.
CI)
... --+-- ..... CI) 20
0
...--- ·2
6 8
Figure 2. Specific activity of /3-galactosidase at various times after growth in sucrose ( ... ), glucose (T) or maltose (.).
A. gtjBIC::lacZ. B. JtJ::lacZ. C. scrA::lacA. D. JruA::lacZ (note that the scale for JruA::lacZ differs from that for the
others). (0) Represents growth in O.D. units.
purB, which is one of the 12 genes belonging to the identified in E. coli as an Enzyme III-independent
pur operon encoding purine biosynthesis enzymes Enzyme II Gle ; as in E. coli [summarized in ref. 45],
[25]. The S. mutans insert, sharing high similarity the ptsG in S. mutans maps to a chromosomal locus
with the oppF gene product of B. subtilis, also gave far from the pts operon (M.G. Cappiello & F. Peruzzi,
a high degree of identity to ATP-binding proteins of unpublished observations).
other organisms, such as L. lactis, S. pneumoniae, S. For many of the clones identified, it is difficult to
typhimurium, H. inJluenzae, E. coli and Bordetella find a relation between their function and sugar
pertussis. metabolism. Among those genes, five belong to the
The S. mutans clones were isolated as showing family of the ABC (ATP-binding cassette) trans-
sugar-responsive gene expression when grown on porters: leuD, livG, oppF, sapR, and uvrA. The
agar. some of the genes identified seem to be clearly substrates for the ABC transporters include oligo-
involved in sugar metabolism (adhE, bglH, fruA, peptides, proteins, sugars, amino acids, and polysac-
gifBIC, gtfD, and tpi). A putative glucose permease charides. With the exception of the uvrA gene product
encoded by the ptsG gene was found whose expres- that is cytoplasmic, the ABC proteins are usually
sion appeared relatively insensitive to sugars. This transmembrane proteins, and they are involved in
protein is part of the PTS system, and it has been many biological processes in bacteria [18]. Many of
161
Cotton MD, Utterback TR, Hanna MC, Nguyen DT, in Escherichia coli: nucleotide sequence determina-
Saudek DM, Brandon RC, Fine LD, Fritchman JL, tion and properties of the plasmid-encoded enzyme. J
Fuhrmann JL, Geoghagen NSM, Gnehm CL, BioI Chern 258: 10586-10593.
McDonald LA, Small KV, Fraser CM, Smith HO, 26. Mayo B, Kok J, Venema K, Bockelmann W, Teuber
Venter JC (1995). Whole-genome random sequencing M, Reinke H, Venema G (1991). Molecular cloning
and assembly of Haemophilus inJluenzae Rd. Science and sequence analysis of the X-prolyldipeptidy-
269: 496-512. laminopeptidase gene from Lactococcus lactis subsp.
13. Fraser CM, Gocayne JD, White 0, Adams MD, cremoris. Appl Environ Microbiol 57: 38-44.
Clayton RA, Fleischmann RD, Bult CJ, Kerlavage 27. Nardi M, Chopin MC, Chopin A, Cals MM, Gripon
AR, Sutton G, Kelley JM, Fritchman JL, Weidman JF, JC (1991). Cloning and DNA sequence analysis of an
Small KV, Sandusky M, Fuhrmann J, Nguyen D, X-prolyl dipeptidyl aminopeptidase gene from
Utterback TR, Saudek DM, Phillips CA, Merrick JM, Lactococcus lactis subp. lactis NCD0763. Appl
Tomb J-F, Dougherty BA, Bott KF, Hu P-C, Lucier Environ Microbiol 57: 45-50.
TS, Peterson SN, Smith HO, Hutchison CA III, Venter 28. Nicholson WL, Setlow P (1990). Sporulation, germi-
JC (1994). The minimal gene complement of nation and outgrowth. In: Harwood CR, Cutting SM
Mycoplasma genitalium. Science 270: 397-403. (eds), Molecular biological methods for Bacillus, pp
14. Fuerst P, Moesch HU, Solioz M (1989). A protein of 391-429. West Sussex, England: John Wiley and
unusual composition from Enterococcus faecium. Sons, Ltd.
Nucleic Acids Res 17: 6724. 29. O'Gaora P, Maskell D, Coleman D, Cafferkey M,
15. Godon J-J, Chopin M-C, Ehrlich SD (1992). Dougan D (1989). Cloning and characterization of the
Branched-chain amino acid biosynthesis genes in sere and aroA genes of Yersinia enterocolitica, and
Lactococcus subsp. lactis. J Bacteriol 174: construction of an aroA mutant. Gene 84: 23-30.
6580-6589. 30. Ogasawara N, Nakai S, Yoshikawa H (1994).
16. Goodlove PE, Cunningham PR, Parker J, Clark DP Systematic sequencing of the 180 kilobase region of
(1989). Cloning and sequence analysis of the fer- the Bacillus subtilis chromosome containing the
mentative alcohol-dehydrogenase-encoding gene of replication origin. DNA Res 1: 1-14.
Escherichia coli. Gene 85: 209-214. 31. Pearce BJ, Yin YB, Masure HR (1993). Genetic iden-
17. Gutierrez JA, Crowley PJ, Brown DP, Hillman JD, tification of exported proteins in Streptococcus pneu-
Youngman P, Bleiweis AS (1996). Insertional muta- moniae. Mol Microbiol 9: 1037-1050.
genesis and recovery of interrupted genes of 32. Perego M (1993). Integrational vectors for genetic
Streptococcus mutans by using transposon Tn917: manipulation in Bacillus subtilis. In: Sonenshein AL,
Preliminary characterization of mutants displaying Losick R, Hoch JA (eds), Bacillus subtilis and other
acid sensitivity and nutritional requirements. J gram-positive bacteria, pp 615-624. Washington, DC:
Bacteriol178: 4166-4175. American Society for Microbiology.
18. Higgins CF, Hiles ID, Salmond GPC, Gill DR, 33. Perego M, Higgins CF, Pearce SR, Gallagher MP,
Downie JA, Evans 11, Holland IB, Gray L, Buckel SD, Hoch JA (1991). The oligopeptide transport system of
Bell AW, Hermodson MA (1986). A family of related Bacillus subtilis plays a role in the initiation of
ATP-binding subunits coupled to many distinct bio- sporulation. Mol Microbiol 5: 173-185.
logical processes in bacteria. Nature 323: 448-450. 34. Perry D. Kuramitsu HK (1981). Genetic transforma-
19. Honda 0, Kato C, Kuramitsu HK (1990). Nucleotide tion of Streptococcus mutans. Infect Immun 32:
sequence of the Streptococcus mutans gtfD gene 1295-1297.
encoding the glucosyltransferase-S enzyme. J Gen 35. Qi HK, Sankaran K, Gan K, Wu HC (1995). Structure-
Microbiol 136: 2099-2150. function relationship of bacterial pro lipoprotein
20. Hudson MC, Curtiss R III (1990). Regulation of diacylglyceryl transferase: Functionally significant
expression of Streptococcus mutans genes important conserved regions. J Bacteriol 177: 6820-6824.
for virulence. Infect Immun 58: 464-470. 36. Reider JL, Macrina F (1976). Plasmid DNA isolation
21. Kiska DL, Macrina F (1994). Genetic regulation of in Streptococcus mutans: Glycine-enhanced cell lysis.
fructosy!transferase in Streptococcus mutans. Infect In: Stiles HM, Loesche WJ, O'Brien TC (eds),
Immun 62: 1241-1251. Proceedings: Microbial aspects of dental caries (a
22. Lane MA, Bayles KW, Yasbin RE (1991). special supplement to Microbial Abstracts, Vol 3), pp
Identification and initial characterization of glucose- 725-736. Washington, DC: Information Retrieval.
repressible promoters of Streptococcus mutans. Gene 37. Rudner DZ, Ledeaux JR, Ireton K, Grossman AD
100: 225-229. (1991). The spoOK locus of Bacillus subtilis is homo-
23. Loeshe WJ (1986). Role of Streptococcus mutans in logous to the oligopeptide permease locus and is
human dental decay. Microbiol Rev 50: 353-380. required for sporulation and competence. J Bacteriol
24. Ludwig W, Weizenegger M, Beltz D, Leidel E, Lenz 173: 1388-1398.
T, Ludvigsen A, Moellenhoff D, Wen zig P, Schleifer 38. Sato Y, Poy F, Jacobson GR, Kuramitsu H (1989).
KH (1990). Complete nucleotide sequences of seven Characterization and sequence analysis of the scrA
eubacterial genes coding for the elongation factor Tu: gene encoded enzyme IIScr of the Streptococcus
Functional, structural and phylogenetic evaluations. mutans phosphoenolpyruvate-dependent sucrose
Arch Microbiol 153: 241-247. phosphotransferase system. J. Bacteriol171: 263-271.
25. Makaroff CA, Zalkin H, Switzer RL, Vollmer SJ 39. Sato Y, Yamamoto Y, Suzuki R, Kizaki H, Kuramitsu
(1983). Cloning of the Bacillus subtilis glutamine HK (1991). Construction of scrA::laxZ gene fusions
phosphoribosylpyrophosphate amidotransferase gene to investigate regulation of the sucrose PTS of
163
Streptococcus mutans. FEMS Microbiol Letts 79: polypeptide chain that functions to energize the
339-346. sucrose permease. J BioI Chern 265: 18581-18589.
40. Shiroza T, Kuramitsu H (1988). Sequence analysis of 46. Tao L, Le Blanc DJ, Ferretti J (1992). Novel strepto-
the Streptococcus mutans fructo-syltransferase gene coccal-integration shuttle vectors for gene cloning and
by flanking regions. J Bacteriol 170: 810-816. inactivation. Gene 120: 105-110.
41. Shiroza T, Ueda S, Kuramitsu HK (1987). Sequence 47. Ueda S, Shiroza T, Kuramitsu HK (1988). Sequence
analysis of the gtfb gene from Streptococcus mutans. analysis of the gtfC gene from Streptococcus mutans
J Bacteriol 169: 4263-4270. GS-5. Gene 69: 101-109.
42. Silhavy TJ, Berman ML, Enquist LW (1984). 48. Yamashita Y, Takehara T, Kuramitsu HK (1993).
Experiments with gene fusions. Cold Spring Harbor, Molecular characterization of a Streptococcus mutans
NY: Cold Spring Harbor Laboratory. mutant altered in environmental stress responses. J
43. Sofia HJ, Burland V, Daniels DL, Plunkett G III, Bacteriol 175: 6220-6228.
Blattner RF (1994). Analysis of the Escherichia coli 49. Yu J, Hederstedt L, Pig got PJ (1995). The cytochrome
genome, V. DNA sequence of the region from 76.0 bc complex (menaquinone: cytochrome creductase) in
to 81.5 minutes. Nucleic Acids Res 22: 2576-2586. Bacillus subtilis has a nontraditional subunit organi-
44. Sun S, Daneo-Moore L, Marri L, Hantman M, zation. J Bacteriol 177: 6751-6760.
Shockman GD (1991). Genetic and physiological
studies of variants of Streptococcus mutans GS-5 that
produce nonmucoid colonies on sucrose-containing
media. In: Dunny GM, Cleary PP, McKay LL (eds), Address for correspondence: Dr Lolita Daneo-Moore,
Genetics and molecular biology of streptococci, lac- Department of Microbiology and Immunology, Temple
tococci, and enterococci, pp 256-260. Washington, University School of Medicine, 3400 N. Broad Street,
DC: ASM Publications. Philadelphia, PA 19140, USA
45. Sutrina SL, Reddy P, Saier MH Jr, Reizer J (1990). Phone: 215-707-3229; Fax: 215-707-7788
The glucose permease of Bacillus subtilis is a single E-mail: mooreld@astro.ocis.temple.edu
Methods in Cell Science 20: 165-179 (1998)
© 1998 Kluwer Academic Publishers.
Abstract. Several techniques are available for the as well as a method for the construction of genetic
exploration of changes in gene expression and fusions by splice-overlap extension PCR (SOE-
protein repertoire in bacteria due to changes in envi- PCR). As genome nucleotide sequences become
ronment. We have been interested in the changes available for the streptococci, PCR will likely be
that S. mutans, a cariogenic oral micro organism, used more in combination with protein databases,
makes in response to growth at low pH values. Two or proteomes, to explore the expression of genes
approaches that we have utilized to evaluate alter- under specific environmental conditions. Therefore,
ations in gene expression are the polymerase chain we include a description of two-dimensional gels
reaction and two-dimensional gel electrophoresis. prepared using protein extracts of cells grown in
The use of degenerate primers in PCR amplification steady-state under different environmental condi-
of genes by homology-based approaches is included tions.
Key words: Dental caries, Oral streptococci, Polymerase chain reaction, Proteomics, S. mutans
Abbreviations: BHI = Brain heart infusion medium; bp = base pairs; kbp = kilobase pairs; PMSF = phenyl
methyl sulfonyl fluoride; rpm = revolutions per minute; ref = relative centrifugal force; TE = Tris EDTA
buffer; TAE = Tris acetate buffer; X-Gal = 5-bromo-4-chloro-3-indolyl- ~-D-galactopyranoside; A, C, T, G =
common deoxyribonucleotides; R = A or G deoxyribonucleotides; Y = C or T deoxyribonucleotides; D = A
or G or T deoxyribonucleotides; N = A or C or T or G deoxyribonucleotides
we refer the reader to well-established treatments of 1 mol/l Tris, pH, 8.3; 100 mmo1l1 MgCI 2; 5%
methods for propagating DNA fragments by cloning NP-40, no. 1-302127 and filter-sterilized.
methodologies [1, 36]. Included here are specific - mineral oil, no. 2705-01Y
descriptions of experimental conditions that we have - E. coli strain DHIOB, no. 18290-015Y
found useful in the amplification and recovery of E. Solutions
streptococcal DNA templates [30, 31,40]. - 20 mmolll Trizma base (Tris), no. T-1530 27 ,
A. Computers and software adjusted to a pH value of 8.0 with HCI.
1. Desktop computer (e.g., Power Macintosh) - 10 mmol/l Tris adjusted to a value of pH 8.0
with the ability to access either a DEC VAX with HCI.
running GCG software or the National Center - Lysis buffer: 10 mmol/l Tris pH 8.0; 0.4 M
for Biotechnology Information database con- sucrose, no. S-501627 ; 50 mg/mllysozyme, no.
taining genetic sequences L-6876.27
2. MacVector software for DNA and protein - TE buffer (10 mmolll Trizma base, pH 8.0; 1
sequence analysis mmol/l EDTA, no. E-5134 27 )
3. Genetics Computer Group software for DNA - TAE buffer (0.04 mol/l Trizma base, adjusted
and protein sequence analysis to pH 8.0 with glacial acetic acid; 1 mmol/l
4. NIH Image v. 1.6 for the Macintosh (avail- EDTA)
able as a public domain resource on the - Glacial acetic acid, no. JT-9508-33. 30
Internet at http://rsb.info.nih.gov/nih-image/) - Phenol, buffer saturated, no. 9712.2
5. Apple Color One Scanner - CHCI 3: isoamyl alcohol (mixed in a 24: 1
B. Equipment ratio), nos. 9180-22 and 9038-01, respec-
1. Gene pulser electroporation apparatus. 5 tively.16
2. Homogenizer, model mini-Bead Beater. 6 - 95% EtOH, no. 9401-25. 16
3. Micro-centrifuge, refrigerated, Eppendorf.30 - 5 mol/l NaCI, no. 3624-15. 16
4. Investigator 2-D gel electrophoresis system. 12 - 10 mg/ml RNAse A in TE buffer, no. R-
5. Adjustable pipettes: nos. P-2; P-20; P-200; P- 4875.27
1000. 26 - 10% (w/v) sodium dodecyl sulfate, no. 15525-
6. Thermal cycler. 23 017Y
7. Thermal cycler, model OmniGene. 14 - PMSF, 10 mg/ml stock solution, in 100%
8. Centrifuge, refrigerated-preparative, no. ethanol, no. P-7626. 27
RC5C. 28 - Bio-Rad dye-binding reagent, 500-0006. 5
9. Rotor locking screw for RC5C centrifuge, no. - Quanti-Gold kit, no. QG-250."
13017.28 - tracking dye: prepared in distilled, de-ionized
10. SS-34 rotor for RC5C centrifuge. 28 water as 0.25% (w/v) bromphenol blue, no.
11. GSA rotor for RC5C centrifuge. 28 161-04045; 0.25% (w/v) xylene cyanol FF, no
12. Shaking water bath.4 161-0423 5; 40% sucrose, no. S-0389. 27
13. Gel apparatus, model Horizon 58Y - ethidium bromide stock solution (10 mg/ml in
14. Pipette-aid, Drummond, VWR. no. 53498- water), no. E-8751.27
001. 30 - ampicillin, 100 mg/ml stock solution in water,
15. UV transilluminator, model no. 3-3000. 31 no. A-9518. 27
C. Culture medium - chloramphenicol, 20 mg/ml stock solution in
- Brain heart infusion medium, no. 0037- 95% ethanol, no. C-0378. 27
07-0.10 - X-Gal, no B-8465. 19 40 mg/ml stock solution
- Todd Hewitt medium, no. 0492-17-6.10 in dimethyl formamide, no. D-8654.27
- Luria-Bertani (LB) agar, as follows: in 1 lof - molecular weight markers, 123 bp ladder, no.
de-ionized water, 10 g tryptone, no. 0123-07- 15613-011 18 and 1 kbp ladder, no. 15615-
5 10 ; 5 g yeast extract, no. 0127-07-1 10 ; 10 g 016. 18
N aCl, no. 3624-15 16 ; 15 g bacteriological F. DNA recovery reagents
grade agar, no. 00-5778KA3. - agarose, 15510-027. 18
D. Reagents for polymerase chain reaction - QIAEX II gel extraction kit, no. 20051. 25
- Deoxyribonucleotides, no. 1-277-049. 7 - glass spin columns, assembled as described
lOx BRL Taq DNA Polymerase Buffer, [15].
included with no. 18038-042. 18 G. DNA cloning reagents
- 50 mmol/l MgCI 2, included with no. 18038- - pGEM-T kit, no. A-3600. 24
042.18 - TA Cloning kit, no. K-2000-01Y
- custom 0ligodeoxyribonucleotides. 18 - CAT GenBlock, no. 27-4895. 31
- Taq DNA polymerase, no. 18038-042.18 H. Glassware and plastics
- lOx modified PCR buffer: made in distilled, - 250 ml centrifuge bottles, no. 3120-0250. 20
de-ionized water with 200 mmol/l (NH4hS04; - Oakridge tubes, no. 3119-0050. 20
167
- 50 ml conical Coming tube, no. 25330-50. 9 sion does not become viscous, place the flask
- 15 ml conical Coming tube, no. 25319-15. 9 in a 60°C water bath for 5-30 min.
- 10 ml disposable plastic pipette, Falcon, no. 5. Transfer the solution to a 50 ml conical
7530. 30 Coming tube and add buffered phenoL Gently
- P-20 Aerosol resistant pipette tips (ART), no. invert the tube several times to extract the
MBP-2149P. 17 proteins into the organic (phenol) layer.
- P-200 Aerosol resistant pipette tips (ART), no. Separate the organic and aqueous phases by
MBP-2069. 17 centrifugation in the Sorvall centrifuge at 2000
- 0.5 ml micro-centrifuge tubes, no. 8508- rpm (478 rcf) for 15 min using the SS-34 rotor,
FT. 17 without the lid. Note: when using the SS-34
- 0.2 ml thin-wall micro-centrifuge tubes, no. rotor without the lid, it is imperative to use the
435.17 rotor locking screw to fasten the rotor to the'
- caps for 0.2 ml thin-wall micro-centrifuge drive spindle. Using a 10 ml plastic pipette and
tubes, no. 440Y the pipette-aid, transfer the aqueous phase
- glass beads, no. G-9018. 27 to fresh tube. Add 20 ml TE buffer to the
- cryovials, with O-rings and screw-caps, no. first phenol layer and re-mix the solution.
4204-S.17 This 'back' - extraction generally results in
improved recovery of DNA left at the inter-
face after the first extraction. Repeat the cen-
3. Procedures trifugation and combine the first and second
aqueous layers.
The procedure for amplifying any fragment of DNA 6. Using the CHC1 3 : isoamyl alcohol solution,
requires a number of preparative steps, including the extract the aqueous phase recovered in step 5
following: preparation of genomic DNA, to act as a (above).
template supporting the replication of a target gene 7. Add 2 1/z volumes of cold 95% EtOH plus
sequence; selection and synthesis of oligodeoxyri- 1/6 volume 5 M NaCl to the aqueous phase.
bonucleotide primers; amplification of the target Mix well and allow the DNA to precipitate
sequences via PCR; recovery of the fragments on ice. Collect the precipitate by centrifuga-
by genetic cloning; and verification of the cloned tion at 19,000 rpm (43,152 ref) for 30 min
fragment by nucleotide sequence determination. Each at 4°C. Carefully invert the centrifuge tubes
of these discrete steps, as performed in our labora- to allow the liquid to pour off. Blot the
tory, will be described below. inverted tubes on paper towel to remove as
A. Preparation of template DNAs: streptococcal much buffer as possible. Resuspend the
DNA isolation (based on [6]) nucleic acid-containing pellet in 10 ml of TE
1. Day one - Inoculate, in the morning, a 25 ml buffer.
culture of Brain Heart Infusion medium with 8. Remove contaminating RNA by the addition
S. mutans. The strains used in these studies of 0.2 ml of the RNAse A solution. Incubate
were UA159 [25] or UR100 [30]. Late in the the suspension for 30 min at room tempera-
afternoon, transfer the 25 ml culture to a 1 litre ture.
flask containing fresh medium, pre-warmed to 9. Add 10 ml of buffered phenol and repeat the
37°C. Allow the culture to grow overnight. extraction process described in step 5. Repeat
2. Day two - Harvest the cells by centrifugation the back extraction on the aqueous phase.
in the Sorvall centrifuge using the centrifuge Repeat Step 6. To the final aqueous phase, add
bottles at 8000 rpm (10,400 rcf) for 10 min 2 1/z volumes of ice-cold 95% EtOH plus 1/6
at 4°C. Wash the collected cell pellets in 150 volume of the 5 M NaCl solution. Collect the
ml of chilled 20 mmolll Tris pH 8.0. precipitate by centrifugation at 19,000 rpm
3. Resuspend the cells in 20 ml of lysis buffer. (43,152 rcf) for 30 min at 4°C. Carefully pour
Resuspend the cells uniformly by vortexing. off the liquid phase and wash the DNA
Transfer the suspension to a sterile flask and by resuspending the pellet in 10 ml of 70%
incubate the mixture with gentle shaking at ethanoL Re-collect the pellet by centrifugation,
37°C for two hours. as before. Gently pour off the liquid and allow
4. Collect the cells by centrifugation in an SS- the pellet to air-dry at room temperature.
34 rotor at 5000 rpm (2,988 rcf) for 15 min at The pellet will become clear as it dries. The
4°C. Resuspend the pellet in 20 ml of TE recovered DNA can be spectrophotometric ally
buffer and add 4 ml of 5 molll, NaCl solution. quantitated by resuspending the pellet in 1-2
Add 2 ml 10% (w/v) sodium dodecyl sulfate ml of TE buffer and measuring the absorbance
solution. The suspensions should become of the material at 260 nm [18]. Typically, we
viscous almost immediately as the cells lyse recover approximately 20 mg/ml of DNA from
and release DNA into solution. If the suspen- this procedure.
168
B. Preparation of DNA templates from E. coli length and distance from each other can be
In addition to PCR-based amplifications of DNA reverse translated, either manually or by
sequences from genomic templates, we have also computer, to create a degenerate nucleotide
found it useful to prepare streptococcal DNA sequence. Typically, we have attempted to
templates from clones maintained in strains of maintain a 50% G:C proportion in degenerate
E. coli [31]. A large number of protocols are sequences and the 3' base must not be degen-
available in the literature for the preparation of erate. For example, the codons for glycine are
plasmid DNAs from E. coli. The protocol accom- GGN, where N represents all four possible
panying the QIA-prep mini-prep kit is reliable bases. if glycine is the last amino acid in
and cost-effective when used according to the the region of conservation, then the N would
manufacturer's instructions. We have also found be deleted from consideration and the last
it useful to use fragments of DNA isolated two bases in the oligodeoxyribo nucleotide
from agarose gels. In those circumstances, the sequence would be GG.
preparation of DNAs has been accomplished D. Synthesis of primers
by separating restriction enzyme digested DNA 1. In the past, we have synthesized primers using
fragments by agarose gel electrophoresis in TAE Model 381A and 391 instruments from
buffer. The position of specific DNA is identified Applied Biosystems [31]. However, a number
following ethidium bromide staining of the gel of commercial suppliers have become avail-
and comparison with molecular weight standards able to provide rapid turnaround times for
during illumination of the stained gel on a UV the custom synthesis of oligonucleotides
transilluminator. Note: UV light can cause skin containing common and uncommon bases.
irritation and long-term exposure may lead to eye Mixtures of oligos are readily prepared by
damage. We invariably use a UV-protective face including all four of the common bases in the
shield when working over the transilluminator. synthesis step at any point in the construction
Appropriately sized fragments are excised from of a custom DNA sequence. Thus, highly
the gel and further purified using the QIAEX II degenerate primer mixes can be obtained rep-
gel extraction kit to recover DNA from agarose resenting all possible combinations of codons
gels. for a given amino acid sequence. The arrows
C. Selection of primer pairs for degenerate PCR in Figure 1 indicate the amino acid sequences
1. The goal of primer sequence selection is to that were used as the basis for synthesis
achieve both the lowest possible degeneracy of ATPase-specific oligodeoxyribonucleotide
and the amplification of a useful fragment primers.
length. Operationally, there is no minimum E. Degenerate PCR conditions
for fragment size, though we have typically 1. The mixture first synthesized for the forward
attempted to generate PCR products between primer consisted of the following sequence: 5'-
500 and 1000 bp. Fragments of this size can GAATTCGTNTTYGCNGGNGTNGGNGAR-
be amplified with highly degenerate primer CGTNACNCGNG-3' and accounted for all
mixes and have sufficient length as to be 262,144 possible sequence variations in the
useful in subsequent insertional mutation amino acid sequence. The reverse primer con-
studies [30]. sisted of the sequence: 5'-GTCGACCATNC-
2. Homologous DNA or protein sequences are CNARDATNGCDATDATRTCYTGNA-3',
collected using the BLAST or FASTA algo- which represented 55,296 possible sequence
rithms in the GCG software package. variations in that segment of ATPase ~
3. In the case of related DNA sequences, the subunit. In later experiments, we also used
nucleotide sequences are translated into much shorter regions of the ATPase sequence.
protein sequence using either the GCG or In those primers, we included restriction
MacVector algorithms. enzyme sites in both primers at the 5' end of
4. Deduced amino acid sequences are then the sequences and the amount of material
aligned using either the multiple sequence homologous to the ATPase was reduced, per-
alignment and PileUp algorithms available in mitting lower degeneracy in the primer and a
GCG or using the ClustalW alignment algo- higher fidelity to the template. The forward
rithm in Mac Vector. primer was as follows: 5'- CTATGACCAT-
5. Deduced amino acid sequences aligned by GAATTCGGNGTNGGNGARCGNAC-
Clustal are formatted to show conserved amino NCGNG-3' (8192 possible combinations),
acid sequences. Typically, it is useful to focus where the underlined sequence represents an
on regions of at least five contiguous amino engineered EcoRI site. The reverse primer
acids, though longer regions of conservation sequence was as follows: 5'-CTGCCG-
are desirable. GTCAGTCGACARDATNGCDATDATRT-
6. Regions fitting the criteria of conserved region CYTGNA-3' (3456 possible combinations),
169
where the double-underlined sequence repre- 2. Commercial kits are available for cloning
sented an engineered SaIl restriction endonu- fragments directly from reactions (e.g., the
clease site. TA-cloning kit or the pGEM-T kit). The kits
2. Transfer into a 500 ~l micro-centrifuge tube are supplied with instructions, which in our
the following: 10 ~l of lOX modified PCR hands have proven reliable.
buffer; 16 ~l of 1.25 mmol/l dNTPs, final con- 3. PCR products can also be separated by agarose
centration of 200 ~mol/l; 2 ~l of sense-strand gel electrophoresis, typically in 1-2% agarose
primer (20 ~mol/l stock) final concentration of in TAE buffer. 10 ~l aliquots of reaction are
1 ~M; 2 ~1 of complementary strand primer mixed with 1 ~l of loading dye and transferred
(20 ~molll stock); 1-10 ~l of template DNA to a well in the agarose gel apparatus.
(range from l30 ng to l30 ~g); adjust to Molecular weight markers are either 123 bp
100 ~l with distilled water; add 100 ~l of ladder or 1 kbp ladder. Voltage is then applied
mineral oil; initiate the thermal cycling, to the gel, usually at 5-10 V/cm of gel,
pausing the instrument when 94 DC is reached depending on the time-frame of your investi-
to add 0.5 ~l of Taq polymerase; resume gation. The length of electrophoresis time is
the cycle at 94 DC for 2 min, 37 DC for 2 min, dependent on the size of the desired PCR
72 DC for 3 min; continue for 30-35 cycles. fragment. However, 1-2 hours is usually suf-
Remove a 10 ~l aliquot and separate the ficient time to resolve fragments.
products by electrophoresis in 1-2% agarose 4. Gels are removed from the apparatus and
in TAE buffer. Note: the difference between placed in a glass tray with approximately
lOx modified PCR buffer and the PCR buffer 200 ml of TAE buffer plus 25 ~l of ethidium
which comes with commercial preparations of bromide dye stock solution (sufficient to cover
Taq DNA polymerase is that the modified form the gel). Allow several minutes for the gel to
contains magnesium at a defined concentra- soak up the stain. Ethidium bromide is a
tion; whereas some commercial buffers are known carcinogen and care, including the use
magnesium-free, permitting the investigator to of latex gloves and a laboratory coat, should
vary the magnesium concentration. The effect be exercised with its use.
of varying the magnesium concentration is to 5. Following the staining interval, the dye
increase the likelihood that base substitutions solution is poured off, and the gel is illumi-
will occur. This is often template and temper- nated on a long-wavelength UV transillumi-
ature dependent. For that reason, we generally nator to reveal the position of the fragments
hold the concentration to 1 mmolli. with respect to the molecular weight markers.
3. We have found that these conditions yield 6. The DNA band with the desired molecular
products visible in stained agarose gels. weight is then carefully excised with a scalpel
However, we have found more consistently, and purified using either a glass wool column
that re-amplification of the material results in [15] or a commercially available purification
significantly greater quantities of product, sim- system (e.g., the QIAEX II gene extraction
plifying recovery of the DNA for cloning. kit).
4. Re-amplify the initial PCR products by placing 7. Purified DNA can then be used directly with
into a 500 ~l micro-centrifuge tube the fol- PCR cloning vectors or can be treated with
lowing: 10 ~l of lOx modified PCR buffer; restriction endonucleases to create 'sticky-
16 ~l of 1.25 mmolll dNTPs, for a final con- ended' DNA for cloning [31]. We have
centration of 200 ~mol/l; 2 ~1 of sense-strand used both approaches and find that the PCR
primer (20 ~molll), final concentration of cloning vectors are generally less troublesome
1 ~molll; 2 ~l of complementary strand in that clones have been readily isolated.
primer (20 ~molll); 1-10 ~l of the initial PCR Digestion of amplified DNAs with restriction
reaction described above; adjust to 100 ~l with enzymes has worked for us, but required at
distilled water; add 100 ~l of mineral oil; start least one and often two digestions of recov-
the thermal cycler, pausing the instrument ered DNA with phenolic extractions between
when 94 DC is reached, to add polymerase, each. The loss of DNA during the extractions,
then resume the cycling program at: 94 DC for coupled with the likelihood of incomplete
2 min, 50 DC for 2 min, 72 DC for 3 min. digestion of recovered DNAs, has in our
F. Cloning of PCR fragments opinion resulted in reduced recovery of clones.
1. PCR products are generally propagated as 8. Cloned PCR fragments must be carried in an
clones carried in E. coli cloning vectors. appropriate E. coli strain. In our hands, the
Cloning of PCR products is accomplished strain has been DHlOB. DHlOB exhibits high
either by directly cloning fragments from the transformation efficiencies by electroporation
reaction mixture or by first purifying a using 0.2 cm cuvettes in the BioRad Gene
fragment by gel electrophoresis. Pulser system. Cells transformed with cloned
170
DNAs are plated on LB agar plates containing desired products. Note that the reaction buffer
appropriate antibiotics and, if desirable, the concentrations included 2 mmolll MgC1 2 •
indicator dye X-Gal. Efficiency of amplification in each reaction
G. Assembly of novel gene fusions using splice- was measured by examining the products of
over-lap extension PCR (SOE-PCR) the reactions on a 1.4% agarose gel in TAE
1. The technique of SOE-PCR, or genetic buffer. Product formation was observed in
knitting, has been used in the past to combine each of the 4 S. mutans promoter region reac-
two distinct regions of DNA, resulting in new tions as well as the reaction designed to
and novel sequences [16, 17]. We have used amplify the CAT gene.
the method to link the promoter region of the 5. For the second set of reactions, a series of
S. mutans ATPase operon to the chloram- dilutions of the step 1 PCR products were used
phenicol acetyltransferase (CAT) gene. The as template, as concentrations of products can
promoter and upstream regions of the S. vary. Serial dilutions of the S. mutans promoter
mutans ATPase possess 2 putative stem-loop fragments and CAT gene reactions were con-
structures immediately upstream of the -35 structed in sterile water. Generally, we have
box of the promoter [40], see Figure 2A. The found that dilutions on the order of 1: 10-
goal was to connect in-frame, in varying incre- 1: 1000 are effective.
ments, the stem-loop structures with the CAT 6. The reactions used were 50 /-11 in size and
gene. The resulting constructs will permit the consisted of the following: 5 /-11 lOx BRL Taq
examination of the role of stem-and-loops in DNA polymerase buffer; 0.8 /-11 25 mmol/l
the regulation of the S. mutans ATPase operon. dNTP's; 2 /-11 50 mmolll MgC1 2 ; 1 /-11 of a 50
2. The first step in SOE-PCR was to design pmolll stock solution of each primer, 1 /-11 of
primers for the construction of the chimeric each template at corresponding dilutions (i.e.,
proteins. The procedure is performed in two 1: 10 dilution of S. mutans promoter piece with
steps. In the first, two fragments of DNA are 1:10 dilution of CAT gene) and 0.5 /-11 of Taq
amplified independently, but with a region of polymerase. The cycling conditions for these
common DNA incorporated into one primer reactions were: 94°C, 2 min for one cycle; 94
for each fragment to be amplified. The incor- °C 1 min, 50°C 1 min, 72 °C 4 mins for 35
porated homologous region, represented in one cycles; and 72 degrees for 10 min for 1 cycle.
end of each amplified DNA product, is then 7. Verification of the correct alignment in the
used to guide annealing of the two products fusions is essential and can only be accom-
during amplification in the second step. The plished by nucleotide sequence determination.
annealing of the two DNAs formed from the Typically, we have cloned the PCR products
reactions in the first step leads to amplifica- by excising the desired band from a gel and
tion of a novel, hybrid DNA during the second purifying the DNA using the QIAEX II kit, as
phase of the process. described above. In the example of SOE-PCR
3. The four 'forward' or 'upstream'S. mutans gene knitting shown here, the products were
primers were designed to create the desired first cloned with the pGEM-T kit. E. coli trans-
portion of stem-loop region (see Figure 2A). formants exhibiting a white phenotype on LB
The 30 residue complementary strand primer medium containing X-Gal (40 mg/l, final con-
(Primer 5, Figure 2A) was synthesized with centration of 40 /-1g/ml) and ampicillin (100
homology to the 15 base pair sequence that is /-1g/ml) were screened for the presence of an
immediately upstream of ATPase c subunit insert. Clones that appeared to have the correct
gene, followed by the first 15 base pairs of insert were screened further by nucleotide
the CAT gene. The pairing of one of the sequencing.
primers (1-4) with primer 5 was used to H. Growth of S. mutans
produce the promoter region of the ATPase. 1. S. mutans strains UA159 [25] and URlOO [30]
4. Primers 6 and 7 (shown in Figure 2A) were were grown at steady-state at pH values of 5
used to amplify the CAT gene fragments. The and 7 using methods described in the paper
5' CAT primer, primer 6, was synthesized to by Chen and Burne [5]. We have also grown
be homologous to the upstream 15 base pairs cells in batch culture using methods described
of the ATPase gene and the first 15 base pairs in the paper by Spatafora [41].
of the CAT gene, inversely complementary to I. Preparation of S. mutans cell-free extracts
primer 5. The 3' primer for the CAT gene, 1. Cells are taken from their growth vessel, either
primer 7, was located downstream of the gene flasks in the case of batch growth or pumped
and was 24 base pairs long with a BamHI site from the chemostat in the case of steady-state
engineered into the 5' end of the primer. Taq growth situations, and collected by centrifu-
polymerase and accompanying buffer (Life gation in pfe-weighed Oakridge tubes at 8000
Technologies, Inc.) was used to obtain the rpm (7,650 rcf) for 10 min at 4°C.
171
2. Supernatant solutions are gently poured off threitol, 2.3% SDS and 0.0625 molll Tris, pH
and discarded. 6.8) the tube gel was sealed to the top of a
3. Cell pellets are suspended at 1 g/ml in stacking gel which is on top of a 10% acry-
breaking buffer: 10 mmolll Tris, pH 7.5; 50 larnide slab gel (0.75 mm thick). SDS slab gel
mmolll NaCl; 1 mmolll EDTA. electrophoresis was then carried out for about
4. One ml of suspension is transferred to a pre- 4 hours at 12.5 rnA/gel. The slab gels were
chilled 2 ml cryovial equipped with a screw- fixed in a solution of 10% acetic acid/50%
cap and O-ring and containing 1 g glass beads. methanol overnight. The following proteins
5. The chilled vials are placed in the holding (Sigma Chemical Co., St Louis, MO) were
compartment of a mini-Bead Beater. The timer added as mw standards to the agarose which
is set to 'timed' for 2 min and the instrument sealed the tube gel to the slab gel: myosin
is turned on to initiate breakage of the cells (220,000), phosphorylase A (94,000), catalase
by the shearing action of the glass beads. After (60,000), actin (43,000), carbonic anhydrase
the cycle is complete, the vials are placed on (29,000) and lysozyme (14,000). These stan-
ice for 1 min, and then re-cycled in the mini- dards appear as horizontal lines on the silver-
Bead Beater for an additional 2 min. Following stained [28] 10% acrylamide slab gel. The gels
the second cycle, the cell extract is clarified were dried between sheets of cellophane paper
by centrifugation of the vials in an Eppendorf with the acid edge to the left.
centrifuge at 8000 rpm (7300 rcf) for 15 min K. Analysis of the two-dimensional gels
at 4°C. 1. Digital images of the 2-D gels are created
6. The supernatant solution is transferred by using the Apple Color One Scanner, or similar
micro-pipette to a fresh vial and the centrifu- device, to scan the dried gels. Scans are con-
gation is repeated for 10 min. The centrifuga- ducted in the 8-bit mode and at a resolution
tion steps function to remove glass beads, of 150 dpi. The images can be saved as either
unbroken cells, and cell membranes. PICT or TIFF files.
7. The extracts are dialyzed overnight in 2 litres 2. The image files are analyzed with NIH Image
of TE buffer at 4 °C with one complete buffer version 1.6 for the Macintosh. Using tools
change after four hours. In the morning, the available in the software, high background
protein preparations are transferred from staining and non-protein streaks can be sub-
dialysis tubing into sterile 15 ml Corning tracted from the image. The position and inten-
tubes to estimate the volume. Using the stock sity of each protein, seen as a spot on the gel,
solution, PMSF is added to a final concentra- can be quantitated and catalogued for com-
tion of 100 Ilg/ml. parison with other gels.
8. Aliquots of dialyzed protein are withdrawn for
the estimation of protein using the Bio-Rad
dye-binding system or the Quanti-Gold kit. 4. Results and discussion
J. Two-dimensional gel preparation and staining
1. We have conducted two-dimensional gel Amplification of genetic fragments from s. mutans
separations in our laboratory using the using degenerate peR. The availability of nucleotide
Investigator 2-D system with the instructions sequences, from a large and growing number of bac-
provided by the manufacturer. An alternative terial genomes, permits the rapid Clustal alignment
to the purchase of sophisticated equipment of deduced amino acid sequences, such as that for
is the use of commercial vendors for the bacterial ATPase ~-subunits shown in Figure 1, Panel
preparation and execution of two-dimensional a. Such alignments provide the background infor-
gel separations. The procedure can be per- mation necessary to begin designing nucleotide
formed rapidly and cost effectively on a primers for use in PCR.
custom-gel basis. We have had separations per- The design of primers can be automated using
formed commercially by Kendrick Labs, Inc. computer alogrithms to determine those regions of
(Madison, WI) according to the method of DNA whose sequence would result in similar melting
O'Farrell [26, 27] as follows: isoelectric temperatures for the forward and reverse primers. Of
focusing (IEF) was carried out in glass tubes the commercially available packages, we most often
of inner diameter 2.0 mm using 2.0% pH 4-8 have used Mac Vector to search for appropriate primer
ampholines for 9600 volt-hour. Fifty ng of an sets and for estimation of primer melting tempera-
IEF internal standard, tropomyosin protein, tures. The algorithm used for computation of Tm
with lower spot of mw 33,000 and pI of 5.2 includes nearest neighbor analysis because of the
was added to the samples. The tropomysin spot effects of base composition on the overall melting
is indicated with an arrow on the 2-D gel or dissociation kinetics of probes. The methods have
pattern. After equilibration for 10 min. in been well described in the literature and we have
buffer '0' (10% glycerol, 50 mmolll dithio- found the temperatures estimated by the program to
172
40
10 20 r-::-'-::-::::-:::--:::---::~-:-1
B. megalenum G I VFAG GERTREG. ' DLVIIE l T D
E. hlrae G I V F T G V G E R T REG ' D L V Y E M8JD
E. coil G[3J V FAG V G E R T REG . DOJV II E •l T D S
B. subtlil s G I V FAG V G E R T REG N D L F Y L.:E::....:.;;M:.....::.S....:D::.....::;....;:'--'-~
50 60 70 80
~
2 50 260 270
YVPVKETV K GFKEILEGKYDHLPED
~
B. megate num GQKG
E. hlfae TG LP G YVP GETV K GFRE I LEGKYD~LPEE
E. coli TG SP G0YV0 L K D TIRGFK~I M EG0YDHLPE()
B. sub l1ll s TG()KGSY PVKETV GYKEIL A GKYDHLPED
Figure 1. Panel a. Computer generated Clustal alignment of deduced amino acid sequences from bacterial ATPase
p-subunits. Arrows indicate the position of degenerate oligodeoxynucleotide primers.
be useful starting points in our hands [33, 34]. We nation of bases in our primer sequences. The prac-
have also found that highly degenerate primers can tical result of using inosine was a large increase in
be used successfully to amplify gene fragments using the amount of primer able to anneal to our template,
fragments encoding portions of the highly conserved, leading to the formation of clear bands at the end of
catalytic subunit of the proton-translocating ATPase the first round of amplification, eliminating the need
(Figure 1, Panel b). Following the amplification of for re-amplification and thereby simplifying cloning
the fragments (Figure I, Panel b), cloning and of our reaction products.
nucleotide sequence determination confirmed the The reaction conditions that we have described
identities of the DNAs [31]. The fragments were then here were successful for amplification of a specific
used as probes to screen for clones of the intact streptococcal DNA fragment. It must be pointed out,
ATPase operon from S. mutans [40]. In subsequent that the results of a given amplification procedure are
studies, we substituted inosine at the wobble base potentially effected by a significant number of vari-
positions of codons. The effect was to greatly relax ables, including the following: annealing tempera-
the requirement for including every possible combi- ture, magnesium concentration, primer: template
173
CD
ATG
1 Step 1
~.:.:..:..:.:..:..:.:::..:.:::..:.:..:..:.:..:..:.:..:..:.>:..:..::..:.>:..:.>:..:.>:..):..:.:..:..:.:..:..:.:..:..:.:..:..:.:..:..:.:..:..:....:..:.>:..:..::..:..::··:·>:··:··::··:·.::··:·.::··:·:":1
CD
0)
~:::..:.:::. :.:::. :.:::. :..-: . :..-: . :..-: . :.:::. :..-: ..:.:::..:..-: . :..-: ..:..-: ..:.:::..:..-: ..:.:::..:.-: . :..-: . :.:::. :.:::..:.-: . :..-: . >::..:.-: . :..-: . :.:::··:·:::··:·:~··:I
Figure 2. Panel a. Splice-overlap extension PCR to create a genetic fusion of the ATPase operon promoter with a
chloramphenicol acetyl transferase gene as the reporter system. In Step 1 of the procedure, the two regions of DNA that
will eventually become fused are amplified independently. In this case, the promoter region of the ATPase operon was
amplified using one each of the primer labeled 1-4 and primer 5. Primer 5 contained 15 bases of sequence that were
precisely homologous to the last 15 bases of the presumptive ATPase promoter region (shown as the open box), up to
the ATG initiation codon for the c subunit in the operon. Primer 5 also contained the first fifteen bases encoding the
CAT gene (shown as the hatched box). The CAT gene was amplified using Primer 6, which contained the same sequence
as Primer 5 but in the reverse orientation, and primer 7. In Step 2 of the procedure, the products from Step 1 are mixed
and used as templates for amplification with one 'forward' primer, either 1, 2, 3 or 4 and Primer 7.
Figure 2. Panel h. Electropherogram of PCR products resulting from the Step 1 reactions described in the legend
for Panel 2a. Lane contents were as follows: Lane 1, 1 kbp DNA molecular weight markers; Lane 2, DNA amplified
with primer 1 and 5; Lane 3, DNA amplified with primer 2 and 5; Lane 4, DNA amplified with primer 3 and 5; Lane
4, DNA amplified with primer 4 and 5. Panel c. Electropherogram of PCR products resulting from the Step 2 reactions,
described in the legend for Panel 2a. Lane contents were as follows: Lane 1, 1 kbp DNA molecular weight markers;
Lane 2, DNA amplified with primer 1 and 7; Lane 3, DNA amplified with primer 2 and 7; Lane 4, DNA amplified with
primer 3 and 7; Lane 4, DNA amplified with primer 4 and 7.
ments. Methodologies, other than PCR, are available unchanged in any base. For the longest fusion, con-
in other bacterial systems for the construction of taining both stem-loops, of two sequenced clones,
genetic fusions; for example, the use of mini-trans- one was a precise product, the other contained a
posable elements to create in-frame fusions . Until single base change. Thus, it can be seen that while
similar approaches are well-established in the oral we had no trouble finding a completely authentic
streptococci, and in the absence of useful restriction clone for each of the four fusions, nucleotide
sites, knitting together diverse DNAs by PCR will be sequence characterization of multiple isolates was
an alternative. The scheme shown in Figure 2a illus- necessary to establish the desired fusion constructs.
trates our approach to linking varying portions of the In terms of the CAT portion of the fusions, we
putative ATPase operon promoter region to a CAT observed CAT gene activity in E. coli for all of the
reporter gene using splice-overlap extension PCR. fusions examined, whether or not they contained a
The result of those procedures are shown in Figure base change in the ATPase promoter region or not
2b, where the predicted laddering effect during (data not shown here). We concluded from those
amplification of the four desired fusions is shown. observations that we had either fortuitously isolated
The figure shows clearly the expected increase in the CAT fusions without changes in the CAT gene or that
molecular weights of the amplification products, as changes were silent. Future experiments will provide
predicted from the scheme in Figure 2a. The results insights into the ability of these promoter fragments
of knitting the operon fragments to the CAT gene to drive CAT synthesis in S. mutans as a function of
are shown in Figure 2c. Subsequent cloning of the external pH value.
amplicons verified the successful alignment of the
CAT gene with the ATPase promoter fragments . Development of additional targets for peR amplifi-
Comment should be made here that we did observe cation using proteomic databases. It has become
single base changes in some of the operon clones . apparent from genomic sequencing studies, that bac-
For example, multiple isolates for each of the fusions terial chromosomes can be catalogued for open-
were examined by nucleotide sequencing. The goal reading frames, but that significant portions of those
was to verify a single correct fusion for each of the reading frames can not be aligned with proteins
four constructs that we were seeking. Our sequencing in existing databases, thus obscuring their function
results showed the following: for the shortest fusion, [10, 12]. Further, the repertoire of proteins expressed
the first clone sequenced was a faithful and precise in varying environmental conditions is not yet
copy of the desired product, completely lacking any understandable from primary nucleotide sequence
changes; of the one and one-and-a-half stem-loop information. As part of the solution to the problem,
fusions, only one of three isolates sequenced was two-dimensional gel electrophoresis of proteins
176
mw
(kD) 4 ••- - - - - - - - - - -.. . . 8 4 ••- - -- - -- - - - - - . 8
220-
94-
60
43
29 -
14-
Figure 3. Panel a. Two-dimensional gel electrophoretic separation of protein extracts prepared from S. mutans strain
UR100 grown at steady-state on BHI medium containing glucose at a pH value of 5. Approximately 100 /lg of protein
was used in the separation. Gels were silver-stained as described in the text. Panel h. Two-dimensional gel electrophoretic
separation of protein extracts prepared from S. mutans strain UR100 grown at steady-state on BHI medium containing
glucose at a pH value of 7. Approximately 100 /lg of protein was used in the separation. Gels were silver-stained as
described in the text.
Figure 3. Panel c. Surface plots of the enclosed square shown in Panel a. Panel d. Surface plots of the enclosed square
shown in Panel b.
prepared from cells grown under specific conditions on the microbe's expressed protein repertoire. The
can be used to establish a given microbe's proteome, amino acid sequences of proteins can be determined
or set of expressed proteins, in a defined environ- directly following transfer of the gel-separated
ment. Proteomic maps have now been established for spots to membrane supports. The resulting 'blots' of
several organisms, grown in varying conditions. The proteins can be processed for sequence determination
most well-defined maps to date are those for E. coli using, in some cases, as little as a femtomole of
[43,44] and for the yeast, Saccharomyces cerevisiae material [19, 38, 39,46]. The amino acid sequences
[29]. The impact of changes in environmental con- resulting from those analyses will be used as the basis
ditions can easily be seen in two-dimensional gel sep- for either direct identification by comparison with
arations of the proteins prepared in extracts from E. existing databases, or the proteins may be novel
coli [42]. observations. If the genome of the organism has been
As genome nucleotide sequences become avail- determined, then the protein sequence can be aligned
able for S. mutans, it will become possible, using with the contigs derived from sequencing projects.
two-dimensional gels, to map the effect of mutations Matches of empirically determined protein sequence
177
with genomic nucleotide data will permit the design proteins for further evaluation is a 2-fold or greater
of homologous primers for use in amplifying the difference between protein samples prepared from
corresponding gene of interest. If the organism's cells grown under differing conditions. Clearly,
genome has not been determined, then the amino acid proteins which appear in one set of extracts and not
sequence can be used as the basis for synthesizing another would be among the first priorities.
degenerate oligonucleotides as we have described The results of two-dimensional gel separations of
above. The degenerate primers can then be used to proteins prepared from S. mutans grown at steady-
amplify all or part of the gene encoding the target state at pH values of 5 and 7 are shown in Figure 3,
protein. Panels a and b. Regions with examples of proteins
Our goal has been to understand the changes in obviously differing between the two gels are indi-
protein expression deployed by S. mutans during cated by the boxes. The differences are more clearly
adaptation to acidic environmental conditions. We seen when surface plots of the regions are made,
have included in this article examples of two-dimen- showing the intensities of the indicated spots in
sional gels prepared with extracts of cells grown in comparison to the same area in the companion gel
two different conditions. The resolution of the gels (Figure 3, Panels c and d). The arrows call the
potentially permits the identification of proteins reader's attention to a protein whose relative abun-
whose expression is shown to be dependent on an dance is increased at pH 5 relative to the gel prepared
environmental variable, in this case culture pH value. with pH 7 extracts. By way of example, we have
Proteins that are shown to be present in varying identified a protein of less than 29 kD that is present
proportion, depending on culture conditions, then in the pH 7 grown organisms and a difference in the
become candidates for micro-sequencing methods to profile of pH 5 proteins, in the region of 14 kD, when
establish their amino acid sequences. compared to the pH 7 grown cells. The proteins so
Comparisons between gels can be readily accom- indicated are examples of potential targets for future
plished with the unaided eye and illumination of gels investigations, using protein micro-sequencing
on a light box. However, quantitation of the gel spots, methods (see, for example, Matsudaira [22, 23]).
representing individual proteins, can be more easily Amino acid sequences developed from these types of
achieved using analytical software. Extraction of gels will permit the development of primers to be
quantitative data from images is dependent on the used in PCR experiments. The goal of those experi-
type of staining used for the gels and on the sensi- ments is to amplify part or all of the genes encoding
tivity of the scanner used to input images of gels pH-regulated proteins from the oral pathogen S.
into a computer. For example, gel fixation with glu- mutans.
taraldehyde results in non-arithmetic deposition of
silver grains on proteins owing to the non-stoichio-
metric interaction of glutaraldehyde with proteins [8]. Ackowledgrnents
Thus, quantitation of silver-stained gels represents an
approximation of the amount of a given protein We thank Nancy Kendrick for helpful discussion.
present in the gel. On the other hand, gels stained This work was supported by DE-10174, P01-
with Coomassie blue can be quantitated more readily DE11549 and the Rochester Cariology Training
because of the established stoichiometry of the dye Program T32-DE07165 (W.L.K).
with protein [4].
Once digitized images of gels have been stored
in the computer, the intensity of a particular protein Notes on suppliers
in the image can be normalized by comparing its inte-
grated density to the total intensity on a gel. By estab- 1. AAPER Alcohol and Chemical Company, DSP-KY-
lishing normalized values for proteins that may be 417, Shelbyville, KY 40065, USA
of interest, comparisons between gels become more 2. Ambion Inc., 2130 Woodward St. #200, Austin, TX
robust and permit the generation of a statistical set 78744, USA
of values. A number of comparative studies showing 3. American Biorganics, Inc., 2236 Liberty Drive,
the profile of proteins prepared from cells grown Niagara Falls, NY 14304-3796, USA
in differing conditions are now in the literature. 4. Bellco Glass, Inc. 340 Edrudo Rd., P.O. Box B,
Regarding the reproducibility of 2-D separations, Vineland, NJ 08360-0017, USA
we have taken the approach of requiring at least 5. BioRad Laboratories, 2000 Alfred Nobel Drive,
Hercules, CA 94547, USA
three separations of the differing preparations of
6. BioSpec Products, Bartlesville, OK 74005-0722, USA
cell extracts to establish a given protein as being 7. Boehringer Mannheim Biochemicals, P.O. Box 50414,
statistically different under two different growth con- Indianapolis, IN 46250, USA
ditions. Our working guideline is to use the Student's 8. Brinkmann Instruments Inc., One Cantiague Road,
t-test to compare the normalized intensities of a P.O. Box 1019, Westbury, NY 11590-0207, USA
protein in question on two sets of three gels each 9. Coming Inc., Science Products Division, Coming, NY
[45]. Our arbitrarily chosen criteria for selecting 14831, USA
178
10. Difco Corporation, P.O. Box 331058, Detroit, MI separated by two-dimensional electrophoresis. Clin
48232, USA Chern 35: 2297-2304.
11. Diversified Biotech, 1208 VFW Parkway, Boston, MA 5. Burne RA, Chen Y-YM (1998). The use of continuous
02132, USA flow bioreactors to explore gene expression and
12. ESA, 22 Alpha Rd., Chelmsford, MA 01824-4171, physiology of suspended and adherent populations of
USA oral streptococci. Methods Cell Sci 20: 181-190 (this
13. Squibb and Sons Inc., Princeton, NJ 08540, USA issue).
14. Hybaid, 8 East Forge Parkway, Franklin, MA 02038, 6. Chassy BM (1976). A gentle method for the lysis of
USA oral streptococci. Biochem Biophys Res Comm 68:
15. Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 603-608.
92008, USA 7. Daspher SG, Reynolds EC (1992). pH regulation by
16. JT Baker Inc., 222 Red School Lane, Phillipsburg, NJ Streptococcus mutans. J Dent Res 71: 1159-1165.
08865, USA 8. Dion AS, Pomenti AA (1983). Ammoniacal silver
17. Laboratory Product Sales, 1665 Buffalo Road, staining of proteins: mechanism of glutaraldehyde
Rochester, NY 14624, USA enhancement. Anal Biochem 129: 490--496.
18. Life Technologies Inc., P.O. Box 6009, 8717 9. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick
Grovemont Circle, Gaithersburg, MD 20877, USA JS (1991). 'Touchdown' PCR to circumvent spurious
19. Molecular Probes, Inc., 4849 Pitchford Ave., Eugene, priming during gene amplification. Nucl Acids Res
OR 97402, USA 19: 4008.
20. Nalgene, 75 Panorama Creek Dr., Rochester, NY 10. Fleischmann RD, Adams MD, White 0, et al. (1995).
14625, USA Whole-genome random sequencing and assembly
21. New Brunswick Scientific, P.O. Box 4005, 44 of Haemophilus inJluenzae Rd. Science 269: 496-
Talmadge Road, Edison, NJ 08818-4005, USA 512.
22. Oxford Molecular Group, 2105 South Bascom Ave., 11. Foster JW, Hall HK (1990). Adaptive acidification
Suite 200, Campbell, CA 95000, USA tolerance response of Salmonella typhimurium. J
23. Perkin Elmer, 850 Lincoln Centre Drive, Foster City, Bacterioll72: 771-778.
CA 94404, USA 12. Fraser CM, Gocayne JD, White 0, et al. (1995). The
24. Promega Corporation, 2800 Woods Hollow Road, minimal gene complement of Mycoplasma genitalium.
Madison, WI 53711, USA Science 270: 397--403.
25. Qiagen Inc., 28159 Avenue Stanford, Santa Clarita, 13. Hamilton IR, Buckley ND (1991). Adapation by
CA 91355, USA Streptococcus mutans to acid tolerance. Oral Micro-
26. Rainin Instrument Company, Mack Road, Woburn, bioI Immun 6: 65-71.
MA 01801, USA 14. Hecker KH, Roux KH (1996). High and low annealing
27. Sigma Chemical Company, P.O. Box 14508, St. temperatures increase both specificity and yield in
Louis, MO 63178, USA touchdown and stepdown PCR. BioTechniques 20:
28. Sorvall, Inc., 31 Pecks Lane, Newtown, CT 06470- 478-485.
2337, USA 15. Heery DM, Gannon F, Powell R (1990). A simple
29. Stratagene, 11011 North Torrey Pines Road, La Jolla, method for subcloning DNA fragments from gel
CA 92037, USA slices. Trends Genet 6: 173.
30. VWR, P.O. Box 626, 200 Center Sq. Rd., Bridgeport, 16. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR
NJ 08014, USA (1989). Site-directed mutagenesis by overlap exten-
31. Pharmacia Biotech, 800 Centennial Ave., P.O. Box sion using the polymerase chain reaction. Gene 77:
1327, Piscataway, NJ 08855-1327, USA 51-59.
32. Fotodyne, Inc. 950 Walnut Ridge Drive, Hartland, WI 17. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR
53029, USA (1989). Engineering hybrid genes without the use of
restriction enzymes: gene splicing by overlap exten-
sion. Gene 77: 61-68.
18. Hotchkiss RD (1957). Methods for characterization of
References nucleic acid. In: Colowick SP, Kaplan NO (eds),
Methods Enzymol III: 708-715.
1. Ausubel FM, Brent R, Kingston RE, et al. (1987). 19. James P (1997). Of genomes and proteomes. Biochem
Current protocols in molecular biology. New York: Biophys Res Comm 231: 1-6.
John Wiley & Sons. 20. Kobayashi H, Suzuki T, Kinoshita N, Unemoto T
2. Belli WA, Marquis RE (1991). Adaptation of (1984). Amplification of the Streptococcus faecalis
Streptococcus mutans and Enterococcus hirae to acid proton-translocating ATPase by a decrease in cyto-
stress in continuous culture. Appl Environ Microbiol plasmic pH. J Bacteriol 158: 1157-1160.
57: 1134-1138. 21. Kramer MF, Coen DM, Dorit RC, et al. (1987). The
3. Bender GR, Sutton SVW, Marquis RE (1988). polymerase chain reaction. In: Ausubel FM, Brent R,
Acid tolerance, proton permeabilities, and membrane Kingston RE (eds), Current protocols in molecular
ATPases of oral streptococci. Infect Immun 53: biology, vol 2, pp 15.0.1-15.8.8. New York: John
331-338. Wiley & Sons.
4. Burgess-Cassler A, Johanson 11, Santek DA, Ide 22. Matsudaira PT (1990). Limited N-terminal sequence
JR, Kendrick NC (1989). Computerized quantitative analysis. Methods Enzymol 182: 602-613.
analysis of coomassie-blue-stained serum proteins 23. Matsudaira PT (1989). A practical guide to protein
179
and peptide purification for micro sequencing. New 37. Sansone C, Van Houte J, Joshipura K, Kent R,
York: Academic Press. Margolis HC (1993). The association ofmutans strep-
24. Miwa T. Esaki H, Umemori J, Hino T (1997). Activity tococci and non-mutans streptococci capable of aci-
of H+ -ATPase in ruminal bacteria with special refer- dogenesis at a low pH with dental caries on enamel
ence to acid tolerance. J Bacteriol 63: 2155-2158. and root surfaces. J Dent Res 72: 508-516.
25. Murchison n, Barrett JF, Cardineau GA, Curtiss III 38. Shevchenko A, Jensen ON, Podtelejnikov AV, et al.
R (1986). Transformation of Streptococcus mutans (1996). Linking genome and proteome by mass spec-
with chromosomal and shuttle plasmid (p YA629) trometry: large-scale identification of yeast proteins
DNAs. Infect Immun 54: 273-282. from two dimensional gels. Proc Natl Acad Sci USA
26. O'Farrell PH (1975). High resolution two-dimensional 93: 14440-14445.
electrophoresis of proteins. J BioI Chern 250: 4007- 39. Shevchenko A, Wilm M, Vorm 0, Mann M (1996).
402l. Mass spectrometric sequencing of proteins from
27. O'Farrell PZ, Goodman HM, O'Farrell PH (1977). silver-stained polyacrylamide gels. Anal Chern 68:
High resolution two-dimensional electrophoresis of 850-858.
basic as well as acidic proteins. Cell 12: 1133-1141. 40. Smith AJ, Quivey Jr. RG, Faustoferri RC (1996).
28. Oakley BR, Kirsch DR, Morris NR (1980). A simpli- Cloning and nucleotide sequence analysis of the
fied ultrasensitive silver stain for detecing proteins in Streptococcus mutans membrane-bound, proton-
polyacrylamide gels. Anal Biochem 105: 361-363. translocating ATPase operon. Gene 183: 87-96.
29. Payne WE, Garrels 11 (1997). Yeast protein database 41. Spatafora GA, Moore MW (1998). Growth of
(YPD): a database for the complete proteome of Streptococcus mutans in an iron-limiting medium.
Saccharomyces cerevisiae. Nucl Acids Res 25: 57-62. Methods Cell Sci 20: 217-221 (this issue).
30. Quivey RG, Faustoferri RC (1992). In vivo inactiva- 42. VanBogelen RA, Kelley PM, Neidhardt FC (1987).
tion of the Streptococcus mutans recA gene mediated Differential induction of heat shock, SOS, and oxida-
by PCR amplification and cloning of a recA fragment. tion stress regulons and accumulation of nucleotides
Gene 116: 35-42. in Escherichia coli. J Bacteriol 169: 26-32.
31. Quivey RG, Faustoferri RC, Belli WA, Flores JS 43. VanBogelen RA, Neidhardt FC (1991). The gene-
(1991). Polymerase chain reaction amplification, protein database of Escherichia coli, 4th ed.
cloning, sequence determination and homologies of Electrophoresis 12: 955-994.
streptococcal ATPase-encoding DNAs. Gene 97: 44. VanBogelen RA, Sankar P, Clark RL, Bogan JA,
63-68. Neidhardt FC (1992). The gene-protein database
32. Roux KH (1994). Using mismatched primer-template of Escherichia coli, 5th ed. Electrophoresis 13:
pairs in touchdown PCR. BioTechniques 16: 812- 1014-1054.
814. 45. Walpole RE, Myers RH (1972). Probability and
33. Rychlik W, Rhoads RE (1989). A computer program statistics for engineers and scientists. New York:
for choosing optimal oligonucleotides for filter Macmillan.
hybridization, sequencing and in vitro amplification 46. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, et al.
of DNA. Nucl Acids Res 17: 8543-8551. (1995). Progress with gene-product mapping of the
34. Rychlik W, Spencer WJ, Rhoads RE (1990). Mollicutes: Mycoplasma genitalium. Electrophoresis
Optimization of the annealing temperature for DNA 16: 1090-1094.
amplification in vitro. Nucl Acids Res 18: 6409-6412.
35. Saiki RK, Scharf S, Faloona F, et al. (1985)
Enzymatic amplification of ~-globin genomic Address for correspondence: Dr Robert G. Quivey, Jr,
sequence and restriction site analysis for diagnosis of Department of Dental Research, Box 611, 601 Elmwood
sickle cell anemia. Science 230: 1350-1354. Ave., University of Rochester, School of Medicine and
36. Sambrook J, Fritsch EF, Maniatis T, eds (1989). Dentistry, Rochester, NY 14642, USA
Molecular cloning, a laboratory manual, 2nd edn. Cold Phone: (716)-275-0382; Fax: (716)-473-2679
Spring Harbor, NY: Cold Spring Harbor Press. E-mail: robert_quivey@urmc.rochester.edu
Methods in Cell Science 20: 181-190 (1998)
© 1998 Kluwer Academic Publishers.
Abstract. Most pathogenic bacteria elicit diseases gene expression with continuous chemostat culture,
as populations colonizing surfaces of a susceptible allowing for the strict modulation of key physiologic
host. In the biofilms that form on host tissues, path- parameters, and ii) for the cultivation of oral strep-
ogenic bacteria can encounter huge variations in tococci as adherent populations which are suitable
environmental conditions that can critically affect for analysis using reporter gene fusions and advanced
persistence and expression of virulence. Gaining a microscopic techniques. These techniques extend the
comprehensive understanding of molecular aspects capabilities of investigators to control key physio-
of pathogenesis by streptococci and enterococci logic parameters and to cultivate the organisms in
will require a detailed dissection of the phenotypic environments that may more closely mimic condi-
capacities of these organisms under conditions that tions in vivo. Thus, these approaches should be
may be experienced in vivo. Described herein are useful in enhancing our understanding of bacterial
technologies that i) merge the study of bacterial pathogenesis.
2. Materials
A. Equipment
Media
1. Homogenizer, model mini-Bead Beater, Bio- Resevoir KOH,
Spec Products. 3 H+
2. Micro-centrifuge, refrigerated, Eppendorf, etc.
VWR. 14
3. Adjustable pipettes: nos. P-20; P-200, P-1000,
Rainin. lO
4. Centrifuge, refrigerated-preparative, Sorvall
RC5C, Sorvall, Inc. 12
5. SS-34 rotor for RC5C centrifuge. 12
~~1~~e = v 1 Xo
......
Constant Volume
Emuent
x
6. Pipette-Aid, Drummond, VWR. no. 53498-
001. 14
7. Beckman DU640 Spectrophotometer, Beck-
man Instruments. 1 F = Flow rate of medium (I/h)
8. New Brunswick Chemostat, New Brunswick V = Liquid Volume in Vessel (Constant)
Scientific.8 Xo = Cell masslliter in vessel (gil)
9. Modified Rototorque, Center for Biofilm X = Cell mass in emuent (gil)
Engineering. 4 Figure 1. A schematic representation of a single-stage
B. Culture medium chemostat for continuous cultivation of microorganisms.
- Base Medium: Tryptone 30 g; yeast extract, 5 A media reservoir delivers sterile medium into the culture
g (Ty).6 vessel at a fixed rate. The volume of the culture in the
E. Solutions vessel is kept constant either by pumping out fluid or by
- 10 mM Trizma base (Tris), no. T-153027, letting it trickle out a port in the side of the vessel.
adjusted to a pH value of 7.8 with HCl. 1O Particular variables (F, V, Xo, and X) are defined and
- Bio-Rad dye-binding reagent, 500-0006. 1 treated in Equations 4-6.
H. Glassware and plastics
- Oakridge tubes, no. 3119-0050.13 mentation for continuous cultivation. In this mode,
- 50 ml conical Corning tube, no. 25330-50. 7 one can achieve 'steady-state' conditions, and this
- 2 ml screw cap microfuge tubes has important implications as detailed below. First,
- glass beads, no. 34007. 13 though, a brief overview of some principles of con-
cryovials, with O-rings and screw-caps, no. tinuous chemostat culture are necessary or useful.
4204-S.7 One runs a continuous chemos tat by introducing
- 0.45 IlM autoclavable filters. 8 fresh medium into the vessel at a fixed rate while
maintaining a constant culture volume. In some
cases, a reflux system that reintroduces expelled
3. Methods, results and discussion bacteria is also employed, but this is unusual in
most applications of the technology to basic research
A. Use of continuous culture to modulate questions. The rate of introduction of the medium,
physiology of oral streptococci: General or dilution rate, D, is expressed as the amount of
Considerations medium introduced per hour as a fraction of the total
culture volume. For example, for a 1 liter culture,
The basic workings of the continuous chemostat are introduction of 100 ml per hour would correspond
depicted in Figure 1. In reality, this is a fairly simple, to a dilution rate, D, of 0.1 h- I . After a number of
albeit not inexpensive instrument. The chemostat generations, 10 appear to be sufficient, the culture
consists of a vessel in which bacteria can be cultured can achieve steady state. At steady state, the optical
in a fixed volume. With existing instrumentation, density of the culture should not change and there-
one can control dissolved oxygen, pH, and rate of fore, the yield, Y, remains constant under steady state
input of nutrient sources, among other chemical and conditions. This occurs because the rate at which the
physical parameters. Operation of the vessel in batch bacteria divide is directly related to the rate of intro-
mode is adequate for examining effects of certain duction of fresh medium at steady state. Another key
environmental influences, but the cells grow as if in point is that some particular nutrient, depending on
batch culture; passing through lag, exponential and medium composition, will always be limiting under
into stationary phase. This means that influences on true steady state conditions. The final yield that is
gene expression as a result of varying growth rates observed will be correlated with the yield achiev-
and growth phase phenomena cannot be excluded. able with the limiting substrate (S). Yield is also
A more appropriate use of the chemos tat for the types related to the maintenance coefficient for that par-
of studies described above is to employ the instru- ticular substrate (Ks), which is usually an empirically
183
determined mathematical expression of the material The really useful aspect of steady state cultures is
and energy spent by the organisms not for growth, that one can modulate a single environmental para-
but to maintain physiologic homeostasis. By way of meter, for example pH, carbohydrate source, carbo-
example, for the oral streptococci, yields will be hydrate availability, or oxygen concentration, among
lower at pH 5.0 compared with pH 7.0 because others, while maintaining the same specific growth
the cells will be spending more energy to pump rate and without necessarily changing the limiting
protons and maintain a comparatively alkaline cyto- nutrient. Conversely, the growth rate can be altered
plasm; higher maintenance. The yields will also vary while keeping constant the aforementioned variables.
depending on whether the cells are grown on sorbitol This is particularly easy with the lactic acid bacteria
or glucose. because respiration is not a factor. With organisms
Working from established equations, one can that can grow on a truly defined medium, it is
demonstrate that at steady state the specific growth possible to define the limiting nutrient. For example,
rate, Il (sometimes Il g ), is directly related to the one can simply obtain a sample of the spent medium
dilution rate D. First, using the standard equation to and measure residual glucose to insure carbohydrate
calculate cell number after a certain period of expo- limitation, or measure ammonia, if that is the sole
nential growth, nitrogen source, to insure that cultures were nitrogen
limited. Practically speaking though, with the oral
(1) streptococci, studies have been restricted to exam-
where X t is the final number of cells present after a ining cultures growing in the presence of limiting or
given time, Xo is the number of cells at time 0, Il the excess carbohydrate. Carbohydrate excess cultures
specific growth rate, and t, the time, usually in hours. have been referred to as nitrogen limiting in some
Taking the natural log of Equation l, past publications, but it is not clear whether the
carbohydrate excess conditions used in those studies
log ,xl = log ,xo + Ilt (2) really resulted in limitation for a nitrogen source
or for some other essential nutrient, e.g., a vitamin.
then taking the derivative of the equation and Nonetheless, since the Gram-positive cocci that are
allowing for one doubling (XI = 2, Xo = 1) focused on in these volumes rely almost solely on
carbohydrate for growth, and have no functional
0.693
--=t (3) respiratory cascades, the modulation of carbohydrate
Il availability is a central physiologic control point and
Equation 3 relates the specific growth rate to the gen- thus highly relevant in many cases. Notably, some
eration time, tg • studies we have conducted indicate a readily metab-
Now, mathematically considering what is hap- olizable nitrogen source, probably glutamine, may
pening with the population under steady state condi- become limiting under carbohydrate excess condi-
tions in a typical chemos tat, the following equation tions with the particular medium we use.
is applicable where the variables are defined in
Figure 1: B. Key tips in maintaining 'long-term' continuous
culture
FXo FX dX
---+!1X-aX=- (4) If one is conducting experiments at low dilution rates,
V V dt
e.g., D = 0.05 h- 1, it takes roughly 5.8 days to achieve
Cells in - Cells out + Growth - Death = Change steady state. So even an experiment designed to
in cell # with time; at steady state = 0. The letter take measurements at three different pH values
(X is a coefficient related to the rate of death of under glucose limiting and glucose excess conditions
cells in the population. can easily extend for 6 weeks. High dilution rates
mandate that media reservoirs and other additives
°ofbecause
In a 'single stage' chemostat, the flow of cells in is be changed frequently. As might be predicted then,
the feed stream is sterile, so that portion one of the most common problems encountered in
the equation cancels. At steady state, dX/dt 0,= continuous culture studies is contamination. This
and if one assumes the contribution to the equation can occur through introduction of more competitive
of cell death to be essentially = 0, which is reason- species during the initial set up of the chemostat or
able for these exponentially growing populations, with changeover of input vessels, from sampling, or
then, as a result of failure of some component of the
systems, generally pump tubing. Some fairly simple
FX
- - + IlX
V
dX
= -dt = ° (5) modifications to the vessel and common aseptic
techniques, as well as some preventive measures can
essentially eliminate contamination problems. Most
F F importantly, thanks largely to techniques developed
Il =-, and D = -, therefore, Il = D (6)
V V in the laboratory of R. E. Marquis (University of
184
Rochester Medical Center), we now routinely employ sugars, respectively, and autoc1aved separately. For
114 inch stainless steel tubing and SwagelockTM oral streptococci, the medium should be completely
brand, stainless steel compression fittings on all of free of sucrose to prevent wall growth or fouling of
the connection points for media and additive feed the pH probe. To remove trace amounts of sucrose
lines. This allows for the connection points of input present in the base medium and the sugar solutions,
lines to be flamed prior to fastening, and to be heated about 1 mg (approx. 1000 U/mg, Sigma) of invertase
directly over a flame for several seconds afterward is added to both the base medium and sugar additives
to decontaminate the connection point. We have prior to autoclaving. The base medium is autoclaved
found this far superior to the 'poke and hope' method for 60 min on a liquid cycle. It is strongly recom-
of connecting tubing using plastic fittings that have mended that the medium be allowed to cool for one
been covered with aluminum foil and autoclaved. hour with the autoclave door open before removing
Use of stainless steel fittings allows for employment the vessel. The vessels can burst, or jarring the hot
of rigorous aseptic techniques, a major advantage container of medium can cause the rapid release
for long term continuous culture. The only other of steam. After the medium cools, additives, e.g.,
suggestion is that tubing, particular any that passes carbohydrates, antibiotics, etc., can be introduced.
through pump heads, should be changed on a regular This is done as shown in Figure 2. The two vessels,
basis. The cost of tubing is minor compared to the with the tubing clamped, are joined using the stain-
cost of media and time devoted to setting up and less steel fittings, wrenches are used to tighten the
maintaining a chemostat. connection, the tube clamps are removed and a flame
is applied for about 10 to 15 s. After the connection
C. The use of continuous culture to modulate has cooled, a hand held pump with tubing that will
physiology of oral streptococci: Growth fit the exposed end of the 0.45 )lm filter on the base
limitations medium vessel is attached and a mild vacuum is
applied. This will cause the sugar solution in the
The following sections detail the media and specifics reservoir to flow into the vessel. If it is desired,
of setting up a chemostat for oral streptococci, in antibiotics can be added to the sugar solution or
particular, we have used these conditions for S. directly to the base medium. We introduce the
mutans and S. salivarius. We, and others who have heat labile compounds by fitting the vessel with
used similar conditions have found that the pre- a curved stainless steel tube that can be covered
scribed base medium will support the growth of oral with aluminum foil. We dissolve the additives and
streptococci using sugar concentrations between 10 filter sterilize them directly into the tube, flaming
and 200 mM. Dilution rates can vary widely, but at afterward.
too slow a rate, the organisms tend to colonize the The preparation of the chemostat culture vessel is
surfaces of the vessel (wall growth), whereas at too fairly straightforward. The culture vessel is fitted
high a dilution rate, the organisms cannot divide fast
enough and the population steadily declines (wash polypropylene
out). D values of 0.05 h- 1 to 0.5 h- 1, or even greater, 0.45 fLm_ / tubing
can be accomplished without unacceptable wall filter 114" stainless steel
.JLJl +--'t-...c: tubing
growth or wash-out, respectively. The lower limits of
pH for growth while still maintaining steady state
is usually around 5 for Streptococcus mutans and Base
/\ +-0.45 fLm
filter
i -----\
autoclave our 1 liter capacity vessels for 30 min. ! .'.'. ~
Be certain to place one, and preferably two 0.45 ~m
exhaust filters on the culture vessel to release
pressure built up with heating of the liquid and do
\.
.
.'
not over tighten the top stainless steel portion of the
vessel onto the glass bottom. After autoclaving and .
, ;
cooling, the chemostat is set up per manufacturers ,i ~
directions, and the media and alkali reservoirs are , \,
50
,
,
,
joined as above. One useful tip is to use a T-con-
,~ ~
nection for the culture vessel medium input. This will , I .,.,
,
allow for the connection of two media reservoirs,
which can be useful for runs that might exhaust one 0.....,.-.-----'"' 0 •.
...............................
vessel in the middle of the night. Both the alkali O~----.---__.-==~~~~
solution and medium source are connected to the o 5 10 15 20
culture vessel through Swagelok tube fittings. Generations
volume of the culture used in the assay. Cell dry in the planktonic phase. Some systems also employ
weights can also be utilized for normalization, or material in the substratum that is more consistent
cell lysates prepared and protein concentration with surfaces found in the oral cavity, such as
used to normalize the data. However, the ease of hydroxylapatite. Although this may be a more
permeablization of the cells and the stability of relevant material, there are pros and cons to using
LacZ to this treatment make it ideal, particularly these types of material. Consideration for the specific
for a large number of samples or kinetic studies of research question must dictate whether one selects a
gene induction. leaching or non-leaching surface. Various continuous
flow systems have been used to examine mixed
I. Biofilm model systems: Considerations population biofilms; composed either of defined
multispecies consortia or used to cultivate popula-
There are a wide variety of biofilm model systems tions following inoculation with plaque samples
currently in use to study adherent populations of oral obtained from humans. The former has been ele-
bacteria from the standpoint of ecology, biological gantly employed to examine the role of various
activities, physiologic concerns and monitoring of environmental parameters, e.g., pH, oxygen, and the
gene expression. These encompass fairly simple presence of particular species on the persistence of
systems in which oral streptococci are cultivated on organisms in mixed populations [2]. Nevertheless, the
wires or glass rods in batch type systems. These purpose of this chapter is not to review available
simpler models usually are composed of a single biofilm models, which has been done quite well in
species and can yield high quality repeatable results several other publications [4, 6, 12]. Instead, we will
for certain applications. Like other systems, when detail methods which we have utilized to examine
using mono species cultures of oral streptococci, monospecies cultures of oral streptococci for the
efficient formation of biofilms that are greater purposes of examining gene regulation and expres-
than a few microns in depth usually requires that sion in these adherent populations.
sucrose is present in the medium as a substrate for
glucosyltransferases, which, in conjunction with J. Cultivation of strains of oral streptococci in a
other glucan binding proteins promote tenaciously commercially available biofilm reactor
adhering popUlations. The next level in complexity
of biofilm model systems contain a single species but We began our studies using a commercially available,
employ continuous flow. These come in a variety of continuous flow biofilm reactor, known as a
shapes and sizes, including micro flow cells, with Rototorque [4]. The vessel was originally designed
working volumes of 50 to 100 III and surface areas for studying biofilms formed by aquatic microor-
of about 1 cm2 that are amenable to microscopic ganisms, and included a reflux loop, which could
evaluation, as well as fairly large (1 L) reactor vessels recycle sloughed and shed microorganisms, and
with more elaborate systems to control environmental planktonic ally growing cells back through the
conditions and fluid flow. The choice of system is systems. The Rototorque was designed with the idea
mainly dictated by the research question, or by pref- that significant shear forces could be generated,
erences among investigators. For example, studies on similar to conditions in water lines, streams,
dynamics of biofilm formation and structure can be etc. However, high shear is neither pertinent or
accomplished on a small scale in microflow cells. favorable to biofilm formation by oral bacteria.
Expectedly, microcells are often the best suited for Consequently, we had the vessel modified by the
these types of studies especially if the substrates to supplier to remove the reflux loop and we operate the
be provided are available in limited quantities, e.g., system at very low shear forces. This allows for the
salivary secretions. Alternatively, systems which can formation of biofilms that can be several hundred
produce substantial amounts of cellular material microns in depth and still has the advantage that
usually utilize media components that are neither fairly large quantities of material can be generated
limiting nor prohibitively expensive. These latter for analyses.
systems are useful to study effects of biofilm Configuration of the Rototorque is as detailed in
heterogeneity on physiologic parameters or on the Figure 4. Specifically, the modified Rototorque is
expression of particular genes, as the larger models roughly 5 cm shorter than the standard instrument
provide sufficient biomass and one can obtain and the port in the bottom of the vessel was sealed,
multiple samples to obtain statistically significant yielding a vessel with a 'working volume' of 0.6 L.
results. Importantly, the continuous flow systems The average surface area of the exposed face of one
incorporate some key features that static, batch of the 12 polystyrene slides in the vessel is approx.
systems do not, including laminar flow, which can 31 cm 2 • Basically, we operate the instrument much
somewhat mimic salivary fluids passing over the like a chemostat. An input pump delivers fresh
plaques, and more flexibility for controlling critical medium at a fixed dilution rate, D, expressed as a
environmental parameters, such as pH, dissolved proportion of the total liquid volume in the vessel.
oxygen, and carbohydrate source and concentration A constant volume is maintained by pumping liquid
188
I
inoculation of the biofilm reactor vessel are grown at
37°C in a 5% CO 2 , aerobic atmosphere in TY with
10 mM glucose, and with antibiotic if necessary. For
cultivation of cells in biofilms, TY medium supple-
1
21.2 em
mented with 10 mM sucrose (TYS) is used. The
Rototorque reactor is filled with about 112 the
working volume of TYS, autoclaved for 30 min,
equilibrated at 37°C, and inoculated with 10 ml of
an overnight culture of the strain to be studied. Fresh
medium is introduced immediately after inoculation.
The instrument can be operated with the motor
connected directly to the shaft of the rotating inner
drum. Rotation of the inner drum is controlled at a
speed determined by a rheostat setting of '20' on the
instrument (approximately 75 RPM). In the past, we
have conducted analyses of biofilm cells after 48 h
Figure 4. A schematic diagram of the modified or for 7 days post inoculation, and visible biofilms
Rototorque. Medium is pumped into the vessel and effluent begin to form in as little as 18 to 24 h [3].
was pumped from the top of the vessel as shown. The
speed of the rotating inner drum was kept constant at 75 L. Sample preparation and assays of reporter
RPM. The temperature of the vessel was maintained by gene product activity
immersion in a 37 °e, circulating water bath. Some dimen-
sions of the vessel, the inner drum, and the width of the The thickness of the biofilms formed is dependent
slides are shown for reference. largely on sucrose concentration, the strain of
bacteria being used, and the length of time the
from the vessel. As with the chemostat, we use biofilms are allowed to form. In the past, we have
Swagelock type fittings to connect the instrument to found that 48 h biofilms are generally about 20 ~m
media feeds. The control of pH in the vessel, unlike in depth, and frequently have properties similar to
a chemostat, is far more technically challenging. their planktonic counterparts. Under the growth con-
Since the biofilms are formed under conditions which ditions we utilize, biofilms of S. mutans formed in 7
promote the colonization of surfaces, use of a pH days achieve thicknesses of of greater than 100 ~m.
probe in the vessel is impractical because the probe It is with these thicker films that we have found that
will quickly become fouled. The use of buffers is the gene expression patterns of the exopolysaccha-
a practical, although less than ideal approach. ride synthases, glucosyltransferase and fructosyl-
However, it must be kept in mind that this does transferase, are altered as compared with their
not really allow for control of the pH in the biofilms, planktonic counterparts [3]. Parenthetically, strains
and that gradients will certainly develop within the of S. sobrinus can form biofilms that are much
surface-growing organisms. The other major consid- thicker under these conditions, while the soft tissue
eration is that attainment of true steady state condi- microorganism, S. salivarius, forms thinner films that
tions, as detailed above, may not be possible in are more easily removed from the polystyrene slides.
biofilms, particularly thicker films. The films thus In the past, we have examined 'quasi-steady state'
formed are heterogeneous and access of all cells in gene expression and induction in a kinetic fashion.
the population to a uniform pool of nutrients is The ability to do this is one major advantage to the
impossible. Moreover, the growth phase of bacteria Rototorque. Specifically, one or more of the twelve
within the films can differ dramatically and biofilm slides can be removed aseptically over a time course.
populations can contain considerable proportions For example, we have looked at induction of the gtf
of apparently dead cells. As a result of these con- and fif genes in cells in 48 h or 7 day cultures and
founding factors, investigators frequently report their then induced with addition of 25 mM sucrose [3]. An
data at 'quasi-steady state', where D has been alternative application might include monitoring
constant for the equivalent of 10 generations, as cal- biomass, polysaccharide content and biochemical
culated above. activities over a 12 day course. Slides are removed
from the vessel, cells are mechanically dissociated
from the slides into a solution of ice cold 10 mM/L
Tris-HCl (pH 7.0) containing 10 ~g of rifampicin and
tetracycline per ml, to arrest transcription and trans-
189
lation, respectively. One can use a rubber policeman chemistry and genetics of prokaryotes. Nonetheless,
or glass microscope slide to assist in removal of there are valid scientific reasons to believe that batch
the cells. The cells are immediately centrifuged at cultivation and the study of planktonic cells may con-
8000x g for 10 min at 4°C. Cells are washed in ice strain the phenotypic capacities of microorganisms.
cold 10 mM Tris-HCl (pH 7.8) and the cell pellets By using alternative methods to alter cellular physi-
kept on ice until all samples are ready for processing. ology, such as tighter control in chemostats or growth
For assays of activities of reporter gene products, of organisms in biofilms, additional key biological
we essentially utilize the protocols described above. activities or behaviors may be revealed that will be
The only key considerations to take into account are central to understanding initiation or progression of
that biofilm cells occur in an aggregated state and the disease. Moreover, the potential biological activities
biofilms themselves can be particularly rich in poly- of consortia of microorganisms should not be over-
saccharides. For these reasons, it is not recommended looked. For example, it is established that degrada-
to utilize dry weights for normalization of activities. tive communities, consisting of populations of
Rather, quantification of protein is the preferred spatially arranged microorganisms, can completely
approach. In the case of cat gene fusions, we homog- catabolize compounds that any single species in the
enize the material in the films as above and simply consortia cannot. There is ample reason, but com-
measure protein by the Bradford assay. We have paratively little solid evidence, to believe that such
confirmed that this assay is suitable for this purpose communities form and play an active role in diseases
by completely hydrolyzing the biofilms, and con- progression. Future studies coupling the use of phys-
ducting amino acid analysis and quantification of iologic tools and studies of adherent populations with
total polysaccharide. The results we have obtained sophisticated and sensitive techniques in molecular
using analytical chemistry correlate well with esti- biology and non-invasive microscopic technologies
mates of protein from the Bradford assay. For promise to open a new era of understanding of the
measuring LacZ activity, we have normalized the pathogenic properties of individual and populations
assay to O.D.600. In these cases, the cells are dispersed of bacteria.
by sonication at 350 W with a microprobe for 30 s,
twice, and then assays are performed and optical
density readings are obtained. Acknowledgments
M. Preparation of cells for microscopy This work was supported by PHS grants DE10362
and DE12236.
For imaging of biofilms, cells are cultured as above.
Slides are removed from the vessel and briefly dipped
in room temperature dH 20 to remove adventitiously Notes on suppliers
bound cells. The polystyrene slides with the biofilms
can then be cut into sections of about 5 cm and 1. Beckman Instruments Inc., 2500 Harbor Blvd.
mounted on a glass microscope slide. A #1 glass slide Fullerton, CA 92634, USA
is then gently placed over the biofilm populations and 2. Bio-Rad Laboratories, 2000 Alfred Nobel Drive,
the adherent populations can be examined by phase Hercules, CA 94547, USA
contrast microscopy. 3. BioSpec Products, Bartlesville, OK 74005-0722, USA
We have also effectively used fluorescence 4. Brinkmann Instruments Inc., One Cantiague Road,
P.O. Box 1019, Westbury, NY 11590-0207, USA
microscopy and scanning confocal laser microscopy
5. Center for Biofilm Engineering, Montana State
to optically section biofilms that have been stained University, Bozeman, MT 59717, USA
with live:dead stains, fluorescein- or rhodamine- 6. Difco Corporation, P.O. Box 331058, Detroit, MI
labeled antibodies, or by providing 4-methylumbel- 48232, USA
liferyl or other fluorescent substrates for LacZ to 7. Laboratory Product Sales, 1665 Buffalo Road,
cells. A detailed description of the methods for Rochester, NY 14624, USA
SCLM will not be presented because more detailed 8. Nalgene, 75 Panorama Creek Dr., Rochester, NY
articles exist [4, 9]. More importantly, it is best 14625, USA
to obtain assistance from individuals who have 9. New Brunswick Scientific, P.O. Box 4005, 44
expertise in the areas of microscopy for these Talmadge Rd., Edison, NJ, USA
studies. 10. Rainin Instrument Company, Mack Road, Woburn,
MA 01801, USA
11. Sigma Chemical Company P.O. Box 14508, St. Louis,
MO 63178, USA
4. Conclusions 12. Sorvall, Inc., 31 Pecks Lane, Newtown, CT 06470-
2337, USA
Batch cultivation of pure cultures of planktonic ally 13. Swagelok Co., Solon, OH, USA
growing bacteria is the cornerstone of virtually all 14. VWR, P.O. Box 626, 200 Center Sq. Rd., Bridgeport,
of our current understanding of the physiology, bio- NJ 08014, USA
190
Abstract. Streptococcus agalactiae or group B strep- epithelial cell tissues by GBS. By utilizing tech-
tococci (GBS) are gram-positive diplococci and are niques to grow primary cultures of both chorion cells
the leading bacterial cause of pneumoniae, sepsis, and amnion cells isolated from human C-section pla-
and meningitis in neonates. Neonatal GBS infections centas, we have established a relevant model to
may occur prior to or during birth. GBS have been investigate certain aspects of GBS adherence and
cultured from the chorioamnionic membrane of invasion into the placental membrane. To identify
pregnant women and have therefore been associated relevant molecules required for GBS to colonize the
with chorioamnionitis and premature labor. A poten- multiple tissues it encounters during an infection, we
tial route for GBS to establish infection of a neonate have applied a variety of biochemical approaches
would be to penetrate the placental membrane of with host cell membrane preparations as well as
colonized pregnant women. In our laboratory, we purified extracellular matrix proteins. These tech-
have constructed in vitro systems to emulate certain niques are enabling us to further characterize the
events during the colonization and invasion of host pathogenic mechanisms utilized by GBS.
Key words: Adherence, Chorioamnion, Extracellular matrix, Group B streptococci, Invasion fibronectin
2. Luria Bertani (LB) medium (Cat. #L3152, C. Radiolabeling GBS and cellular adherence assays
Sigma Chemical CO.).2 1. Reagents
3. Agar select (Cat. #A5054, Sigma Chemical - Bovine serum albumin (Cat. #A7030,
CO.).2 Sigma Chemical CO.).2
B. Primary cell isolation - PBS containing 5.0% wtlvol bovine serum
1. Reagents albumin (BSAlPBS) bovine serum albumin
- Normal term human placentas of C-section (Cat. #A7030, Sigma Chemical Co.)?
procedures. - S35 Translabel (Cat. #51006, ICNt or 3H
- Phosphate buffered saline (PBS), pH 7.5 leucine (Cat. #TRI51O, Amersham).5
• Sodium phosphate, monobasic, anhy- - RPMI without leucine, methionine, gluta-
drous (Cat. #S8282, Sigma Chemical mine, lysine, or sodium bicarbonate (Cat.
CO.).2 #R7130, Sigma Chemical Co.)?
• Sodium chloride (Cat. #S7653, Sigma - Sodium bicarbonate (Cat. #S5761, Sigma
Chemical CO.).2 Chemical CO.).2
- Penicillin G (Benzylpenicillin, Cat. #P3032, - Lysine (Cat. #L5501,SigmaChemicaICo.).2
Sigma Chemical CO.).2 - Leucine (Cat. #L5652, Sigma Chemical
- Streptomycin sulfate (Cat. #S9137, Sigma CO.).2
Chemical CO.).2 - Methionine (Cat. #M6039, Sigma Chemical
- Vancomycin (Cat. #V1130, Sigma Chemical CO.).2
CO.).2 - Glutamine (Cat. #G7029, Sigma Chemical
- Amphotericin B-solubilized (Cat. #A9528, CO.).2
Sigma Chemical CO.).2 - Sodium hydroxide (Cat. #VW3247-1,
- Dulbecco's modified Eagle's medium VWR-SP).6
nutrient mixture F-12 Ham (DMEI F-12 1:1 2. Miscellaneous supplies
mixture) (Cat. #D6421, Sigma Chemical - 24-well tissue culture plates.
CO.).2 - Beckman Ready Caps (Cat. #534181,
- Fetal bovine serum (Cat. #F-4135, Sigma Beckman Instruments).?
Chemical CO.).2 - Beckman Scintillation Counter (Model
- L-glutamine-penicillin-streptomycin solu- #LS6001C, Beckman Instruments)?
tion (Cat. #G1146, Sigma Chemical 3. Invasion assays
CO.).2 - 24-well tissue culture plates.
- ITS liquid media supplement, 100x (Cat. - Penicillin G.
#13146, Sigma Chemical Co.)? - Gentamicin sulfate (Cat. #G 1264, Sigma
- Trypsin 1:250 (Cat. #T4799, Sigma Chemical CO.).2
Chemical CO.).2 - Trypsin-EDTA solution, Ix.
- Trypsin-EDTA solution, Ix (Cat. #T3924, - Triton X-lOO (Cat. #XlOO, Sigma Chemical
Sigma Chemical CO.).2 CO.).2
- Trypan blue solution (0.4%) (Cat. #T8154, D. Solid phase adherence assays
Sigma Chemical CO.).2 1. Reagents
2. Miscellaneous supplies - Radiolabeled bacteria (see above supplies).
- Sterile surgical gloves. - Bovine serum albumin.
- Forceps. - Non-fat dry milk.
- Scissors. - PBS.
- 50 ml volume disposable conical centrifuge - Sodium hydroxide.
tubes. - Fibronectin (Cat. #F0895, Sigma Chemical
- Plastic 25 ml volume pipettes (Cat. CO.).2
#431032, Fisher Scientific).! - SuperSignal Chemiluminescence Substrate
- Falcon large (150 mm x 15 mm) sterile petri (Cat. #34080, Pierce).8
dishes (Cat. #1058, Fisher Scientific).! - FN-15 monoclonal antibody to human
- Corning 24-well tissue culture plates (Cat. fibronectin (Cat. #F7387, Sigma Chemical
#25820, Fisher Scientific).! CO.).2
- Falcon 75 cm2 flat bottom flasks (Cat. - Anti-mouse immunoglobulin-horseradish
#3013, Fisher Scientific).! peroxidase conjugate (Cat. #A9917, Sigma
- Hemacytometer (Clay Adams, New York). Chemical CO.).2
- 0.22 J.lm pore size 500 ml volume filter unit - Tween-20 (polyoxyethylene monolaurate,
(Cat. #450-0020, Nalgene)? Cat. #P7949, Sigma Chemical Co.)?
- Tissue culture incubator. 2. Miscellaneous supplies
- Jouan GR412 swinging bucket centri- - Beckman Ready Caps® (Cat. #534181,
fuge. Beckman Instruments).?
193
pletely separated so that one cell type will not Cell densities are then determined using a
contaminate the other in subsequent culture. standard hemacytometer and staining the cells
The respective layers are cut into 1 cm x 5 cm with trypan blue. Cells are seeded into 75-cm2
strips using surgical scissors and forceps, and then tissue culture treated flasks or 24-well tissue
placed into separate large sterile petri dishes culture treated plates at a density of 1 x 105
containing 50 ml DME/Ham's F-12 medium with cells/cm2 • Allow 24-48 h incubation time at
0.5% wt/vol trypsin (Sigma 1 :250 trypsin) and 37°C, 5% CO 2 for initial cell adherence and
100 )lm EDTA. Petri dishes are then incubated for growth. An inverted microscope is necessary to
45 min at 37°C, 5% CO 2 in a tissue culture incu- assess cell growth. Cell layers are then rinsed
bator. Amnion membrane strips are then removed gently with PBS using a pipette to remove
with forceps and placed into a petri dish con- residual red blood cells and fresh medium is then
taining fresh DME/Ham's F-12/trypsin solution added. Cell growth medium is subsequently
and incubation continued for an additional 1.5 h. changed every three days. Cell monolayers should
Chorion strips are similarly transferred to fresh reach confluence within about 5 days. The purity
trypsin solution and incubation continued for an of the cell cultures is generally determined by
additional 2.5 h. morphological observation. Chorion cells are
Membrane tissues are then asepticly transferred oblong whereas amnion cells more cuboidal. The
to 50 ml conical centrifuge tubes containing 20 purity of the cell cultures has also been confirmed
ml DME/Ham's F-12 medium with 5% fetal on several experiments by transmission electron
bovine serum (FBS) which has been heat treated microscopy.
for 10 min at 65°C (HI). Total volume should not Growth rate will vary among placental prepa-
exceed approximately 30 ml. Tubes are vortexed rations. Cells are not usually passaged more than
thoroughly to release cell mono layers from the one time. Alternatively, cells grown in 75-cm 2
basement membranes. Chorion cells require flasks may be frozen in liquid nitrogen for later
thorough vortexing (4-6, 30-sec bursts) at the use. Cells are removed from the flask by incu-
highest setting to be released from the extracel- bating in Hank's Balanced Salt's Solution con-
lular matrix (ECM). A major cause of poor cell taining 0.5 gil porcine trypsin and 0.2 gil EDTA
recovery is insufficient vortexing. In contrast, (trypsiniEDTA solution). Cells are transferred to
amnion cells should be released by vortexing for a conical centrifuge tube and pelleted by cen-
3, 20-sec bursts. Amnion cell isolation is other- trifugation at 1500 Xg for 10 min at 6 0c. Pellet
wise identical to that of the chorion cell proce- is suspended in 5 ml of growth medium con-
dure. The ECM proteins will clump together, taining 40% FCS (HI) and 10% DMSO and
while the cells remain in the supernatant. The aliquoted in cryogenic vials. Cells are frozen at
sample is poured into a large petri dish and the -70°C overnight before storing in liquid nitrogen.
ECM clumps removed with forceps. Liquid Frozen cells are rapidly thawed in a 37°C water
containing the cells is transferred to a new 50 ml bath, and then immediately added to fresh chorion
conical tube and centrifuged at 1500 xg for 10 cell growth medium. The isolation of chorion and
min at 6 °C in a Jouan swinging bucket cen- amnion cells was initially based on modifications
trifuge. The supernatant is decanted and both of previously described protocols [3, 4, 6].
amnion and chorion cell pellets are individually C. Radiolabeling of GBS
suspended in fresh cell growth medium. Cell Deficient RPMI 1640 is made by reconstituting
growth medium is DME/Ham's F-12 medium one bottle of medium with 1 liter of distilled water
with 5% vol/vol FBS (HI), penicillin (10 U/ml) , and adding 300 mg of glutamine, 50 mg of
streptomycin (100 )lg/ml), amphotericin B (2.5 leucine, 40 mg of lysine, 2 g of sodium bicar-
)lg/ml), Ix ITS (Sigma mixture of 5 )lg/ml insulin, bonate, and either 50 mg of leucine (for methio-
5 )lg/ml transferrin, and 5 ng/ml selenium), and nine-deficient media) or 15 mg of methionine (for
2 mM L-glutamine. If bacterial contamination leucine-deficient media). The medium is then
becomes a problem, vancomycin (40 )lg/ml) can filter-sterilized using a 0.2 )lm pore size filter
be added as an additional antibiotic as flask. The pH of the RPMI should be adjusted to
Staphylococci are often the culprit. There will be approximately 7.0, as a more alkaline pH may
a significant number of red blood cells in the result in reduced labeling efficiency.
chorion cell pellet, but they do not seem to inter- Four hundred microliters of a stationary-phase
fere with the initial adherence and growth char- bacterial culture is grown to log phase (OD at
acteristics of the chorion cells. Far fewer red 600 nm = 0.5-0.6) in 10 ml ofTHB (streptococci)
blood cells are usually present in the amnion or LB (E. coli) by incubation at 37°C with
preparation. There will be some small clumps of aeration for approximately 2 h. A 1 ml aliquot of
cells following centrifugation which can be dis- bacteria is washed twice by centrifugation in a
persed by drawing the sample up and down in a Hermle Z230M microcentrifuge, and suspended
25 ml pipette. in 2 ml of RPM I 1640 without L-Ieucine or L-
195
methionine, depending on the radiolabel to be are then washed 6x with PBS. It is essential that
used. One to two hundred ~Ci of 3H leucine or the plates are vortexed between each wash to
35S_ Trans label are then added, and GBS are incu- eliminate high background. The PBS is aspirated
bated for 30 to 60 min at 37°C in a shaking at each wash steps. Two hundred microliters of
incubator. Bacteria are pelleted by centrifugation, 2 M NaOH are then added to each well and incu-
suspended in 4 ml of TH or LB broth, and incu- bated for 20 min at 65°C to lyse cells and
bated for 1 h to an aD of 0.5-0.6 at 600 nm. adherent bacteria. Fifty microliters of the lysate
Bacteria are then washed once with PBS and sus- suspension are added to Ready-Caps (any method
pended in 4 ml of TH or LB broth with 30% for scintillation counting may be used but we have
glycerol. Radiolabeled bacterial preparations are found the Ready-Caps to be very convenient) for
assessed for both incorporated activity (cpm) by determination of cell associated cpm of radio-
using Beckman Ready Caps (a solid-phase scin- activity.
tillant) and a Beckman LS6001C Scintillation The number of CFU associated per well is
Counter, and for colony forming units (CFU) per determined by converting the total cell associated
ml by plating dilutions of 10-5, 10-6 and 10-7 onto cpm per well into CFU s as described in the above
TH or LB agar plates. A CFU:cpm ratio is derived radiolabeling bacteria section. This number is an
for each preparation. Radiolabeled bacteria are approximation since it assumes that all of the
then frozen down in 0.5 ml aliquots at -70°C. adherent counts are from viable bacteria. All
Bacterial viability and bacterial adherence are samples are performed in triplicate.
minimally affected by freezing in TH or LB broth E. Invasion assays
with 30% glycerol. Qualitative determination of intracellular bacteria
Care should be taken to avoid inhalation of has been performed by a modification of the
volatile compounds in 35S_ Trans label. All proce- procedure described [9]. Primary cell cultures are
dures involving uncapped 35S_ Trans label samples grown to confluence in 24-well tissue culture
should be performed in a certified fume hood until treated plates as described above. Cell monolayers
unincorporated label has been removed. are washed 3 times in PBS followed by the
D. Bacterial adherence assays addition of 340 ~l cell growth medium per well.
Assessment of bacterial attachment to cell Stationary phase cultures of bacteria are washed
monolayers is performed using a modification one time with sterile PBS and suspended in
of a previously described protocol [11]. Radio- chorion cell growth medium. Bacteria are then
labeled bacteria are used (see above procedure) diluted to 10-2 so that approximately 5 x 105 CFU
to determine the adherent characteristics of in a 60 ~l vol are added per well. Initial contact
GBS. Aliquots of radiolabeled bacteria are of the bacteria with the cell monolayer is aided
thawed by the addition of 1 ml THB and then by centrifugation at 800 Xg for 10 min, at 6 °C
incubated at 37°C for 15 min. Bacteria are in a Jouan swinging bucket centrifuge. Plates are
pelleted by centrifugation in microtubes and then incubated at 37°C, in 5% CO 2 , for 2 h.
washed Ix in PBS. The cell pellets are then Non-adherent, extracellular bacteria are
suspended in PBS containing 1.0% wt/vol bovine removed by washing cell monolayers four times
serum albumin (BSAIPBS) and placed on ice for with sterile PBS. The plates are gently swirled
2 h. between washes and the PBS is then removed by
Chorion or amnion cell monolayers are grown aspiration. Five hundred ~l of cell growth medium
to confluence in 24-well tissue culture plates (as containing 10 ~g/ml penicillin and 100 ~g/ml
described above) and washed 3x with PBS. The gentamicin are added to each well to kill adherent
PBS is removed by aspiration. Five hundred extracellular bacteria. Plates are again incubated
microliters of BSA/PBS is added per well and at 37°C, in 5% CO 2 , for 2 h. Following the
plates are incubated at 4 °C for 2 h. Incubation incubation period, an aliquot of the medium can
of both the bacteria and tissue culture wells with be spread on TH agar plates to verify that the
1.0% BSA is necessary to reduce non-specific extracellular bacteria have been eliminated.
interactions of bacteria with both the cell mono- Growth medium is aspirated off the cell mono-
layer as well as the plastic plate. The BSAlPBS layer and the cells are subsequently washed five
is aspirated off of cell monolayers and dilutions times with sterile PBS as described above.
of bacteria in 200 ~l volumes of BSA/PBS are Two hundred microliters of trypsin/EDTA
added. Bacteria are allowed to settle onto the solution are added per well to release the adherent
monolayer for 6 h at 4°C or alternatively, bacteria cell layer. The plates are incubated at 37°C for
can be brought into contact with the cell surface 30 min or until the cell layers are no longer
by centrifugation at 800 xg prior to a 2 h incu- visibly adherent (an inverted microscope may aid
bation at 4°C. Incubation at 4 °C will inhibit in assessing the degree of cells still attached). This
potential bacterial invasion into the cells and step is followed by the addition of 800 ~l of
prevent bacterial growth during the assay. Plates 0.025% vol/vol Triton X-I00 to lyse the eukary-
196
otic cell membranes. Lysis may take up to 30 min G. Solid-phase ECM binding assays
at 37 DC. Samples are then drawn up and down Fibronectin or other ECM proteins are diluted
in a 1 ml pipette to break up small clumps of with PBS for a range of concentrations in 50 III
cellular debris, added to individual microfuge volumes and incubated in round bottom microtiter
tubes, and vortexed thoroughly for at least 12 sec wells at 4 DC for 18 h to allow proteins to adhere.
each. Fifty microliter aliquots of the lysis mixture Wells are then incubated for 2 h with 5%
are plated on TH or LB agar plates to determine BSA/PBS to reduce non-specific binding of
the number of intracellular CFU. Agar plates are bacteria. Fifty microliter aliquots of radiolabeled
incubated at 37 DC overnight and individual GBS (1-3 x 10-6 CFU well-I) are added. The plate
colonies counted. is centrifuged at 800 xg for 10 min at 4 DC and
The CFU count is multiplied by 20 to deter- incubated for 1 h at 4 DC. Supernatant is then
mine the total number of intracellular bacteria per aspirated from the wells. Wells are washed three
well. This number is divided by the number of times with 5% non-fat dry milk in PBS, and then
CFU initially added to the cell monolayer and three times with PBS. Following each addition of
multiplied by 100 to determine the percent wash buffer, GBS bound by low avidity interac-
invasion of the inoculum. All samples are per- tions are removed from the plate by shaking the
formed in triplicate and several dilutions of each plate on a Vortex Genie 2 (VWR Scientific) at
inoculum can be used to determine at what con- level 2 for 10 sec. To determine the extent of high
centration of bacteria saturates the system. avidity bacterial binding, 50 III of 2 N sodium
F. Detection of liquid-phase fibronectin binding to hydroxide is added to each well and the plate is
GBS by immunoblot analysis incubated at 65 DC for 1 h.
One ml of a log-phase culture (generated as The sodium hydroxide lysates are then dis-
described above) is pelleted by centrifugation in pensed onto solid scintillant (Ready Caps), dried
a microcentrifuge, washed twice with 1 ml of at RT, and well-associated cpm are quantified with
PBS, and suspended in 1 ml of 5% BSAlPBS. Ten a scintillation counter. Initial input cpm are also
micrograms of fibronectin is added to the tube quantified. Background counts from scintillant
containing bacteria and the mixture is incubated without lysate added are also quantified. All
on an Orbitron rotating platform for 1 h at 4 DC. experiments are performed in duplicate. The
Bacteria are pelleted, washed twice with 1 ml of percent adherence is calculated as follows: (cpm
5% BSA/PBS, and three times with 1 ml of PBS. bound - cpm background)/cpm added x 100. The
Bacterial pellets are then boiled for 5 min with error bars shown are 95% confidence intervals for
100 III of SDS-PAGE sample buffer. Bacteria are the mean; 95% confidence interval = (standard
pelleted, and proteins contained in the super- deviation/square root (N») x 1.962, where N is the
natants are separated by electrophoresis on a 5% number of replicates.
SDS-polyacrylamide gel using a BioRad Mini-
Protean II electrophoresis cell following the
manufacturer's instructions. One hundred nano- GBS adherence to eukaryotic cell membrane
grams of fibronectin is loaded onto the gel as a proteins
positive control.
Proteins are then transferred to a PVDF Isolation of A549 membranes
membrane (Immobilon-P) using a Bio-Rad ATCC A549 cells are grown in one to three 150 mm2
Laboratories Mini Trans-Blot Cell and the tissue culture flasks using RPMI -1640 supplemented
protocol established by the manufacturer. Non- with 10% fetal bovine serum and 2 mM L-glutamine
specific binding to the membrane is prevented by as growth medium. Generally, cells will divide to
soaking the membrane in PBS containing 5% non- form confluent monolayers in 3-5 days. Adherent
fat dry milk and 0.1 % Tween-20 (Blotto). The cells are removed from the flask by adding 10 ml of
membrane is then reacted with anti-fibronectin 1 mM EDTA in PBS and incubating the flask for
FN-15 ascites diluted 1:1000 in Blotto and 15 min at 37 DC. The flask is then struck vigorously
washed three times for 1 min in Blotto. The onto a countertop to loosen the cells. If cells remain
membrane is then incubated with rabbit-anti- adherent, they are incubated for additional 5 min
mouse IgG-horseradish peroxidase conjugate intervals and checked for up to 45 min until cells can
diluted 1: 1000 in Blotto for 1 h. The blot is then be loosened. EDTA treatment may dissociate an
washed once for 20 min, twice for 5 min with indeterminate proportion of fibronectin and other
Blotto, and three times for 5 min with PBS con- ECM proteins from cell surface receptors during this
taining 0.1 % Tween-20. Fibronectin reacting cell removal process. Cells are then removed with a
bands were visualized with an enhanced chemi- Pasteur pipette and pelleted by centrifugation at
luminescence (ECL) system and exposed to XAR- 200 xg for 10 min at 4 DC. The supernatant is aspi-
2 film for 0.2 sec to 1 min. rated and the cells are suspended in 10 ml of ice-cold
lysis buffer (10 mM HEPES, pH 7.4, 50 mM
197
mannitol). For the remainder of the procedure all the membrane during later steps. The side of the
solutions are kept on ice to prevent proteolytic degra- membrane which faced the gel will be referred to as
dation of membrane proteins. the 'top-side'.
Cells are homogenized in a Dounce homogenizer The membrane is placed into the bag containing
and with 30 strokes of the plunger. The proportion of 5% BSAIPBS with forceps. Care should be taken to
disrupted cells is assessed by trypan blue staining. avoid allowing any portion of the membrane except
Fifty microliters of the cell suspension is mixed with the edges to touch the forceps, and to avoid touching
50 /-ll of trypan blue solution, and 10 /-ll of this the membrane with gloves or fingers, as this may
mixture is placed in a hemacytometer. Intact cells result in excessive non-specific binding. The bag is
(which exclude trypan blue) and disrupted cells are then sealed and incubated for 2-24 h at 4 dc. Longer
counted separately. If the proportion of lysed cells or shorter incubation times may result in excessive
is less than 90%, the homogenization procedure is background or decreased specific binding, respec-
repeated until greater than 90% lysis is achieved. tively.
Forty microliters of 0.5 M Phenyl-methyl-sulfonyl An aliquot of 35S-labeled GBS is thawed (see
fluoride (PMSF) in DMSO is then added (for a final radiolabeling of bacteria) and bacteria are pelleted by
concentration of 2 mM PMSF) to inhibit serine pro- centrifugation in a microcentrifuge for 1 min. The
teases. GBS are suspended in 1 ml THB and incubated for
The homogenate is centrifuged at 200 Xg for 10 30 min at 37°C. Bacteria are again pelleted by
min at 4 °C to pellet the nuclei and unlysed cells. The centrifugation, washed with 1 ml of 5% BSAIPBS,
supernatant, which contains the majority of mem- and then suspended in 1 ml of 5% BSA/PBS.
branes is removed. Some membrane material will Bacteria are then incubated for 2-4 h on ice. Shorter
remain in the pellet. The pellet fraction is suspended incubation times result in increased background
in 10 ml of lysis buffer, and the centrifugation binding.
step repeated. The supernatant is again saved and The following steps should be carried out on a
combined with the initial supernatant. The pellet is bench dedicated to radioactive materials, which has
then discarded. been covered with at least two layers of absorbent
To separate the membranes from soluble cellular bench-top paper with a water-tight backing. A corner
components, the mixture is centrifuged in a swinging of the sealed bag containing the blotting membrane
bucket rotor (SW40) at 70,000 Xg for 1 h. The super- is cut open, and the radiolabeled GBS are added to
natant is discarded and the pellet, containing the bag. The bag is then sealed with care taken to
membrane material, is suspended in 0.5 ml of lysis insure that no bubbles remain inside the bag. The
buffer. The membranes are stored in aliquots of edges of the bag should then all be heat-sealed
25-100 /-ll at -70°C, and are stable for at least adjacent to the membrane. This insures that all
2 years. Repeated freeze-thaw cycles can result in bacteria will be brought into contact with the
degradation of membrane proteins and should be membrane, and that leakage of the bag during sub-
avoided. sequent centrifugation will be avoided. The bag is
then placed in the lid of a 96-well microtiter plate
Bacterial blots which has been lined with 3 mm Whatman filter
Purified extracellular matrix proteins and eukaryotic paper. The membrane must be placed with the top-
cell membrane proteins are separated by elec- side up. If the membrane is placed top-side down,
trophoresis in a 10% SDS-polyacrylamide gel as no signal will be seen as minimal protein passes com-
described by Laemmeli. A Mini-Protean II pletely through the membrane during electrotransfer
Electrophoresis System from Bio-Rad Laboratories of proteins. The GBS are brought into contact with
and protocols provided by the manufacturer are used the membrane by centrifugation at 800 xg for 10 min
in this experiment. Proteins are then transferred to a at 4 dc. The blotting membrane is then incubated for
PVDF membrane (lmmobilon-P), as described by 1 hat 4°C.
Towbin, using a Bio-Rad Laboratories Mini Trans- One corner of the sealed bag is carefully opened
Blot Cell following manufacturer's protocols. and the liquid decanted into a radioactive waste
Ten milliliters of 5% BSA/PBS are placed in a container. The bag is then placed on several layers of
plastic heat sealable bag, which should then be paper towel and cut on all four edges with a razor
rotated to coat the entire interior surface of the bag. blade. The upper portion of the bag is then removed.
Allowing the PVDF membrane to touch an uncoated Fifty milliliters of ice-cold 5% non-fat dry milk in
portion of the bag may result in excessive back- PBS is placed in a container large enough to hold the
ground binding of GBS to the membrane. After blot. The membrane is then placed in the container
electro transfer of proteins is completed, the portion top-side up. The container is placed on ice and gently
of the membrane containing the standards should be shaken on an orbital platform at approximately 60
cut off, rinsed briefly with water, and saved. The rpm for 5 min. The blot should move freely in the
lower right hand corner of the membrane should then container. Placing the blot upside-down during
be cut off with a razor blade to allow orientation of washing or allowing the blot to stick to the container
198
during washing may result in higher backgrounds or cells grown in vitro (data not shown). Allowing
lower signals. The washing step is repeated twice bacteria to settle onto the cell monolayer as opposed
with 5% non-fat dry milk in PBS, and then three to centrifugation of the bacteria gives consistently
times with ice-cold PBS alone. Initial wash solutions 2-fold less adherent bacteria over the range of inocu-
should be discarded as radioactive waste. lums. Background radioactivity is generally at or near
A sheet of Glad-Wrap is placed on the bench top. undetectable levels if the wash steps are followed as
The blotting membrane is then placed top-side down described in Procedures.
on the Glad-Wrap and the edges folded. 35S is a low- Subsequent to bacterial attachment, invasion into
energy beta-emitter and therefore signals cannot be the intracellular environment of chorion cells could
detected through the blotting membrane or through allow the bacteria to penetrate the first protective
multiple layers of Glad-Wrap. Care should therefore layer of the chorioamnion and enable GBS to
be taken to insure that the top-side of the membrane establish an infection at a deeper site. Following GBS
faces the X-ray film, and that only a single layer of inoculation onto chorion cell monolayers, viable GBS
Glad-Wrap is between the membrane and the film. are recovered from the intracellular environment,
The membrane should not be placed directly on the indicating that these bacteria have the ability to enter
film as it may adhere tightly to the film, making film chorion cells (Figure 2). Up to 6.0% of the GBS
development impossible. Thicker food-wraps, such inoculum can be recovered. The degree of invasion
as Saran Wrap, partially block the signal and result is variable from strain to strain, but is a shared GBS
in slightly longer exposure times. The film should be phenotype. Interestingly, although E. coli DB5a are
developed after exposure times of 1-24 h, depending adherent in this system (see Figure 1), they are
on the strength of the signal. relatively non-invasive, suggesting that there may be
specific factors expressed by GBS for adherence and
invasion which enable the bacterium to enter the
Results and discussion intracellular chorion cell environment.
We routinely see a minimum of one to four percent
GBS adherence and invasion of chorion cells GBS invasion using the inoculums indicated in
Colonization of host tissues is the initial phase of Figure 2. The numbers of intracellular CFU recov-
bacterial and host interaction. GBS have been ered among triplicate samples are very similar (see
cultured from infected placental membranes in vivo error bars in Figure 2); however, results do vary
[1] and GBS have been shown to adhere to intact among experiments. Additionally, certain batches of
chorioamniotic membranes in vitro by electron chorion cells are more susceptible to bacterial
microscopy [5]. The adherence assay performed invasion than are other preparations. Therefore, E.
here demonstrates that primary human chorion cells 5.000
grown in vitro also provide an analogous colonizable
surface as shown in Figure 1. The two clinical
4.000
isolates of GBS tested, strain A909 and strain COB1,
bound to the chorion cell monolayer in a saturable
manner. Similar results were observed with amnion c: 3.000
0
2.500E-o<i '00
<0
>
c: 2.000
2.000E-o<i
-;;R,
0
'0
c: 1.500E-o<i 1.000
:>
til
:::> 1.000E+6 A909 Bound
LL
o 0.000
COH-l Bou nd U')
±
~
5.000E+5 I
DH5 Bound o o
o
OOOOE~+----,----.----,----,
'+ '"+ Strain
§
w
§
ui
Figure 2. GBS invasion into primary human chorion cells.
CFU Offered GBS strain A909 and strain CORl, and E coli DR5-alpha
were inoculated onto confluent chorion cell monolayers
Figure 1. GBS and E. coli adherence to primary human at 5.0 x 105, 4.1 X 105 , 5.8 X 105 CFU per well, respec-
chorion cells. Primary chorion cells were isolated and tively. E coli DR5-alpha is a non-invasive bacterium in
grown to confluence in 24-well tissue culture plates. this system. The percent invasion is defined as the number
Dilutions of radiolabeled bacteria were added to mono- of CFU recovered from cellular lysates divided by the
layers and assessed for adherence as described in initial extracellular inoculum multiplied by 100. Error bars
Procedures. Error bars indicate standard deviations. N =3. indicate standard deviations. N = 3.
199
coli DH5a should be included as a non-invasive above background wells that were coated with BSA
control in every experiment. Streptococcus gordonii, alone.
an oral streptococci, has also been used reproducibly GBS binding is very reproducible from well to
as a gram-positive non-invasive control (data not well (see error bars, Figure 3). Furthermore, binding
shown). from experiment to experiment also shows minimal
Similarly, adherence assay results will vary variation if the same strain of GBS and the same
somewhat from experiment to experiment and among manufacturer's lot of fibronectin is used. However,
various placenta cell preparations; however, relative adherence can vary greatly depending on both the
binding trends among both GBS strains and other manufacturer and the lot of fibronectin used. GBS
bacterial species are qualitatively consistent. adherence to fibronectin from Sigma Chemical Co.
GBS have previously been shown to invade has ranged from 5% to 45%. Fibronectin from
lung epithelial carcinoma cells [9], and have been GIBCO/BRL gave approximately 30% adherence,
observed in lung tissue sections of infected Macaca while fibronectin obtained from Calbiochem resulted
nememstrina primates [8]. This intracellular invasion in no GBS adherence « 1%). Background levels can
has been postulated to enable GBS to reach deeper also vary greatly depending on a number of factors,
sites which could result in bacteremia. The chorion including the manufacturer of BSA and of microtiter
and amnion cells form the outermost and innermost plates. Pilot experiments should be performed to
protective barriers, respectively, of a neonate from identify optimal reagents. Non-specific binding of
bacterial infection. It is possible that the invasive GBS to microtiter wells should be less than 0.2%. It
interaction described here may enable GBS to reach is important that blocking of both plates and bacteria
deeper sites of the human placental membrane with 5% BSA be performed for at least 2 h prior to
leading to premature rupture of the membrane or addition of the bacteria to the micro titer wells.
infection of the amnionic fluid. The results of a typical experiment assaying GBS
binding of soluble fibronectin using strain COHI is
Adherence of GBS to fibronectin in solid phase and shown in Figure 4. Group A streptococcal (GAS)
in solution strain STRA-B498 was included as a positive control.
Bacterial pathogens often bind to extracellular GAS bound to material which reacted with the anti-
matrix proteins in order to colonize a host or to coat fibronectin FN-15 ascites and was of the predicted
itself with a host molecule to evade host immune molecular weight of fibronectin (-220 kDa). No
surveillance. We examined the ability of GBS to reactive protein was eluted from GBS after incuba-
bind to purified ECM proteins using the protocols tion with fibronectin, suggesting that GBS do not
described above and determined that GBS do bind adhere to fibronectin in solution. These results have
to immobilized fibronectin [10]. GBS adherence to been very reproducible. For complete elution of
laminin, tenascin, vitronectin, or fibrinogen was bound proteins, the bacterial samples should be
not observed. The results of a typical fibronectin boiled for at least 5 min in SDS-PAGE sample buffer.
binding experiment are shown in Figure 3. The
degree of binding is concentration-dependent, as Ligand blot analysis of GBS adherence to
expected. Significant adherence is seen even at very fibronectin and to A549 cell membrane proteins
low concentrations. As little as 100 ng of fibronectin Our laboratory has previously demonstrated that GBS
added per well shows GBS adherence significantly adhere to cultured epithelial cells. In lane A of the
ligand blot shown in Figure 5 we show that radio-
50 labeled GBS adhere to two specific A549 cell
membrane proteins. GBS adherence to fibronectin
~
c: 40 was included as a positive control. Non-reducing
~ sample buffer (without 2-mercaptol-ethanol) was
~ 30
"0 used in this experiment. GBS predominately bound
« 20 two proteins bands in lane B containing fibronectin,
?f. which correspond to variants of fibronectin which are
10
migrating at a slightly higher predicted molecular
,(
I I I I I 1111 I I I 111111 weight than that of full length fibronectin. No binding
o 10 100 is observed when reducing sample buffer is used
(data not shown).
Fibronectin Coating Concentration (J1g/ml)
Binding to specific ECM proteins is very repro-
ducible from blot-to-blot when using the same lots
Figure 3. Adherence of radio labeled GBS strain CORI
to immobilized fibronectin. Polystyrene microtiter plates of membranes and the same GBS strain. However,
were coated with varying concentrations of fibronectin and significant variability in binding can be seen among
incubated with log-phase 3R-Iabeled strain CORI. Results membrane preparations and among different GBS
are percentages of input adherent inocula. Error bars rep- strains. It should also be noted that these membrane
resent 95% confidence intervals for the mean. protein labeling experiments can and have been
200
kDa
A B c A B
kDa
200- 200 -
110 -
74 -
45 -
Cindy L. Munro
Virginia Commonwealth University, School of Nursing, Richmond, Virginia, USA
Abstract. The rat model of endocarditis is a well rat model of endocarditis, damage to the aortic valve
established experimental protocol which closely and sterile vegetation formation is accomplished by
approximates human native valve endocarditis. The insertion of a polyethylene catheter through the
rat model of endocarditis has been used to examine carotid artery into the left ventricle. Following
the role of particular streptococcal virulence factors, catheter insertion, an inoculum of streptococci are
to assess immunoprotective strategies, and to injected intravenously. Vegetations removed from the
evaluate the efficacy of selected antibiotic treatment heart valves during thoracotomy of euthanized
regimens for streptococcal endocarditis. Like animals are qualitatively cultured for streptococcal
humans, rats are generally susceptible to endocarditis infection. The method, including investigator safety
only if the cardiac valves have been damaged. In the considerations, is described in detail.
1. Introduction 2. Materials
The viridans streptococci are part of the normal oral A. Anesthesia and procedure
flora of humans, but can cause serious infections - Animal scale
when introduced into the bloodstream. One of the - Ketamine hydrochloride, provided through
most serious infections caused by the viridans institutional animal resources department.
streptococci is native valve endocarditis. The rat - Methoxyflurane, provided through institutional
model of endocarditis is a well established experi- animal resources department.
mental protocol which closely approximates the - Conical centrifuge tube, 50 ml, disposable,
infection in humans [1, 4]. Fisherbrand #05-539-6, Fisher.!
Roughening of the heart valve, such as is caused - Syringes, B-D tuberculin, 1 ml with 25 G
by the presence of a catheter in the heart, facilitates !/2 inch needles, #BD309626, VWR?
the establishment of infection. Like humans, rats are - Rat restrainer, Model 700R, Roboz Surgical
generally susceptible to endocarditis only if the Instrument Company, Inc. 3
cardiac valves have been damaged. In the rat model B. Procedure
of endocarditis, a closed catheter tube (made of - Electric clippers with #40 blade, such as Oster
polyethylene tubing sealed with silicone) is inserted animal clippers, #lO749-020, VWR,z
through the carotid artery into the left ventricle, - Delta phase ™ Operating Board, Model 390P,
where it remains for the duration of the experiment Braintree Scientific. 4
and induces damage to the aortic valve and sterile Deltaphase isothermal pads, warmed (37°C) ,
vegetation formation. Aseptic technique and general Model 39DP, Braintree Scientific. 4
anesthesia are used for the procedure of catheter - Instrument soak, such as Lysol IC-instrument
insertion. Following catheter insertion, an inoculum disinfectant presoak solution, #C6320-16,
of streptococci are injected intravenously. Vegeta- VWR.2
tions removed from the heart valves during thoraco- - Ethanol for use as skin cleansing solution.
tomy of euthanized animals are qualitatively cultured - Scalpel, sterile disposable with #10 surgical
for streptococcal infection. blade, Fisherbrand #08-927-5A, Fisher.!
The rat model of endocarditis was first described - 2 curved toothed forceps, such as Micro
by Santoro and Levison [11]. The model has been Dissecting Forceps, 4 inch, I X 2 teeth, #RS-
used to examine the role of particular streptococcal 5153, Roboz Surgical Instrument Company,
virulence factors [3, lO, 17], to assess immunopro- Inc?
tective strategies [16], and to evaluate the efficacy of 2 curved serrated forceps, such as Micro
selected antibiotic treatment regimens for strepto- Dissecting Forceps, 4 inch, #RS-4136, Roboz
coccal endocarditis. Surgical Instrument Company, Inc. 3
- Iris scissors, such as Vannas Ultra Micro
204
Scissors, very delicate, 3 inch, curved, #RS- grams, catheters should be 5 cm long. To prepare
5611, Roboz Surgical Instrument Company, catheters, cut PElO Clay Adams polyethylene
Inc. 3 tubing to 5 cm lengths and melt one end by
- Baby Diffenbach serrafine clamps, 11/8 inch, pressing the tip with a pair of hemostatic forceps
curved, very delicate, #RS-742l, Roboz which have been flamed. Verify that the end of
Surgical Instrument Company, Inc. 3 the catheter is sealed by placing the open end in
- Suture, 3-0 silk, cut in 7 mm lengths, Braintree instrument disinfectant solution; if the solution
Scientific. 4 rises in the catheter by capillary action, the seal
- Catheters,5 cm, cut from PElO Clay Adams is inadequate. Cut suture material to 7 cm lengths.
polyethylene tubing, #63019-003, VWR,z Soak all instruments, catheters, and suture in
- Sterile 4 x 4 gauzes, such as Johnson and instrument disinfectant for the time period rec-
Johnson #B3063-9, VWR,z ommended by the manufacturer.
- Michel skin clips, 7 mm, #RS-9270, Roboz On the evening before catheterization, remove
Surgical Instrument Company, Inc. 3 food from cages. On the morning of the proce-
- Michel skin clip applying forceps, #RS-9292, dure, water should be withheld. Each animal
Roboz Surgical Instrument Company, Inc. 3 should be weighed to determined correct anes-
- Surgical gloves. thesia dose. Calculate the dosage for induction of
- Sterile PBS. anesthesia with ketamine HCI (100 milligrams
- Bench paper for surgical field. ketamine/kg weight). Pick the rat up by the base
C. Necropsy supplies of his tail and place his front feet on the edge of
- Ethanol. the rat restraint; the animal should enter the
- Sterile 4 x 4 gauze. restraint willingly. Secure the restraint. Following
- Tissue grinders, sterile disposable, one per cleansing of the animal's abdomen with an
animal, # 3505, Sage Products.s alcohol swab, inject correct dose of ketamine
- Sterile PBS. intraperitoneally using a 1 cc syringe with 1/2 inch
- Syringe, B-D tuberculin, 1 ml with 27 gauge 25 gauge needle. Return the animal to his cage,
2 inch needle, #BD309623, VWR,z with supervision, until the anesthetic takes effect.
- Mayo scissors, 6 3/ 4 inch, straight, #RS-6872, When the animal has become very groggy, shave
Roboz Surgical Instrument Company, Inc. 3 the neck area from chin to just below the sternum
- Operating scissors, 4% inch, curved, sharp, using an electric clipper with a close blade (for
#RS 6703, Roboz Surgical Instrument example, a #40 blade).
Company, Inc. 3 Monitor anesthesia level throughout the pro-
- Hemostatic forceps, such as Rankin-Kelly cedure by checking the pedal reflex; anesthesia
forceps, 6 1/4 inch, straight, #RS-7140, Roboz is adequate when the animal does not respond to
Surgical Instrument Company, Inc.3 brisk foot rubbing. If a deeper level of anesthesia
- Toothed forceps (see 2b above). is required, supplement with whiffs of methoxy-
- Serrated forceps (see 2b above). flurane. Deliver the methoxyflurane by placing a
- Surgical gloves. few drops of methoxyflurane on a gauze pad
- Bench paper for necropsy field. placed inside a 50 ml conical tube; the conical
tube will fit snugly over the rat's nose. Control
administration of inhalation anesthetic by
3. Procedures replacing or removing the conical tube containing
methoxyflurane from the animal's nose. Always
A. Preoperative Preparation and Anesthesia prepare and administer methoxyflurane under a
Animal investigations must comply with all hood. Anesthesia should not be so deep as to
applicable legal and institutional requirements. diminish respiratory effort. If respiratory diffi-
All animal experiments must be reviewed and culties occur or if respiratory effort diminishes,
approved by the institutional animal use com- discontinue methoxyflurane immediately; blow
mittee prior to initiation of the work. The insti- gently on the rat's nose to encourage respiratory
tutional committee and animal resource personnel effort and dissipate methoxyflurane. If respiratory
can be valuable sources of information and assis- arrest occurs, attempt resuscitation by holding the
tance to the investigator. rat by the thorax and shaking gently. If gentle
Order male Sprague-Dawley rats, weighing shaking does not elicit respiratory effort, pick the
225-250 grams, so that they will arrive at least rat up and drop him onto the operating board from
one week prior to the procedure; this enables a height of about 3-4 inches.
observation of the animals for pre-existing illness B. Surgical procedure
and minimizes pre-procedure stress. Use sterile technique throughout the surgery;
Prepare catheters and assemble supplies prior instruments should be thoroughly cleaned and
to the procedure. For rats weighing 225-250 disinfected before use on another animal. Wear
205
gloves throughout the procedure. When the rat is tional suture at the caudal end of the artery.
well under the influence of anesthesia, lay him on Remove toothed forceps supporting the artery,
his back on an operating board (with thermal pad allowing it to return to the neck cavity, and check
in place in board and clean bench paper covering) again for hemostasis. Clip excess suture ends.
with the animal's head toward you. Cleanse the Tuck the loose end of catheter under skin of the
neck area with 70% isopropanol applied to a neck. Close the skin with skin clips.
4 x 4 gauze pad. Using a sterile disposable scalpel C. Postprocedure
with a #10 blade, make an incision vertically Place the rat in his cage on a warmed isothermal
through only the skin layer of the neck. Begin just pad; the presence of the pad during surgery and
above the level of the sternum and end below the recovery counteracts anesthesia induced hypo-
rat's chin. The incision should be 1/2 to 1 inch in thermia. Be certain water is available to replace
length. Gently pull the fascia of the neck apart. volume lost during surgery and to prevent dehy-
The carotid artery lies underneath and lateral to dration. Food may be replaced at this time. Keep
the sternohyoideus muscle and medial to the handling to a minimum to avoid distress to the
rectus capitus muscle. Locate the right carotid animal. The animal must not be left unattended
artery. Gently dissect tissues away from the artery until recovered from the anesthesia. Check the rat
with 2 pairs of curved toothed forceps. Slide one frequently during recovery from anesthesia;
pair of curved toothed forceps under the artery. observe for return of the righting reflex, intake
Gently pull the artery up out of neck cavity, of oral fluid, and movement about cage. Observe
stretching gently to free it from associated tissues. for hematoma or bleeding at site. Assess post
Throughout the remainder of the procedure, keep surgical animals at least daily. Observations
the artery elevated out of the neck cavity by sup- should include the skin wound, activity level,
porting it on the open blades of one pair of intake of food and water, and behavior. Deaths
forceps; the pressure provided by the blades of the beyond the immediate postoperative period may
forceps will keep the artery occluded and prevent be related to ventricular arrhythmias secondary
unnecessary bleeding. to catheter irritation of the ventricular muscle, or
Tie off the right carotid artery at the exposed to cerebrovascular accidents from emboli origi-
cephalic end with a 7 cm length of 3-0 silk suture. nating from the sterile vegetations on the aortic
Place two 7 cm long pieces of suture under the valve.
artery at the exposed caudal end. Make a small D. Inoculation
slit (less than one third the radius of the vessel) Inoculation can be performed between I and 5
in the top half of the artery with iris scissors. This days post operatively, but consistency within
slit will provide an entry hole for insertion of the experiments is critical. Prepare an inoculum of
catheter. A correct cut will form small drop of organisms at the desired cell count in 0.5 ml
blood on the top of the slit. Be careful not to cut volume; draw the inoculum into a I cc syringe
too deeply, or the artery will tear and the animal with 27 gauge 1/2 inch needle. Pick the rat up by
will exsanguinate. If the artery becomes dry the base of his tail and place his front feet on the
during manipulations, rewet with sterile saline edge of the rat restraint. The animal should enter
using a sterile transfer pipette. Grasp a catheter the restraint willingly. Secure the restraint.
with serrated forceps in your dominant hand and Cleanse the tail with 70% isopropanol; if needed,
thread the catheter (unsealed end first) through the place the tail between warmed (37°C) isothermal
slit in the artery, steadying the artery with a pads to dilate the tail veins. Steady the tail with
second pair of curved forceps in your nondomi- your nondominant hand, being careful not to
nant hand. Push down on the toothed forceps sup- induce a sharp bend in the tail. Locate the lateral
porting the artery to open the vessel. Advance the tail vein, just below the skin surface. Inject the
catheter toward the heart until resistance is met. inoculum intravenously. Proper injection will not
The artery will bleed while the catheter is being raise a bleb and minimal resistance to fluid injec-
advanced; work quickly to minimize blood loss. tion will be encountered. Hold pressure at the site
When the catheter is in place (as determined by until hemostasis occurs before returning the rat to
advancement of a majority of its length, resistance his cage.
to further advancement, and pulsation of the E. Necropsy
catheter in synchrony with the heartbeat), place a Sacrifice rats by carbon dioxide inhalation. Place
baby Diffenbach serrafine clamp on the artery the rat on his back with feet toward you. Wipe
above the two lengths of sutures placed at the down the chest with 70% ethanol. Make a V-
caudal end of the artery. Tie each of the two loose shaped incision in the chest below sternum.
sutures around the caudal end of artery, securing Undermine skin from the chest muscle using
the catheter in place and preventing further blood Mayo scissors. Extend the skin incision to the
loss from the artery. Remove the clamp and check level of the neck on each side with Mayo scissors.
for hemostasis. If bleeding occurs, tie an addi- Make a horizontal incision below the sternum
206
through the peritoneum. Clip the diaphragm away. Table 1. Endocarditis cases in rats inoculated with wild
Cut the cartilage of ribs on each side of sternum, type and mutant strains of S. mutans
from base to apex. With a pair of hemostatic
forceps, pull the flap of sternum back to expose Log \0 Wild type V403 Mutant Vl996
the heart. Position the hemostatic forceps above inoculum
Casesa Totalb Cases Total
the animal's head so that the weight of the forceps
attached to the tip of the sternum keeps the chest 7 11 19 4 24
cavity exposed. Expose the vena cava by lifting 6 4 6 0 6
the heart gently with a pair of toothed forceps. If 5 4 8 0 3
desired, take a blood sample from the inferior 4 0 3 0 2
vena cava using a 1 cc syringe with 27 gauge
1/2 inch needle. Pull the heart up gently (to avoid a Number of animals with streptococci recovered from
displacement of the catheter). Using curved vegetations.
b Surgical survivors with correct catheter placement at
scissors, clip through the heart tissue close to the
aorta and dissect up to free the heart. Check necropsy.
visually for catheter placement. Cut off the apex
of the heart. Locate the right ventricle. The right
and left ventricles can be distinguished by wall to use the minimum number of animals necessary to
thickness, with the right ventricle having a thinner generate statistically valid results. Several in vitro
wall. Place one blade of the curved scissors into methods to examine particular aspects of strepto-
the left ventricle through the cut at the apex, coccal virulence relevant to endocarditis have been
exiting through the aorta. Make a lateral cut described [5-7, 10, 13-15, 17, 19]. These in vitro
through the left ventricular inner wall, the septum, methods include assays to measure binding to com-
and the right ventricle to expose the aortic valve. ponents commonly found in sterile vegetations,
Place the open heart on a sterile 4 x 4 gauze and including fibrin and platelets [8-10, 13, 18], or to
examine valve leaflets for vegetation. Check assess avoidance of host immunodefenses [10, 19].
catheter placement over the valve. If desired, However, the interaction of microorganisms with
place the catheter tip in appropriate medium for living animals is so complex that it is impossible to
qualitative culture. Remove vegetations from the adequately model the entire system in vitro, and it is
valve with curved scissors and serrated forceps. often essential to study the streptococci in live ver-
Place the vegetations in a sterile disposable tebrate animals in order to understand the importance
homogenizer tube with 2 ml sterile saline. of particular virulence factors in human infection.
Homogenize by rotating pestle in homogenizer Rats are a preferred species for the endocarditis
tube. The sample is now ready for quantitative model because of similarities between human and rat
culture on appropriate media. Identity of organ- endocardial infections, small size, surgical hardiness,
isms recovered should be determined, and should and relatively low purchase and housing costs.
be the same as the inoculating strain. The combination of injected ketamine for induc-
tion of anesthesia and inhalation of methoxyflurane
for anesthesia maintenance is appropriate for survival
4. Results and discussion surgery in rodents when the procedure is of short
duration. The effects of ketamine last 30-40 minutes,
Representative results which can be obtained using while the procedure generally takes less than 20
the rat model of endocarditis are presented in minutes; methoxyflurane has potent analgesic effects
Table 1. In the example provided (which is taken which extend into the immediate post operative
from previously published data [10D , virulence of a period. However, attention to investigator safety is
wild type organism, S. mutans V403, is compared to an important aspect in the use of inhalation anes-
virulence of an allelic exchange mutant defective in thetics. Methoxyflurane has been associated with
exopolysaccharide synthesis (VI996). Infectivity is hepatic and renal toxicity in humans [12], and the
strikingly different between wild type and mutant National Institute for Occupational Safety and Health
strains. At the highest inoculum (107 organisms per recommends limiting exposure to halogenated
animal), 57% of animals inoculated with the wild anesthetics such as methoxyflurane to 2 ppm during
type organism develop endocarditis, while only 17% anesthetic administration [2]. In addition, increased
of animals inoculated with the mutant strain devel- levels of methoxyflurane in the investigator's imme-
oped endocarditis. diate environment can lead acutely to hypoxia which
The rat model of endocarditis is an excellent may be experienced as dizziness. It is imperative,
method for in vivo evaluation of virulence of the therefore, that rat catheterizations be performed under
viridans streptococci. As with any animal use, every a functioning fume hood. The 50 ml conical tube
effort should be made to use in vitro methods if used to deliver whiffs of methoxyflurane to the rat
acceptable, and to carefully design the experiments should be stored inverted and toward the back of the
207
hood to prevent release of anesthetic vapors into the with bacterial endocarditis. Infect Immun 58: 515-
investigator's work space. 522.
The rat model of endocarditis requires specific 8. Manning JE, Geyelin AJ, Ansmits LM, Oakey HJ,
psychomotor skills that are perfected over time. Knox KW (1994). A comparative study of the aggre-
Surgical survival rates are highly dependent upon the gation of human, rat and rabbit platelets by members
of the Streptococcus sanguis group. J Med Microbiol
technical expertise of the investigator. Success in the
41: 10-13.
technique can be measured by the number of animals 9. Manning JE, Hume EB, Hunter N, Knox KW (1994).
that survive the surgery, have correct catheter place- An appraisal of the virulence factors associated with
ment at time of necropsy, and have vegetations streptococcal endocarditis. J Med Microbiol 40:
present on cardiac valves. Additionally, the organ- 110-4.
isms recovered from vegetations should be the same 10. Munro CL, Macrina FL (1993). Sucrose-derived
as those used in the inoculum. Assuming technical exopolysaccharides of Streptococcus mutans V403
competence in the rat surgery, results generated with contribute to infectivity in endocarditis. Mol
the rat model of endocarditis are highly reproducible. Microbiol 8: 133-142.
11. Santoro J, Levinson ME (1978). Rat model of exper-
imental endocarditis. Infect Immun 19: 915-918.
12. Stimpel TM, Gershey EL (1991). Selecting anesthetic
Notes on suppliers
agents for human safety and animals recovery surgery.
FASEB J 5: 2099-2104.
1. Fisher, 711 Forbes Ave., Pittsburgh, PA 15219, USA 13. Sullam PM, Costerton JW, Yamasaki R, Dazin PF,
2. VWR, P.O. Box 483, Bridgeport, NJ 08014, USA Mills J (1993). Inhibition of platelet binding and
3. Roboz Surgical Instrument Company, Inc., 1000 aggregation by streptococcal exopolysaccharide. J
Connecticut Avenue, NW, P.O. Box 19148, Washing- Infect Dis 167: 1123-1130.
ton, DC 20036, USA. Telephone 202-393-1234 14. Sullam PM, Frank U, Yeaman MR, Tauber MG,
4. Braintree Scientific, Inc., P.O. Box 850929, Braintree, Bayer AS, Chambers HF (1993). Effect of thrombo-
MA 02185-0929, USA. Telephone 617-843-7932 cytopenia on the early course of streptococcal endo-
5. Sage Products, Cary, IL, USA carditis. J Infect Dis 168: 910-914.
15. Tart RC, van de Rijn I (1991). Analysis of adherence
of Streptococcus defectivus and endocarditis-associ-
References ated streptococci to extracellular matrix. Infect Immun
59: 857-862.
1. Baddour LM, Christensen GD, Lowrance JH, Simpson 16. Viscount HB, Munro CL, Burnette-Curley D, Peterson
W A (1989). Pathogenesis of experimental endo- DL, Macrina FL (1997). Immunization with FimA
carditis. Rev Infect Dis 11 (3): 452-463. protects against Streptococcus parasanguis endo-
2. Burkhart JE, Stobbe TJ (1990). Real-time measure- carditis in rats. Infect Immun 65: 994-1002.
ment and control of waste anesthetic gases during 17. Wells VD, Munro CL, Sulavik MC, Clewell DB,
veterinary surgery. Am Ind Hyg Assoc J 51: 640-645. Macrina FL (1993). Infectivity of a glucan synthesis-
3. Burnette-Curley D, Wells V, Viscount H, Munro CL, defective mutant of Streptococcus gordonii (Challis)
Fenno JC, Fives-Taylor P, Macrina FL (1995). FimA, in a rat endocarditis model. FEMS Microbiol Lett 112:
a major virulence factor associated with Streptococcus 301-305.
parasanguis endocarditis. Infect Immun 63: 4669- 18. Willcox MD (1995). Potential pathogenic properties
4674. of members of the 'Streptococcus milleri' group in
4. Carbon C (1993). Experimental endocarditis: A relation to the production of endocarditis and
review of its relevance to human endocarditis. J abscesses. J Med Microbiol 43: 405-410.
Antimicrob Chemother 31 (Suppl D): 71-85. 19. Wu T, Yeaman MR, Bayer AS (1994). In vitro resis-
5. Douglas CW, Brown PR, Preston FE (1990). Platelet tance to platelet microbicidal protein correlates with
aggregation by oral streptococci. FEMS Microbiol endocarditis source among bacteremic staphylococcal
Lett 60: 63-67. and streptococcal isolates. Antimicrob Agents
6. Ford I, Douglas CW, Preston FE, Lawless A, Chemother 38: 729-732.
Hampton KK (1993). Mechanisms of platelet aggre-
gation by Streptococcus sanguis, a causative organism
in infective endocarditis. Br J Haematol 84: 95-100. Address for correspondence: Cindy L. Munro, RN, PhD,
7. Herzberg MC, Gong K, MacFarlane GD, Erickson PR, Virginia Commonwealth University, Box 980567,
Soberay AH, Krebsbach PH, Manjula G, Schilling K, Richmond VA 23298-0567, USA
Bowen WH (1990). Phenotypic characterization of Phone: 804-828-3410; Fax: 804-828-7743
Streptococcus sanguis virulence factors associated E-mail: cmunro@vcu.edu
Methods in Cell Science 20: 209-216 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. Methods are described for the identifica- isolated membranes. Incubation of intact cells with
tion and extraction of lipoproteins and other cell- wall-hydrolyzing enzymes effects release of proteins
surface proteins from streptococci. Polypeptides linked covalently to cell-wall peptidoglycan. Surface
loosely-associated with the cell surface may be proteins have been shown to be important for the col-
extracted from intact cells with detergents, while onization and virulence properties of streptococci and
lipid-linked and other membrane-integral proteins are thus are potential targets for new anti-microbials and
efficiently solubilized only from disrupted cells or components of novel vaccines.
Key words: Anchorage of polypeptides, Lipoproteins, Polypeptide release, Streptococcus surface proteins,
Wall-associated protein extraction
Abbreviations: aa = amino acid; LTA = lipoteichoic acid; PMSF = phenyl methyl sulfonyl fluoride; SDS =
sodium dodecyl sulfate; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLS =
sodium lauroylsarcosinate
aspirate off the supernatant and wash the cells using the method of Towbin et al. [32]. For
by suspension and centrifugation as before. efficient transfer of high molecular mass
3. To the bacterial cell pellet add 0.12 ml proteins > 180 kDa blotting times of 90 min
spheroplasting buffer, suspend cells by at 20 V cm- l are recommended. Transfer effi-
vortex-mixing and add 6 III of 10000 units ciency may be improved further by reducing
mutanolysin ml- l . Mix gently and incubate the the methanol concentration to 5% (vol/vol) in
suspension for 30 min at 37°C with occasional the transfer buffer. Immunoblots are developed
gentle mixing. Collect the spheroplasts by as described in detail elsewhere [10].
centrifugation (5000 g, 4°C, 10 min), transfer
carefully 0.1 ml supernatant to a clean tube,
add 0.1 ml sample buffer and heat for 10 min 4. Results and discussion
at 70°C in preparation for SDS-PAGE. To
extract wall-associated proteins from Entero- A. Lipoproteins
coccus faecalis cells, lysozyme (final concn.
0.2 mg ml- l ) must be added together with The presence of a L-(S,A)-(A,G)-C-(S,G) consensus
mutanolysin. sequence within the signal peptide of a polypeptide
F. Preparation of culture supernatant proteins precursor is the 'signature' for lipid-modification of
1. Prepare late-exponential phase culture as the mature protein. The simplest and most widely-
described in Procedure C, 1, except in TY- utilized means of confirming that a mature protein
glucose medium. Pellet cells by centrifugation is lipid-modified is to demonstrate that following
(10,000 g, 4°C, 10 min), collect the super- growth of bacterial cells in radioactively-labeled fatty
natant, transfer to a clean polycarbonate acid (usually palmitate), the polypeptide carries cova-
culture tube and re-centrifuge. lently-bound radiolabel. Lipoproteins are thought to
2. Remove 2 ml clear supernatant into a 15 ml be held at the cell surface through intercalation of
glass Corex tube. To precipitate proteins, add their lipid moiety into the cell membrane. In support
8 ml acetone at -20°C, mix by inversion, and of this notion, lipoproteins are effectively released
incubate tube without shaking for 16 h at following incubation of cells with ionic detergents,
-20°C. by not by treatment of cells with solutions of salts
3. Centrifuge the suspension (10,000 g, 4°C, 10 or chaotrophes. The nature of the detergent utilized
min), drain the tube carefully so as not to affects significantly the efficiency of extraction.
disturb the loose sediment, then place the tube Thus incubation of intact cells with SLS at ambient
under vacuum for 10 min to dry the precipi- temperature (Procedure C) extracts upwards of 20
tate. Add 0.2 ml sample buffer, vortex mix to polypeptides from the S. gordonii cell surface [7]. A
suspend, transfer to a 1.5-ml microfuge tube major component of this extract is a phosphocarrier
and heat the suspension for 10 min at 70°C. protein (HPr) which is poorly extracted by incubating
In preparation for SDS-PAGE, centrifuge for cells with SDS at elevated temperatures [7]. By
5 min at 10,000 g, collect the supernatant and contrast, SLS treatment of intact cells does not effec-
add 20 III sample loading dye. tively remove lipoproteins, and is 50% less efficient
G. Protein separation and detection than SDS for solubilizing eH]palmitate-labeled
1. Separate proteins by SDS-PAGE [15]. The lipoproteins from membranes of disrupted cells [8].
sensitive method of staining proteins with Following metabolic labeling of S. gordonii DLl
silver [21] is necessary to visualize protein (Challis) cells with eH]palmitate and extraction of
profiles of SLS extracts (Procedure C), proteins with SDS (Procedure D), 13 lipopolypeptide
mutanolysin extracts (Procedure E), and bands may be discerned by fluorography of SDS-
culture supernatant fluid (Procedure F). Care PAGE gels (Figure 1). Roughly similar numbers of
should be taken to not overload gels with radioactively-labeled protein bands are detected in
Procedure F samples since these contain extracts of Streptococcus sanguis, Streptococcus
elevated salt levels that may cause distortion parasanguis and Streptococcus mutans cells grown
of protein banding patterns. in the presence of radioactively-labeled palmitate
2. To detect [3H]palmitate-Iabeled lipoproteins by [8,31]. The three slowest-migrating lipopolypeptide
fluorography following SDS-PAGE, first fix bands in Figure 1 of apparent molecular masses 78
the gel for 30 min in 10 volumes of 25% kDa, 78 kDa (co-migrate) and 76 kDa, which are
(voVvol) methanol, then transfer to 10 volumes expressed during growth of S. gordonii cells in BHY
of Amplify and shake gently for 30 min. medium, are oligopeptide-binding proteins serving
Vacuum-dry the fluor-impregnated gel onto the Hpp oligopeptide uptake system [12]. Insertional
Whatman 3 mm paper at 60°C and then inactivation of the hppA gene in S. gordonii OB285
expose the dried gel to X-ray film for up to abolishes production of the 76-kDa labeled band
7 d at -80°C. (Figure 1, lane 2), confirming that HppA is a lipopro-
3. Electroblot proteins onto nitrocellulose by tein.
213
1 2 2 3 1 2 3 1 2 3
218 -
100 - 100 -
72 -
72 - 43 -
43 - A B c
Figure 2. SDS-PAGE patterns and immunoblot analysis
of proteins released following mutanolysin treatment of
S. gordonii wild-type (lane 1), OB277 cshA31 cshB2 (lane
2), or OB219 sspA sspB (lane 3). A, profile of silver-
stained proteins; B, Western blot of panel A reacted with
28 - rabbit antiserum to CshA poylpeptide diluted 1:500; C,
Western blot reacted with antibodies to antigen 1111
Figure 1. Fluorogram of [3H]palmitate-Iabeled proteins polypeptide (SpaP) of S. mutans diluted 1:200. Primary
extracted from S. gordonii DLl wild-type (lane 1) or antibody binding was detected with peroxidase-linked
OB285 hppAl mutant (lane 2) cells. Positions of molec- swine antibodies to rabbit immunoglobulin G and devel-
ular mass markers (in kDa) are indicated on the left. oped with chloronapthol [10]. Positions of molecular mass
marker proteins (kDa) are indicated.
Although streptococcal lipoproteins are firmly reproducible for a given strain and its isogenic
anchored at the cell surface and fractionate along mutants (Figure 2). The CshA (259 kDa) and CshB
with isolated cell membranes [8] there is evidence (245 kDa) oligospecific adhesins [19] are recovered
that some release may occur, perhaps as a result of more or less intact from the surface of S. gordonii
lipid turnover [30]. The B-Iactamases of bacilli are wild-type cells and are detected by reacting Western
in fact cleaved from the cell surface late in the growth blots with CshA antibodies (Figure 2B, lane 1). Also
cycle and released into the culture fluid as released intact from S. gordonii cells are the SspA
hydrophilic products lacking the lipid modification (171 kDa) and SspB (160 kDa) salivary glycopro-
[25]. Functional studies of streptococcal lipoproteins tein adhesins of the antigen 1111 family [9], detected
have been hampered by findings, from a number of by reacting Western blots with antigen 1111 family
laboratories, that expression of even relatively small polypeptide antibodies (Figure 2C, lane 1). This
coding segments of the genes on multi-copy plasmids method is generally applicable to extracting these
can be deleterious to growth of Escherichia coli kinds of polypeptides from most other species of oral
cloning hosts. By contrast, expression of Bacillus streptococci. Isogenic S. gordonii mutants in which
licheniformis B-Iactamase is well tolerated by E. coli, cshA and cshB genes are inactivated do not produce
and thi& has permitted detailed analyses of the struc- Csh-antibody reactive bands (Figure 2B, lane 2), but
tural requirements for lipoprotein processing and still retain the SspA and SspB adhesins (Figure 2C,
membrane localization [6]. lane 2). Isogenic sspA sspB double mutants of S.
gordonii that do not express antigen llII-antibody
B. Wall-associated proteins reactive bands (Figure 2C, lane 3) are unaffected in
expression of CshA and CshB proteins (Figure 2B,
Proteins that are covalently-linked to cell wall lane 3). These data demonstrate clearly that Csh and
peptidoglycan in oral streptococci are released from Ssp adhesins are independently expressed and pre-
cells following treatment with the hydrolytic enzyme sented on the streptococcal cell surface.
mutanolysin in the presence of an osmotic stabilizer Sequence analyses have revealed that the afore-
to minimize cell lysis (Procedure E). Fifty or more mentioned S. gordonii adhesins all possess a C-
polypeptide bands ranging in molecular mass from terminal anchorage region, viz. the LPxTGx motif,
> 250 kDa to < 30 kDa are detected upon silver- hydrophobic sequence and charged tail. However the
staining of SDS-PAGE gels of mutanolysin extracts precise structural and sequence requirements neces-
from S. gordonii DLl cells (Figure 2). Lipoproteins sary for determining anchorage are still being eluci-
do not appear to be released in significant amounts dated, and the enzyme(s) involved in cleavage and
by this treatment. The SDS-PAGE patterns are highly transpeptidation is/are currently uncharacterized. It
214
appears that the exact location of the anchorage still carries an single LPxTGx sequence motif, is also
region within a polypeptide may not be critical, since secreted intact into the culture fluid and not anchored
when the anchorage region of Staphylococcus aureus at the cell surface (Figures 3A and 3B, lane 3). The
protein A is translocated by using recombinant phenotypic effects of these mutations are to render
technology to a central position within a hybrid the mutant cells deficient in CshA-mediated adhesion
polypeptide, it still becomes covalently linked to the properties [19]. Interestingly, a human oral isolate
wall [23]. On the other hand, evidence suggests that of S. mutans was shown recently to release 'natu-
the LPxTGx motif alone might not be sufficient for rally' a truncated form of PAc adhesin (antigen 1111)
recognition by the sorting machinery. This was protein into the culture supernatant. Sequence
demonstrated by analyzing the cell-surface versus analysis suggests this occurs as a result of a
culture supernatant distributions of CshA protein in frameshift mutation within the C-terminal coding
S. gordonii wild-type or isogenic mutant cells in region of the gene causing premature termination of
which various C-terminally truncated forms were translation [22]. Although PAc is required for
generated by gene disruption. The extracellular fluid adhesion of S. mutans cells to salivary glycoprotein
of stationary phase cultures of S. gordonii contains pellicle in vitro [14], the consequences of protein
in excess of 70 polypeptides as detected by SDS- anchorage failure on bacterial growth and survival in
PAGE of proteins collected by precipitation vivo are not yet known. It has been suggested that
(Procedure F) (Figure 3A, lane 1). Small quantities release of specific surface protein antigens by strep-
of intact CshA protein are released into the culture tococcal cells may assist in evasion of host immune
supernatant during growth of wild-type cells as defences [17].
shown by immunoblot analysis (Figure 3B, lane 1).
Isogenic mutant S. gordonii OB 186 carries an inser- C. Novel cell-surface protein anchorage
tion within the cshA gene that C-terminally truncates mechanisms
the CshA protein by 250 aa residues [18]. Ensuing
loss of the anchorage region results in release of the Currently there is much interest in determining the
truncated protein from the cell surface into the roles of various surface proteins in epithelial cell
culture extracellular fluid (Figures 3A and 3B, invasion by Listeria monocytogenes. While a major
lane 2). It is of note that anchorage failure occurs invasion-associated surface protein InlA carries the
despite there being present, internally within the trun- characteristic gram-positive bacterial LPxTGx motif
cated polypeptide, four additional LPxTGx motifs and anchorage region [16], two additional invasion-
[19]. A more extensive C-terminally truncated associated proteins with novel cell-surface anchorage
polypeptide produced by S. gordonii OBI93, which sequences have been recently described. The first of
these, ActA, is required for actin polymerization fol-
lowing entry of Listeria into host cells. ActA protein
1 2 3 1 2 3 carries at the C-terminus a trans-membrane helix of
22 aa residues and a short charged cytoplasmic tail,
218 - but no LPxTGx motif [13]. ActA can be extracted
from intact Listeria cells with SDS which is consis-
100- tent with it being non-covalently linked to the cell
surface. A second protein, designated InlB, which is
72 - also involved in bacterial entry into the host cell [2],
carries three 80 aa residue repeat blocks at the C-
43- terminal end. These repeat block sequences, which
possibly bind to cell wall polymers, are essential for
surface localization of InlB, and are sufficient for
allowing re-association of secreted or exogenously-
added InlB protein with the Listeria cell surface [2].
This mode of cell-surface association of InlB is
reminiscent of that of PspA which is held at the
A B Streptococcus pneumoniae cell surface via interac-
tion of C-terminal aa residue repeat blocks with the
Figure 3. SDS-PAGE patterns and immunoblot analysis choline-containing LTA [33]. As new streptococcal
of culture fluid proteins present in early stationary phase
surface antigens and proteins with novel biological
cultures of S. gordonii wild-type or isogenic mutant strains
expressing truncated forms of CshA. Lane 1, S. gordonii activities are continually being discovered, it is likely
DLl wild-type; lane 2, OB185 cshA8; lane 3, OB193 that additional surface protein-anchorage sequences
cshA5. Panel A, profile silver-stained proteins; Panel B, and mechanisms will be revealed . For example, a
corresponding Western blot reacted with antibodies to protein with glyceraldehyde-3-phosphate dehydroge-
CshA (see legend to Figure 2). Positions of molecular mass nase actIVIty is present on the cell surface of
marker proteins (kDa) are indicated. Streptococcus pyogenes [26] and is sensitive to
215
trypsin. The primary aa sequence reveals no obvious 18. E. I. du Pont de Nemours & Co. Inc., Wilmington DE
surface-anchorage determinant, and the protein is not 19898, USA
removed from cells with SDS, so a novel anchorage 19. Samco Scientific Inc., San Fernando CA 91340,
mechanism might be predicted. USA
antigen mutants of Streptococcus mutans serotype c. RJ (1980). Association of protein with the cell wall of
Infect Immun 58: 289-296. Streptococcus mutans. Infect Immun 28: 118-126.
15. Laemmli UK, Favre M (1973). Maturation of the head 25. Nielsen JBK, Lampen JO (1982). Membrane bound
of bacteriophage T4. I. DNA packaging events. J Mol penicillinases in gram-positive bacteria. J BioI Chern
BioI 80: 575-599. 257: 4490-4495.
16. Lebrun M, Mengaud J, Ohayon H, Nato F, Cossart P 26. Pancholi V, Fischetti VA (1992). A major surface
(1996). Internalin must be on the bacterial surface to protein on group A streptococci is a glyceraldehyde-
mediate entry of Listeria monocytogenes into epithe- 3-phosphate dehydrogenase with multiple binding
lial cells. Mol Microbiol 212: 579-592. activity. J Exp Med 176: 415-426.
17. Lee SF (1995). Active release of bound antibody by 27. Russell RRB (1979). Wall-associated protein antigens
Streptococcus mutans. Infect Immun 63: 1940-1946. of Streptococcus mutans. J Gen Microbiol 114:
18. McNab R, Jenkinson HF (1992). Gene disruption 109-115.
identifies a 290 kDa cell-surface polypeptide confer- 28. Schatz G, Dobberstein B (1996). Common principles
ring hydrophobicity and coaggregation properties in of protein translocation across membranes. Science
Streptococcus gordonii. Mol Microbiol 6: 2939-2949. 271: 1519-1526.
19. McNab R, Jenkinson HF, Loach DM, Tannock GW 29. Schneewind 0, Fowler A, Faull KF (1995). Structure
(1994). Cell-surface-associated polypeptides CshA of the cell wall anchor of surface proteins in
and CshB of high molecular mass are colonization Staphylococcus aureus. Science 268: 103-106.
determinants in the oral bacterium Streptococcus 30. Sutcliffe IC, Russell RRB (1995). Lipoproteins of
gordonii. Mol Microbiol14: 743-754. gram-positive bacteria. J Bacteriol 177: 1123-1128.
20. Medaglini D, Pozzi G, King TP, Fischetti VA (1995). 31. Sutcliffe IC, Tao L, Ferretti JJ, Russell RRB (1993).
Mucosal and systemic immune responses to a recom- MsmE, a lipoprotein involved in sugar transport in
binant protein expressed on the surface of the oral Streptococcus mutans. J Bacteriol 175: 1853-1855.
commensal bacterium Streptococcus gordonii after 32. Towbin H, Staehelin T, Gordon J (1979).
oral colonization. Proc Natl Acad Sci USA 92: Electrophoretic transfer of proteins from polyacry-
6868-6872. lamide gels to nitrocellulose sheets: Procedure and
21. Morrissey JH (1981). Silver stain for proteins in poly- some applications. Proc Natl Acad Sci USA 76:
acrylamide gels: A modified procedure with enhanced 4350-4354.
uniform sensitivity. Anal Biochem 117: 307-310. 33. Yother J, White JM (1994). Novel surface attachment
22. Murakami Y, Nakano Y, Yamashita Y, Koga T mechanism of the Streptococcus pneumoniae protein
(1997). Identification of a frameshift mutation PspA. J Bacteriol 176: 2976-2985.
resulting in premature termination and loss of cell wall
anchoring of the PAc antigen of Streptococcus mutans
GS-5. Infect Immun 65: 794-797. Addressfor correspondence: H. F. Jenkinson, Department
23. Navarre WW, Schneewind 0 (1994). Proteolytic of Oral and Dental Science, University of Bristol, Dental
cleavage and cell wall anchoring at the LPXTG motif Hospital and School, Lower Maudlin Street, Bristol BSI
of surface proteins in gram-positive bacteria. Mol 2LY, UK
Microbiol 14: 115-121. Phone: 0044 117 928 4304; Fax: 0044 117 928 4428
24. Nesbitt WE, Staat RH, Rosan B, Taylor KG, Doyle E-mail: howard.jenkinson@bristol.ac.uk
Methods in Cell Science 20: 217-221 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. Microbial pathogens require iron to reconstituted with manganese and/or magnesium.
survive and promote disease. Reports in the literature The final metal ion concentrations in this medium
indicate that iron can potentiate infection in the were confirmed by inductively coupled argon plasma
human host by triggering the expression of bacterial (ICAP) analysis. Importantly, the iron-depleted
virulence determinants [8]. Streptococcus mutans is medium supported the growth of S. mutans only
a prevalent microorganism in dental plaque and the when supplemented with 0.01 to 10 f.1M iron, con-
principal causative agent of human dental caries. This centrations which are consistent with those found in
oral pathogen may respond to the changing avail- human saliva. The practical applications of this
ability of iron in the plaque environment brought medium are far-reaching and include the identifica-
about by conditions of feast or famine. Our under- tion of iron sources, and iron-responsive genes and
standing of a role for iron in S. mutans-induced caries their products which may promote streptococcal
formation is limited, however, by the lack of an pathogenesis. Other trace metals may be altered in
appropriate test medium. In this study we applied this medium to promote investigations of their
batch chromatography to remove iron from a chem- putative effect(s) on streptococcal growth and
ically-defined medium [7] which we subsequently expression.
Abbreviations: FMC = a conventional chemically-defined medium; FMC- = FMC prepared without the iron
component; mdFMC cx = metal-depleted FMC, FMC prepared without the metal components followed by
treatment with Chelex-lOO; FMCcx- = iron-free mdFMC cx supplemented with manganese and/or magnesium
FMC c/ = FMC cx- supplemented with exogenous ferric chloride; ICAP = inductively coupled argon plasma
analysis
Table 1. Media components (gIL) this iron-repleted medium (FMC c/ ) for subse-
quent growth experiments.
Solution A (Ix): D. To prepare modified FMC agarose plates
KH 2P 04 0.44 Agar can carry with it residual iron which cannot
K 2HP04 0.30 be monitored by conventional ICAP analysis.
Na2HP0 4 anhydrous (dibasic) 3.15
Thus, we recommend that ultrapure agarose be
NaH2 P04 • H20 (monobasic) 2.36
Na acetate· 3H20 9.95
used as the solidifying agent when preparing
(NH 4hS 04 0.60 plates, and that the following protocol be
NaCI 0.01 applied to minimize iron carry-over.
L-cysteine 0.10 Dissolve the components of Solution A
DL-tyrosine 0.20 (Table 1) in 830 ml of ddH 20 and adjust pH to
L-glutamine 0.005 6.9 with concentrated HCl. To this solution add
Na2C0 3 2.10 100 ml 5% tryptone and 50 ml 20% glucose.
Solution B (200x): Transfer this solution to a 2 L flask containing
MgS0 4 ·7H20 4.0 30 g Chelex-100 and shake vigorously on an
MnS0 4 ·H20 0.15 orbital shaker (350-400 rpm) at 4 °c overnight.
FeCI3 ·6H20 0.04 Pass the medium through a 0.2 ~m filter to
Solution C (200x): remove the resin.
pantothenate hemicalcium 0.16 Reconstitute the medium with MgS0 4 • 7H20
p-amino benzoic acid 0.02 and MnS0 4 • H2 0 to a final concentration of
thiamine· HCI 0.08 20 ~M and I ~M, respectively. Supplement, if
nicotinic acid 0.40 desired, with exogenous iron and confirm the
pyridoxine monohydrochloride 0.16 metal ion concentration by ICAP. Add 15 g of
D-biotin 0.002 ultrapure agarose and autoclave.
riboflavin 0.08 Cool the medium to 45-50 °c and add 5 ml
folic acid 0.02 solution C and 10 ml solution D (Table 1). Stir
Solution D (lOOx): gently and pour plates.
adenine 1.0 E. Inductively coupled argon plasma (lCAP) analysis
guanine 1.0 Perform the general operation of the ICAP
uracil 1.0 analyzer according to the recommendations of
the manufacturer. Standardize the analyzer for
magnesium, manganese and iron using stock
ml solution C and 10 ml solution D. Mix gently solutions containing these metals at 10 ppm. After
and store at 4 °c in aIL Pyrex laboratory bottle. a 15 minute equilibration period, adjust the
Under these conditions, the medium is stable for sample flow rate into the plasma using 1000 ppm
up to 2 weeks. Yttrium to visualize the argon flame. Program the
C. To Prepare Modified FMC Medium machine (ThermoSpec software) to perform four
To solution A, aseptically add 100 ml 5% readings per sample and to report the data as an
tryptone, 50 ml 20% glucose,S ml solution C, and average. Analyze the media samples in 5 ml
10 ml solution D. Transfer this medium to the aliquots at t = 30 seconds post-uptake. Rinse the
sterilized Chelex-lOO resin and shake vigorously uptake tubing thoroughly with 5% HCI and then
on an orbital shaker (350-400 rpm) at 4 °c with ddH 2 0 between readings. Convert metal ion
overnight. Pass the medium through a 0.2 ~m concentrations provided in ppm to ~M units.
bottle top filter to remove the resin and perform F. Bacterial growth
ICAP analysis. Reserve 50 ml of this metal- Inoculate 14 ml of Todd Hewitt broth contained
depleted FMC (mdFMC cx ) for subsequent washes. in a 15 ml polypropylene tube with 150 ~l of a
Reconstitute the remammg medium with S. mutans cell concentrate and incubate at 37°C
MgS0 4 ·7H 20 and MnS0 4 • H 20 to a final con- and 5% CO 2 for 16 hours. Harvest the cells by
centration of 20 ~M and 1 ~M, respectively centrifugation at 7000 rpm for 10 minutes and
(FMC cx-). Confirm the final metal ion content of pour off the supernatant. Wash the cell pellet with
this iron-depleted medium by ICAP analysis. 1 ml of mdFMC cx and repeat two more times to
Supplement FMC cx- with various concentra- remove all traces of iron. Resuspend the pellet in
tions of an appropriate iron source (e.g., 0.01-10 1 ml mdFMC cx • Use 100-150 ~l of this cell
~M FeCI 3 ). Specifically, transfer 50 ml aliquots concentrate to inoculate each of five sterile 15 ml
of the FMC cx- to separate sterile 50 ml poly- polypropylene tubes containing 14 ml of iron-
propylene tubes and supplement each with iron. repleted FMC (FMC c/ ) . Incubate all tubes
Aliquots should be derived from a single batch standing at 37°C and 5% CO 2 • To monitor
of medium to ensure that any differences observed growth, obtain OD600nm values for individual tubes
are due to variations in iron concentration. Use at t = 8, 10, 12, 16 or 24 hours post-inoculation
220
(i.e., use one culture per timepoint). These early, magnesium (20 IlM). Therefore, we conclude that S.
mid-, late-log and stationary phase cultures may mutans has nutritional requirements for iron as well
then be applied to nucleic acid and/or protein as for magnesium.
isolation protocols to characterize putative iron- Preliminary experiments in our laboratory suggest
regulated expression. that manganese can support the growth of S. mutans
Furthermore, a S. mutans transposon library in iron-free FMCcx-. Since S. mutans can grow in the
may be replica-plated onto FMC- and FMC c/ presence of manganese or iron, it appears that either
agarose plates to identify mutants altered in iron metal can satisfy the nutritional requirements for this
uptake and/or storage. oral microbe. Consistent with this finding are reports
in the literature suggesting an interchangeability of
manganese and iron in promoting the growth of S.
4. Results and discussion mutans [6]. Finally, surface protein profiles for S.
mutans cultures grown in FMCcx - supplemented with
This work describes the generation of an iron- manganese were different from those derived from
depleted, chemically-defined medium which supports cultures grown in FMC cx - supplemented with iron
the growth of streptococci upon supplementing with (data not shown). This indicates that manganese has
exogenous FeCI3 • The final metal ion concentrations a unique effect on S. mutans expression.
in this medium were confirmed by ICAP analysis. The reproducibility of this protocol is sound but
Alternatively, atomic absorption spectroscopy or can vary with water quality. For instance, trace
inductively coupled plasma mass spectroscopy (lCP- amounts of iron can persist in ddH 2 0 as well as on
MS) may be applied to confirm metal concentrations. glassware. A well-maintained water still and purifi-
As shown in Figure la, the treatment of FMC- cation system is required, and the use of disposable
with Chelex (FMC cx- ) markedly reduced the residual plasticware is recommended whenever possible.
iron content of the medium from 1.6 to 0.005 IlM. Moreover, the preparation of iron-free magnesium
The growth of S. mutans was also restricted in this and manganese stock solutions is particularly tedious.
medium as measured after a 24 hour incubation at Thus, we recommend that only ultra-pure reagents be
37°C and 5% CO 2 (Figure Ib). Growth was restored used and that highly concentrated metal solutions
to this medium when 0.01 to 10 IlM iron was added (at least 100 mM) be prepared to limit the carry-over
(FMC cx+). Importantly, these iron concentrations are of iron into the supplemented medium. Since we
comparable to those present in human saliva (unpub- found the removal of iron from manganese stock
lished observations). As the concentration of iron in solutions to be especially challenging, we often
FMC c/ was increased from 0.01 to 10 IlM, S. mutans omitted manganese from the preparation of FMCc/
growth increased in parallel. This growth pattern so that the sole effect(s) of iron on S. mutans growth
leveled off, however, when iron was provided at and expression could be assured.
concentrations greater than 10 IlM (data not shown). In conclusion, the medium described in the present
S. mutans did not grow in any of the media described study can be used to investigate the effect(s) of
herein unless it was supplemented with exogenous multiple iron sources on the expression of strepto-
a) b)
14 1.2
12 - -
~ 10 10.:
OJ
~ 8
~0.6
10.2
0
:E
v 6
v
·5r;,;. 4 0.4
2 vi
0 n 0 D ~
FMCcx- FMCcx+
FMC FMC- FMCc:x- FMCcx+ FMC FMC-
Media Media
Figure la. Iron concentrations in FMC, FMC without iron Figure lb. Effect of iron on S. mutans growth. S. mutans
(FMC-), Chelex-treated FMC- (FMC cx -), and FMC cx - was grown to stationary phase (t = 24 h) in the media
repleted with 10 ~M FeCl 3 (FMC c/ ) as determined by described in Figure la. While each of these media condi-
ICAP analysis. While each of these media conditions was tions was tested in triplicate, the results of a single
tested in triplicate, the results of a single representative representative experiment are shown. All media were
experiment are shown. All media were supplemented with supplemented with manganese and/or magnesium as
manganese and/or magnesium as described in the text. described in the text.
221
Abstract. A PCR based, reverse capture checker- digoxigenin-labeled amplicons from up to 45 samples.
board hybridization methodology was designed Consequently, 1350 hybridizations are performed
to rapidly detect and enumerate oral species of simultaneously using a single membrane. Hybridiza-
Streptococcus and other genera in plaque samples to tion signals are detected using chemifluorescence
assess the bacterial etiology of root surface caries or procedures. Bacterial numbers are determined by
other oral diseases. The procedure circumvents the analyzing hybridization signals using a Storm
need for in vitro bacterial cultivation. Up to 30 system. A genus-specific probe is used to assess the
reverse capture probes that target regions of 16S total number of streptococci and universal probes are
rRNA genes are deposited on a nylon membrane in used to assess total bacterial counts. Use of this pro-
separate horizontal lanes using a MiniSlot apparatus. cedure enables laboratories to analyze thousands of
16S rRNA genes of DNA from plaque or pure clinical or environmental samples with many probes
cultures are PCR amplified using a digoxigenin- in a relatively short period. This methodology has
labeled primer. Hybridizations are performed in broad range applications in microbiology to monitor
vertical channels in a Miniblotter apparatus with population distributions in a given environment.
Key words: 16S rRNA, Checkerboard hybridization, DNA probes, Phylogeny, Streptococcus
(% Difference)
I I I I I I I
Streptococcus mitis
Membrane
Streptococcus pneumoniae
Streptococcus oraUs
Streptococcus mitis bv II
Streptococcus strain H6
Streptococcus strain 7A
Streptococcus sanguis
Streptococcus parasanguis Capture probes applied to Nylon
Streptococcus gordonii membrane using Minislot
Streptococcus strain B5Se
Streptococcus salivarius
Streptococcus intermedius
+
Streptococcus constel/atus Probes
Streptococcus anginosus
Streptococcus sobrinus
Streptococcus mutans
Veillonelln parvula
Veillon ella dispar
Capture probes UV
Bijidobacterium dentium
crosslinked to membrane
+
Bijidobacterium breve
Stomatococcus mucilaginosus
Rothia dentocariosa
r - - - - - Actinomyces odontolyticus
' - - - - Actinomyces viscosus
+
neighbor-joining method of Saitou and Nei [18] was used
for phylogenetic tree construction. The scale bar represents
a 10% difference in nucleotide sequence as determined
by measuring the lengths of horizontal lines connecting •c •
two species.
•
c
assess their role in the etiology of root surface caries.
•
c
Briefly, the procedure is as follows. DNA probes are
synthesized with mUltiple thymidines (T) at the 5' • •
end of the oligonucleotide. The poly-T tails are
crosslinked to the membrane support via UV irradi- Chemifluorescence or chemiluminescence
ation, which leaves the specific probe available for detection
hybridization. The 16S rRNA genes of DNA isolated
from bacterial plaque are amplified using universal
primers (the forward primer is labeled with digoxi-
genin). The digoxigenin-Iabeled amplicon is Figure 2. Schematic representation of the PCR-based,
reverse-capture, checkerboard hybridization methodology.
hybridized to the capture probes bound to the
The Minislot is made of aluminum, whereas the
membrane. The digoxigenin residues are complexed Miniblotter is made of plastic. Note that the bottom bases
to anti-digoxigenin antibody covalently bound to of the Minislot and Miniblotter are not shown. Actual
alkaline phosphatase. Standard chemifluorescence number of slots on the Minislot is 30, and number of
(Storm system) or chemiluminescence (X-ray film) hybridization channels on the Miniblotter is 45 (thus, a
procedures are used for detection. Figure 2 represents potential of 1350 simultaneous hybridizations on a single
a schematic representation of the procedure. solid support).
225
using a Stratalinker 1800 (Stratagene) on c) Wrap apparatus in Saran wrap and place in
autocrosslink position. The thymidine tails ziplock bag with two wet paper towels.
are preferentially UV crosslinked to the Seal bag well and place on tilter (slow
nylon which leaves the specific probe avail- speed) at 55 DC for 2 to 4 h.
able for hybridization. d) Heat 200 ml of Ix SSPE, 0.1 % SDS at
d) Place membrane in container and wash the 55 DC, and take out blocking buffer to thaw,
membrane in 5x SSPE, 0.5% SDS for 30 then put on ice.
min at 55°C on shaker (gentle shaking). 7. Washing and detection
e) Rinse in distilled water and proceed to the a) Carefully remove Miniblotter from the bag
prehybridization step or dry the membrane by keeping it level. Position the washing
(probe side up on scrap membrane). Wrap manifold (that comes with the apparatus) in
in Saran Wrap for later use. Store dried the slots where the loading holes are and
membranes at room temperature in a dark, press in firmly. Using appropriate-sized
dry environment. tubing, connect one side of the manifold to
4. Prehybridization a vacuum source and the other side into the
a) Prepare prehybridization solution (must be 200ml of Ix SSPE, 0.1 % SDS and then
made fresh daily). Place membrane in 60 aspirate the samples (it takes approximately
ml of prehybridization solution in a 1 min).
Tupperware container. b) Disassemble the Miniblotter. Remove
b) Close container's lid tightly and place membrane with a forceps and place the
container on a shaker (gently shaking) at membrane face down in buffer 1 (conjugate
55 DC for 1 h. Prehybridization can go buffer) for 1 min.
overnight, but ensure that the seal is c) Pour off buffer 1 and put in 10 ml of
secured tightly. blocking buffer and 50 ml of buffer 1.
c) Pre-warm Miniblotter apparatus at 55 DC. Block the membrane for I h with gentle
5. Setting up Miniblotter shaking at room temperature.
a) Position one foam pad on top of the bottom d) Pour off the blocking solution and put in
plate of the Miniblotter-45. 60 ml of buffer 1 + 6 III of anti-digoxi-
b) Lift membrane (using forceps) from pre- genin-AP Fab fragments (antibody). Gently
hybridization solution and drip off excess shake for 30 min at room temperature.
prehybridization solution. e) Pour off the antibody solution and wash
c) Place the membrane face up on top of the with 60 ml of buffer 1 for 10 min (gently
foam pad (with cut corner facing upper left) shaking at room temperature). Repeat this
and mark with a marker on the edge of the step once.
foam where the first well and last well f) Pour off second wash and put in buffer 2
begin and end. (substrate buffer). Pour in just enough of
d) Place the top of the Miniblotter apparatus buffer 2 so as to cover membrane and let
on the top of the membrane making sure sit for 2 min without shaking. Keep probe-
that the top part of the Miniblotter channels side down.
are about 3-4 mm above the first Minislot g) Lift membrane from buffer 2 and drip
well and that the bottom part of the excess buffer off. Place membrane (probe
Miniblotter channels are below the last side up) on top of a piece of Saran wrap.
Minislot well. Clamp the whole apparatus Pour about 3 ml of Attophos on membrane.
tightly. Cover the top of membrane with Saran
6. Preparing and applying samples wrap. Spread the Attophos evenly for 1
a) Heat 5 ml of 1.25x hybridization buffer to minute using a 10 ml pipette (roll pipette
55°C. Add 9.6 III denaturing solution to back and forth). Drip excess Attophos off
9.6 III DNA sample (PCR product), and membrane and place membrane between
then add 108.1 III of the heated hybridiza- two mylar sheets (Saran wrap may be used
tion buffer. if wrinkles are avoided).
b) Heat sample to 55 DC. Just prior to loading h) Expose membrane for 2-4 h away from
Miniblotter, add 7.7 III neutralizing solution. direct light (in a drawer between two sheets
Mix. Load 135 III of each sample into a of paper). May have to expose for longer
hybridization channel on the Miniblotter time - as much as overnight (longer
via the small holes at the end of each exposures often results in higher back-
channel (avoid bubbles). For empty lanes, ground).
load 135 III of 1.25x hybridization buffer. i) Scan the membrane using the Storm
With practice, 45 samples can be loaded in system. Scanning time is about 5 min. If a
15 min. Storm system (or equivalent system which
229
measures chemifluorescence) is not avail- The assay works well for the identification of pure
able, then replace Attophos with any of the cultures and for the detection and enumeration of
commercially available chemiluminescence specific species or higher taxa directly in clinical
substrates (e.g., CDP Star) and expose blot samples, without the need for in vitro bacterial
to X-ray film for 15 to 30 min (the optimal cultivation.
time of exposure should be tested empiri- In the pilot study shown in Figure 3, the standards
cally). Comparable qualitative results are represent 5 x 106 cells of each specific target and the
obtained using chemiluminescence detec- clinical samples represent bacterial dental plaque
tion. from 7 healthy volunteers. One sample (BK-4) was
j) Quantitation of the hybridization signals a saliva specimen from one of the patients. The limit
can be obtained on the Storm system using of detection for the assay was about 1 x 103 cells.
the provided software, ImageQuant. We are Many of the sites had high levels of S. sanguis, the
presently optimizing our protocol to obtain S. oratis cluster (S. oratis, S. pneumoniae or S. mitis),
quantitative data using ImageQuant. species of Rothia or Stomatococcus, a potential new
Quantitation of the data can be obtained species B5SC, S. mitis biovar II, and species of
from chemiluminescence data using laser Veillonella. Only some of the sites had A. viscosus,
densitometry; however the level of sensi- S. gordonii, and S intermedius. No or very few sites
tivity is only about 2 orders of magnitude. had detectable levels of S. mutans, S. sobrinus, S.
ImageQuant can achieve about 5 levels of salivarius, Bifidobacterium sp. or A. odontolyticus.
magnitude. In the analysis of the BK-4 saliva sample, the
relatively high level of hybridization with the all
streptococci probe and the relatively low levels of
4. Results and discussion hybridization with specific streptococcal probes
indicated that an unknown species of Streptococcus
The PCR based, reverse capture checkerboard (for which there was no probe) was present.
hybridization methodology described in this study Some species could not be differentiated ade-
can be used to rapidly detect and enumerate oral quately on the basis of 16S rRNA sequence base
species of Streptococcus and other genera (Figure 3). comparisons (Figure 1). For example, S. oratis, S.
--- --
---
--- -- -
--- S. unJ,:i"o.:ms'X. J..'1)rJtNtii
-
S. ""l{ilw.\:,I.)'·X. In/l'l'nk"lli ll.\
---- -- - - -
DNA
Probes
..--_ .... _.
II~S('
-
-
All !II1'l'fllncuCl:I
-
- ---_._.--
-
. -_._--
1\11 RljiduhlJf 'In1ltlll
1\\1 'i·llkm,·lItJ
Uniwool· 1(»(\I
Ul'liwrsal.)41
Figure 3. Blot demonstrating hybridization of digoxigenin-Iabeled, peR products of standards and clinical samples
with reverse-capture, oligonucleotide probes. This figure illustrates the population distribution of streptococcal species
and other genera in plaque or saliva samples from 7 patients.
230
pneumoniae, and S. mitis are essentially indistin- probes are to be developed, significant time must be
guishable, and thus a single probe (D38) was spent in optimizing probes for standard hybridization
designed to target all three 'species' - the S. oraUs conditions.
cluster (Table 1; Figure 3). Other species required One major advantage of utilizing probes that target
more than one probe for identification. For example, l6S rRNA genes is that they can be used to monitor
no single probe for S. sanguis could be designed. species that can not be presently cultivated. The
Consequently, probe D48 that targeted S. sanguis, S. phylogenetic identity of these 'uncultivable' organ-
salivarius, and a potentially new species (designated isms can be determined by sequencing cloned l6S
as H6) was used to determine the levels of all three rRNA genes that are amplified directly from envi-
species. Levels of hybridization with individual ronmental samples [7, 14]. These procedures have
probes to S. saUvarius and H6 can then be subtracted been used to deduce the identity of many unknown
from the levels obtained with probe D48. Thus, as cultivable and not yet cultivable bacterial species
described above, little or no S. saUvarius or H6 were from free-living and host-associated sources [5, 11,
present in any of the clinical samples, but S. sanguis 16,20,23]. Probes to these not yet cultivable species
was present in high numbers. can be designed just easily as they can be for cul-
Although not presented here, we have shown in tivable species.
preliminary experiments that quantitative data can be One of our initial goals in developing this assay
obtained from hybridization blots. The Storm system was to measure bacterial population densities of the
allows for the direct quantitation of chemifluores- predominant microflora on dental root surfaces.
cence samples without the need of X-ray film. Signal Although it is widely accepted that mutans strepto-
intensities obtained from the Storm system for each cocci play a major role in caries production, there
sample can be expressed as microbial counts by is growing evidence indicating that 'low pH' non-
reference to standards run at known concentrations. mutans streptococci and other genera, such as
In addition, percent of total streptococci or percent Actinomyces, may also playa role in the disease
of total bacteria can be obtained by dividing the [21, 22]. With the use of validated probes, it is now
signal intensities from the individual probes by the possible to analyze a statistically sufficient number
signal intensities obtained from the all streptococci of sites to assess the role of the low pH, nonmutans
probe or universal probes, respectively. streptococci, mutans streptococci, and other bacteria
Another advantage of the reverse capture format in root surface caries. Such a comprehensive analysis
is that the crosslinked probes can be preserved of the bacterial flora on root surfaces has not been
indefinitely (i.e., years) on dry membranes stored in previously performed. We are also utilizing checker-
the dark at room temperature. Consequently, 'stock' board methodology to determine the bacterial
membranes with probes can be made prior to population densities of predominant microorganisms
processing of the clinical or bacterial samples. involved in oral health, periodontitis, acute necro-
Another advantage is that short hybridization times tizing ulcerative gingivitis, and refractory periodontal
(from 2 to 4 h) of the oligonucleotide probes disease. This methodology has obvious utility beyond
(compared to overnight with whole genomic probes) that of the oral cavity - it can be used for ecological
allow for same day hybridization. studies of many environments that harbor a diverse,
The reproducibility and accuracy of the reverse cultivable and not-yet-cultivable bacterial population,
capture, checkerboard hybridization methodology is such as extraoral areas of the body, animals, insects,
dependent upon the extreme care by the researcher soil, and deep sea sediments.
with regard to the following precautions. Gloves
must be used for all steps of the hybridization. The
nylon membrane must not be touched with bare or Acknowledgments
gloved hands. The membranes should be picked up
with clean forceps by grabbing only at its corners. It These studies were supported by Public Health
is important to filter all solutions since particulate Service grant P50-DE-070009 from the National
matter (e.g., dust) may appear as aberrant spots after Institute of Dental Research.
development. We have recently detected different We thank Dr W. G. Weisburg and Dr D. J.
background levels of hybridization with different lots Lane for their helpful discussions on DNA-DNA
of Nylon membrane - some lots have no detectable hybridization.
background, whereas others have some background.
Nevertheless, these background signals, as shown in
Figure 3, can be subtracted out during quantitative Notes on suppliers
analysis. If validated probes are used and the pre-
cautions are heeded, good results can be achieved the 1. Amersham Corp., Arlington Heights, IL, USA
first time through. With practice, hybridization blots 2. Benton Dickinson and Co., Cockeysville, MD, USA
such as shown in Figure 3 are achieved routinely in 3. Bio-Rad Laboratories, Hercules, CA, USA
about 8 hours (not including time for PCR). If new 4. Boehringer Mannheim Corp., Indianapolis, IN, USA
231
5. Brinkmann, Westbury, NY, USA molecules. In: Munro HN (ed), Mammalian Protein
6. Cole Palmer Instrument Co., Chicago, IL, USA Metabolism, vol. 3 (pp 21-132).Academic Press, Inc.,
7. Coy Laboratory Products, GrassLake, MI, USA New York.
8. Fisher Scientific, Pittsburgh, PA, USA 10. Kawamura Y, Hou XG, Sultana F, Miura H, Ezaki T
9. FMC BioProducts, Rockland, ME, USA (1995). Determination of 16S rRNA sequences of
10. Immunetics, Inc., Cambridge, MA, USA Streptococcus mitis and Streptococcus gordonii and
11. J.T. Baker, Phillipsburg, NJ, USA phylogenetic relationships among members of the
12. Life Technologies, Gaithersburg, MD, USA genus Streptococcus. Intern J System Bacteriol 45:
13. Marsh Biomedical Products, Inc., Rochester, NY, 406-408.
USA 11. Liesack W, Stackebrandt E (1992). Unculturable
14. Molecular Dynamics, Sunnyvale, CA, USA microbes detected by molecular sequences and probes.
15. Northeast Labs, Waterville, ME, USA Biodiversity Conserv 1: 250-262.
16. Operon Technologies, Alameda, CA, USA 12. Morrissey DV, Collins ML (1989). Nucleic acid
17. Owl Scientific, Inc., Woburn, MA, USA hybridization assays employing dA-tailed capture
18. Perkin Elmer, Foster City, CA, USA probes. Single capture methods. Molec Cell Probes
19. Promega, Madison, WI, USA 3: 189-207.
20. Rainin, Emeryville, CA, USA 13. Nelms LF, Odelson DA, Whitehead TR, Hespell RB
21. Sigma Chemical Co., St. Louis, MO, USA (1995). Differentiation of ruminal and human
22. Stratagene, LaJolla, CA, USA Streptococcus bovis strains by DNA homology and
23. USA/Scientific Plastics, Ocala, FL, USA 16S rRNA probes. Current Microbiol 31: 294-300.
24. VWR Scientific Products, S. Plainfield, NJ, USA 14. Pace NR, Stahl DA, Lane DJ, Olsen GJ (1986). The
analysis of natural microbial populations by ribosomal
RNA sequences. Adv Microb Ecol 9: 1-55.
References 15. Paster BJ, Dewhirst FE (1988). Phylogeny of campy-
lobacters, wolinellas, Bacteroides gracilis, and
1. Bentley RW, Leigh JA (1995). Development of PCR- Bacteroides ureolyticus by 16S ribosomal ribonucleic
Ba~ed hybridization protocol for identification of
acid sequencing. Inter J System Bacteriol 38: 56-
streptococcal species. J Clin Microbiol 33: 1296- 62.
1301. 16. Paster BJ, Dewhirst FE, Cooke SM, Fussing V,
2. Bentley RW, Leigh JA, Collins MD (1991). Poulsen LK, Breznak JA (1996). Phylogeny of not-
Intrageneric structure of Streptococcus based on com- yet-cultured spirochetes from termite guts. Appl
parative analysis of small-subunit rRNA sequences. Environ Microbiol 62: 347-352.
Intern J System Bacteriol 41: 487-494. 17. Saiki RK, Walsh PS, Levenson CH, Erlich HA (1989).
3. Bentley RW, Leigh JA, Collins MD (1993). Genetic analysis of amplified DNA with immobilized
Development and use of species-specific oligonu- sequence-specific oligonucleotide probes. Proc Natl
cleotide probes for differentiation of Streptococcus Acad Sci 86: 6230-6234.
uberis and Streptococcus parauberis. J Clin Microbiol 18. Saitou N, Nei M (1987). The neighbor-joining
31: 57-60. method: A new method for reconstructing phyloge-
4. Cangelosi GA, Iversen JM, Zuo Y, Oswald TK, netic trees. Mol BioI Evol 4 :406-425.
Lamont RJ (1994). Oligonucleotide probes for mutans 19. Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst
streptococci. Mol Cell Probes 8: 73-80. FE, Levin AE (1994). 'Checkerboard' DNA-DNA
5. Choi BK, Paster BJ, Dewhirst FE, Gobel UB (1994). hybridization. Biotechniques 17: 788-792.
Diversity of cultivable and uncultivable oral spiro- 20. Stackebrandt E, Liesack W, Goebel BM (1993).
chetes from a patient with severe destructive peri- Bacterial diversity in a soil sample from a subtrop-
odontitis. Infect Immun 62: 1889-1895. ical Australian environment as determined by 16S
6. Dewhirst FE, Paster BJ (1991). DNA probe analyses rDNA analysis. FASEB J 7: 232-236.
for the detection of periodontopathic bacteria in 21. van Houte J, Lopman J, Kent R (1994). The predom-
clinical samples. In: Hamada S, Holt SC, McGhee inant cultivable flora of sound and carious human root
JR (eds), Periodontal disease: Pathogens and surfaces. J Dent Res 73: 1727-1734.
Host Immune Responses (pp 367-377). London: 22. van Houte J, Lopman J, Kent R (1996). The final pH
Quintessence Publishing Co., Ltd. of bacteria comprising the predominant flora on sound
7. Giovannoni S (1991). The polymerase chain reaction, and carious human root and enamel surfaces. J Dent
p. In: Stackebrandt E, Goodfellow M (eds), Nucleic Res 75: 1008-1014.
Acid Techniques in Bacterial Systematics (pp 23. Ward DM, Weller R, Bateson MM (1990). 16S rRNA
177-203). John Wiley & Sons, New York. sequences reveal numerous uncultured microorgan-
8. Jacobs JA, Schot CS, Bunschoten AE, Schouls LM isms in a natural community. Nature 345: 63-65.
(1996). Rapid species identification of 'Streptococcus
milleri' strains by line blot hybridization: Identifica-
tion of a distinct 16S rRNA population closely related Author for correspondence: Bruce Jay Paster, Forsyth
to Streptococcus constellatus. J Clin Microbiol 34: Dental Center, 140 Fenway, Boston, MA 02115, USA
1717-1721. Phone: (617) 262-5200 x288; Fax: (617) 262-4021
9. Jukes TH, Cantor CR (1969). Evolution of protein E-mail: bpaster@forsyth.org
Methods in Cell Science 20: 233-239 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. Enterococci (Enterococcus faecium and spread. Pulsed-field gel electrophoresis (PFGE) has
Enterococcus faecalis) and streptococci such as emerged as the one of the most widely applicable,
Streptococcus pyogenes (Group A streptococcus), reproducible, and stable methods to examine strain
Streptococcus agalactiae (Group B streptococcus), identity in bacterial organisms. The procedure used
and Streptococcus pneumoniae are increasing in in our laboratory for PFGE typing of whole cell DNA
importance as both hospital-acquired and community digested with SmaI for enterococci, S. pneumoniae,
pathogens. Emerging resistance and increasing inci- S. pyogenes, and S. agalacatiae is presented. Issues
dence of these organisms has necessitated the regarding interpretation are also reviewed and dis-
analysis of their epidemiologic mechanisms of cussed.
Abbreviations: PFGE = pulsed-field gel electrophoresis; CHEF =contour-clamped homogenous electric fields
This review will focus on PFGE typing procedures C. Glassware and Plasticware
for the above-mentioned organisms: enterococci - Sterile pipet tip (USA Scientific Plastics, Cat.
(including Enterococcus Jaecium and E. Jaecalis), #1011-1006).18
Group B streptococci, Group A streptococci, and S. - Graduated conical centrifuge tubes with caps,
pneumoniae. (Sarstedt, Inc., Cat. #62.553.002PS).19
- 125 ml Erlenmeyer flask (Baxter Diagnostics,
Order #F4253-125).13
2. Materials - Petri dish lid (Fisher Scientific, Order #8-
757-12).10
A. Equipment and supplies - Pasteur pipet (WWR Scientific Products, Order
- Sterile inoculating loop (Secure Medical #TXATIl4673-0).20
Products, Inc., Cat. #58335).1 - Microcentrifuge tube (Fisher Scientific, Cat.
Test tube rack (Bel Art Products, Cat. #05-407-10).10
#F18746-0002).2 D. Chemicals/Reagents
Pipetter (Ulster Scientific, Inc., Mfr. - Trypticase soy agar with 5% sheep blood
#V200TE).3 (Becton Dickinson & Company, Order
Tabletop centrifuge (IEC, Cat. #2438).4 #21261).2 1
Sterile transfer pipet (Samco Scientific, Order - Trypticase soy broth (TSB) (Becton Dickinson
#202-15).5 & Company, Order #4397342).2 1
5.0 McFarland Equivalence Turbidity Standard - Brain heart infusion broth (Becton Dickinson
(Remel, Cat. #20-415).6 & Company, Order #21812).2 1
Sterile cotton-tipped applicator (Citmed, Order - Todd-Hewitt broth (Difco Laboratories, Order
#22-9589).1 #0492-17 _6).2 2
- Microwave oven (Sears, Roebuck & Company, - 5% yeast extract (lCN Biomedicals, Inc., Order
Kenmore Model #721.89652).8 #103303).23
- 50°C water bath (Precision Scientific, Order - Glucose (ICN Biomedicals, Order #901521).23
#66800).9 - Choline chloride (Sigma, Order #C-1879).15
- Lead ring (Fisher Scientific, Order #05- - NaCI (EM Science Industries, Inc., Order
869-1).10 #SX0420-1).24
- Plug molds (BioRad, Cat. #170-3622 )Y - Tris-base (Sigma, Cat. #T-8524).15
- Kimwipes (Kimberly Clark Corp., Order - Low melting point agarose (Boehringer
#34155).12 Mannheim, Order #100-450).25
- Bulb (Baxter, Cat. #P5215).13 - TRIZMA hydrochloride (Tris), (Sigma, Cat.
- Teflon spatula (Fisher Scientific, Cat. #14- #T-7149).15
356-8).10 - Ethylenediamine tetraacetic acid (EDTA)
- 37°C water bath (Precision Scientific, Cat. (Sigma, Cat. #E-1644).15
#66800).9 - Polyoxyethylene 20 cetyl ether (Brij 58)
- Rack for microcentrifuge tubes (Fisher (Sigma, Cat. #P-5884).15
Scientific, Cat. #03-213-2).10 - Deoxycholic acid (deoxycholate) (Sigma, Cat.
- Contour-clamped homogeneous electric fields #D-6750).15
system (CHEF DRII System Cat. #170-3612 or - N-Iauroyl sarcosine (sarcosyl) (Sigma, Cat.
CHEF DRIll System, BioRad Laboratories, #L-9150).15
Cat. #170-3695).1l - Ribonuclease A (Sigma, Cat. #R-6513).15
- Gel casting stand and comb (BioRad - Lysozyme (Sigma, Cat. #L-6876).15
Laboratories, Cat. #170-3620 and 170-4326).1l - Mutanolysin (Sigma, Cat. #M-9901).15
- UV transilluminator (254-360 nm) (Fotodyne, - Proteinase K (Sigma, Cat. #P-6556).15
Inc., Cat. #3-3000, 120V).14 - Pulsed-field certified agarose (BioRad
- Polaroid camera and photographic hood Laboratories, Cat. #162-0137).1l
(Fotodyne, Inc., Cat. #5-5330 and 5-5343).14 - Tris/borate/EDTA buffer (lCN, Aurora, OH,
- Polaroid film (Sigma, Cat. #F-3390).15 Cat. #816202).2 3
B. Optional equipment: - Ethidium bromide (Sigma, Cat. #EI51O).15
- Computerized scanner (HP ScanJet II cx,
Hewlett Packard) or gel documentation system
(Gel Doc 1000, Biorad Cat. #170-7266).1l 3. Procedures
- Molecular epidemiology analysis software
program (Dendron software, Solltech, Inc. 17 or U sing sterile technique, streak a single colony for
Molecular Analyst Fingerprinting software, isolation onto a trypticase soy agar plate with 5%
Biorad Laboratories, Cat. #170-7637).1l sheep blood using a sterile inoculating loop and
incubate the plate at 37°C with 5% CO2 overnight.
235
For enterococci, Group A streptococci, and Group The contents should also be examined for complete
B streptococci, transfer 2-3 isolated colonies to melting of the agarose.
5 ml of brain heart infusion broth and incubate at Add weight to the Erlenmeyer flask with a lead
37°C overnight in a test tube rack. ring (to prevent tipping) and place into a 50°C water
For Streptococcus pneumoniae, transfer 2-3 bath. Place a Petri dish lid over the opening of the
isolated colonies to 5 ml TSB, add choline chloride flask. Also place the test tube rack with the cell
to 1%, and 50 ~l of lysed horse blood (LHB). suspensions into the 50 °C water bath.
Incubate overnight at 37°C in the presence of 5% Clean the plug molds with sterile dH 20 and
CO2 , Kimwipes and assemble.
Beginning with the first graduated tube in the front
LHB preparation of the rack, move left to right, examining the tube
Aseptically distribute 250 ml of defibrinated horse to determine the volume of the cell suspension. Add
blood (Remel order #54-096)6 to 4 or 6 250 ml an equal volume of the low melting point agarose to
capacity Nalgene centrifuge bottles. Add an equal make a 1:2 dilution using a 5 3/ 4 inch Pasteur pipet
volume of sterile distilled water to each bottle. and bulb. Take care not to contaminate the flask
- Run each bottle through 5 freeze-thaw cycles as of agarose with any of the cell suspensions. Mix
follows: thoroughly by pulling the mixture up into the pipet
Tilt bottles slightly and store overnight at and expelling it back into the tube several times.
-20°C. Remove bottles from freezer in AM Finally, draw the cell/agarose suspension up into the
and allow to thaw at room temperature during pipet. Fill three wells of the plug mold, from right
the day. Return to freezer at the end of the day. to left, three isolates per mold, leaving the last well
- Centrifuge at 12,000 xg, 5°C, 20 min. blank. Fill the wells of the mold slowly to avoid
- Decant supernatant (LHB) into appropriate bubble formation in the wells. Refrigerate at 4 °C for
containers and store at -70 or -20°C. 20 minutes.
Pour the overnight growths into sterile 15 ml gradu- Prepare fresh centrifuge (or other sterile) tubes by
ated conical centrifuge tubes with caps and centrifuge labeling them and organizing them in the same order
at 4000 rpm for 20 minutes. Pour off the supernatants as those in the 50°C water bath. Fill each with 5 ml
completely. Resuspend the cell pellets completely in lysis buffer.
PIV buffer. Any clumps of bacteria in the cell sus-
pension will result in a white streak in the lane of the Lysis buffer, pH 7.6
gel. - 6 mM TRIZMA hydrochloride (Tris)
- 1.0 M NaCl
PlV buffer, pH 7.6 - 100 mM ethylenediamine tetraacetic acid (EDTA)
- 1.0 M NaCl 0.5% polyoxyethylene 20 cetyl ether (Brij 58)
- 10 mM Tris-base - 0.2% deoxycholic acid (deoxycholate)
The amount of buffer added to each cell pellet will - 0.5% N-Iauroyl sarcosine (sarcosyl)
depend on the size of the pellet (typically between - 50 ~g/ml ribonuclease A
0.5 ml and 1 ml). Pour a small quantity of the PIV - 1 mg/mllysozyme
buffer stock into a separate sterile tube and add the For S. pneumoniae and Group B streptococcus,
PIV buffer drop-wise with a sterile transfer pipet. The add 200 U mutanolysin per ml of lysis buffer.
resulting suspension should have the opacity of a 5.0 When the plugs are cool, remove them from the
McFarland standard. The same optical density should molds when cooled with the curved end of a Teflon
be used forsolates being compared to each other. spatula. Move from left to right, keeping the blank
Arrange the tubes in a test tube rack in rows of space next to third isolate of each group, and insert
three. There will be three isolates per ten-sample plug in tubes containing lysis buffer.
mold and three plugs (wells) per isolate. Incubate overnight in a 37°C water bath with
If results are urgently needed, the cell suspensions gentle shaking at approximately 65 rpm (add weight
can be made by taking colonies directly from an agar to the test tube rack with a lead ring).
plate of the isolated bacteria using a sterile cotton- Pour off the lysis buffer and replace with 5 ml of
tipped applicator and suspending them in 0.5-1.0 ml ESP buffer.
of PIV buffer.
In a glass 125 ml Erlenmeyer flask, prepare a ESP buffer, pH 9.0-9.5
quantity of 1.6% low melting point agarose in PIV - O.4M EDTA
buffer equal to the volume of all cell suspensions - 1% sarcosine
combined plus some dead volume (plus 5-10 mls - 0.5 mg/ml proteinase K
extra). A microwave oven, at 30% power for approx- Incubate overnight in a 50°C water bath with
imately 2-3 minutes, can be used to melt the agarose. gentle shaking (65 rpm).
The agarose should be checked at 30-second inter- Pour off the ESP buffer completely and replace
vals, and the contents swirled to ensure even heating. with 5 ml of TE buffer.
236
TE buffer, pH 7.5 tube. Add lml of TE buffer to the tube. Incubate the
- 20 mM Tris tubes at 37°C for 1 hour.
- 5 mM EDTA
Incubate at 37°C for 30 minutes with gentle Running the gel. Make 120 ml of 0.8% pulsed-field
shaking. Repeat 5x. certified agarose in 0.5x Tris/borate/EDTA buffer.
Place plugs in fresh TE buffer and store at 4 °C Allow the agarose to cool in a 50°C water bath.
until used. (Weight the flask and cover the opening of the flask
with a Petri dish.)
Digestion of the agarose plugs prior to elec- Clean the gel casting stand and comb with
trophoresis. Create a legend of the samples including Kimwipes and dH 20. Attach the end plates securely
an appropriate DNA size standard. This should with the screws. Place the casting stand on a level
indicate which position each plug will have in the surface. Adjust the comb to 2 mm above the casting
gel. It should also contain data regarding the para- stand surface and 5-10 mm away from one of the end
meters at which the gel will run (i.e., pulse interval, plates. Pour the agarose gently into the casting stand
voltage, duration of run.) so that no bubbles are formed. Inspect the agarose
Label a 1.5 ml snap cap microcentrifuge tube for for bubbles. If there are any, pop them with a Pasteur
each plug on your proposed gel with a permanent pipet before the agarose solidifies. Allow the agarose
marker. Place the tubes in the same order as they will to set for 1 hour. Prepare the contour-clamped
appear on the gel in a rack for microcentrifuge tubes. homogenous electric fields instrument by pumping
Add 270 ~l of sterile dH 20 to each of the micro- any residual buffer from the lines and draining the
centrifuge tubes. instrument. Gently wipe the inside of the CHEF with
Then add 30 ~l of the lOx reactionlincubation Kimwipes. Be careful in the area of the electrodes;
buffer intended for use with the restriction endo- wipe them very gently in a single motion along the
nuclease selected for digestion of the DNA to each length of the platinum wire. Plug the drain tubing of
of the microcentrifuge tubes. For enterococci, S. the instrument, and adjust the CHEF so that it is
pneumoniae, Group B streptococci and Group A level. Use a leveling instrument to ensure this is done
streptococci, SmaI, which recognizes the sequence accurately.
CCCGGG can be used. These recognition sites are Add 2 liters of 0.5x TBE buffer to the CHEF, and
infrequent in most Gram-positive organisms which turn on the pump and chiller at least 30 minutes prior
have A-T rich genomes [6]. to the run. Be sure that the buffer is circulating at one
Next, cut a third of a single plug for each isolate liter per minute and that the chilling device keeps the
to be run on the gel with the Teflon spatula by buffer at 14°C during the electrophoresis.
placing the blade perpendicular to the plug and Remove the comb from the solidified agarose
pressing down in a single motion. The plugs are and add some 0.5x TBE buffer to the wells. This
semi-soft and should be cut easily. Then, using the will facilitate loading of the DNA inserts and reduce
spatula, lift the piece of plug, wick the excess TE the possibility of the formation of bubbles in the
buffer with a Kimwipe from the plug and place it into wells. Using the Teflon spatula, place each DNA
the labeled microcentrifuge tube. The remaining two- insert into the well designated on your template
thirds and extra two plugs are returned to a labeled for that plug. If bubbles form in the wells, use the
tube containing fresh TE buffer and refrigerated for spatula to gently manipulate the DNA insert until the
future use. bubble is eliminated. It may be necessary to add more
Add 3 ~l of the restriction endonuclease chosen 0.5x TBE buffer to the well in order to accomplish
for digestion (SmaI) to each microcentrifuge tube. this.
The enzyme must remain on ice or in a chilled con- Move the casting stand to the CHEF instrument
tainer during this time, and returned to the freezer and loosen the screws of the casting stand. Remove
as soon as possible. Close the microcentrifuge tubes the end plates and be careful that the gel does not slip
and mix the contents of each by inverting gently 4 out of the casting stand. Position the gel on the
or 5 times. Then gently tap the pointed end of the casting stand over the surface of the buffer in the
tube on the benchtop to ensure that the plug is CHEF and lower the casting stand and gel into the
completely covered by the enzyme/buffer mixture. buffer leading with the end of the gel that contains
Examine the tube to check that the plug is not stuck the wells. It is necessary that it be done this way so
in the cap, and to make sure that it is indeed present that the plugs are not popped out of the wells by the
in the tube. Return the tube to the rack and incubate pressure of the buffer under the gel. Gently push the
the tubes overnight at the temperature recommended gel off the casting stand into the buffer and position
by the manufacturer for the restriction endonuclease it behind the gel stops. Seat the gel gently by pushing
(20°C for SmaI). down lightly on the gel. Check to be sure that the
After the incubation period is complete, pour off gel is sitting flat on the bottom of the gel box.
the enzymelbuffer mixture and keep the plug in the Replace the lid of the CHEF and take care not to
237
bend the contacts of the interlock. Set the pulse times 4. Results, interpretation, and discussion
(initial and final), run time and voltage.
There should be at least 10 bands in the PFGE pattern
Pulse times. See Table 1 for pulse times. Settings for optimal evaluation and comparison to other
may need to be adjusted due to individual instrument patterns . PFGE patterns may be compared visually
variation and conditions. Use shorter pulse times or with the use of a computerized scanning and
for resolution of smaller DNA fragments. Use software system. A computerized system for analysis
longer pulse times for resolution of larger DNA is quite useful for a large number of isolates or for
fragments. comparison of isolates over a long period of time.
After the electrophoresis is complete, stain the gel Standard guidelines exist for the interpretation of
in 0.5 ~g/ml ethidium bromide for 20-30 min. It may strain relatedness in PFGE [16, 17]. In general, iden-
be necessary to decolorize the gel in dH 2 0 for 30 min tical PFGE patterns among isolates indicate that those
to 1 hour. The bands can be visualized (using eye isolates are the same strain. Choose the predominant
protection) on a UV transilluminator (254-360 nm) pattern or the pattern from the index patient of an
and photographs can be taken with a Polaroid camera outbreak to be the index PFGE pattern. PFGE pattern
and photographic hood and Polaroid film. Dispose of differences of only 2 to 3 bands compared to the
the used ethidium bromide as directed by your insti-
tutional safety committee.
....._
.. -----
-..... .- ..... --
.. -
.........
--=
- -" •
I ..
.:
Table 1. Pulse times applied for PFGE of total cell DNA
-= ..
-... -- •
-
from different species of enterococci and streptococci
-- - - -
-
.- • - -- -
Organism Pulse time
-
- - i-- • =-~ -- II
- -
145.5
-
Enterococcus faecium 1-20 seconds for 15 hours 97
at 200 volts
.:
Enterococcus faecalis 1-20 seconds for 15 hours
at 200 volts
Group B streptococcus 1-30 seconds for 15 hours Figure 2. Pulsed-field gel electrophoresis (PFGE) of
at 200 volts Group B streptococcus (Streptococcus agalactiae) whole
cell DNA digested with Sma!. The PFGE patterns suggest
Group A streptococcus 1-20 seconds for 16 hours a heterogeneous group of strains. Asize standard is shown
at 200 volts at left.
Streptococcus pneumoniae 1-30 seconds for 15 hours
at 200 volts 23456789Kb
-1000
2 3 4 5 6 7 8 9 10 Kb - 436.5
1000 - 339.5
388
- 242 .5
291 - 194
194 - 145.5
145.5 - 97
97 - 48.5
48.5
Figure 3. Pulsed-field gel electrophoresis (PFGE) of
Group A streptococcus (Streptococcus pyogenes) whole
cell DNA digested with Sma!. There are four very different
Figure 1. Pulsed-field gel electrophoresis (PFGE) of patterns present, but the strains providing the pattern in
Enterococcus faecium whole cell DNA digested with Lanes 1 and 2 are highly related (Pattern A), as are those
SmaI. The PFGE patterns of isolates in Lanes 1-10 suggest providing the pattern in Lanes 3, 4 and 5 (Pattern B);
highly related, if not identical, strains. A size standard is Lanes 6 and 7 (Pattern C); and Lanes 8 and 9 (Pattern D).
shown in Lane 11. A size standard is shown in the right lane.
238
index PFGE pattern may indicate a single genetic whole cell DNA, which resolves larger fragments of
event, and these isolates may be closely related to the linear double-stranded DNA (75-600 kb range).
index PFGE pattern. PFGE pattern differences of 4 Examples include vancomycin resistance in entero-
to 6 bands are possibly related to the index PFGE cocci, and macrolide-lincosamide-streptogramin,
pattern. PFGE pattern differences of ~ 7 bands tetracycline, or clindamycin resistance in enterococci
suggest that the isolates are distinct strains. If a com- or staphylococci. In addition, correlate serotyping
puterized analysis is done, computerized dendograms results if available for pneumococcal isolates, since
can be generated . PFGE patterns with coefficients capsular transformation has been reported to occur,
of similarity ~ 85% are examined closely for strain and thus isolates with the same PFGE type may occa-
relatedness [5, 8]. sionally vary by serotype [1, 3]. Most importantly,
Since PFGE of whole cell DNA examines the results of strain identity should be reviewed in the
chromosome, a relatively stable genetic marker, it is context of, and not independently of, existing epi-
a reproducible and accurate method of determining demiologic data for the isolates [16, 17]. Examples
bacterial strain identity. Be careful to correlate anti- of the types of PFGE patterns produced by highly
biotic susceptibility results, since isolates that are the related, as well as divergent, strains of bacterial
same by PFGE type can occasionally vary by anti- species are provided in Figures 1 (E. faecium), 2 (S.
biotic susceptibility results, due to acquisition or loss agalactiae), 3 (S. pyogenes), and 4 (S. pneumoniae).
of an antibiotic resistance trait encoded by an extra-
chromosomal element such as a plasmid. Differences
based on small, usually circular, extrachromosomal Notes on suppliers
genetic elements will not be detected in PFGE of
1. Secure Medical Products, Inc., 1000 Allanson Rd.,
1 2 3 4 5 6 7 8 9 10 11 Mundelein, IL, 60060, USA
kb 2. Bel Art Products, Pequannock, NJ 07440-1992, USA
3. Ulster Scientific, Inc., PO Box 819, New Paltz, NY
-388 12561-0819, USA
-291 4. IEC, 300 Second Avenue, Needham Heights, MA
02194, USA
-194
5. Samco Scientific, 1050 Arroyo Avenue, San
-145.5 Fernando, CA, USA
-97 6. Remel, 12076 Sante Fe Dr., Lenexa, KS 66215, USA
-48.5 7. Citmed, Cottontail Medical Products, 18601 South
Main Street, Citronelle, AL, USA
8. Sears, Roebuck & Company, Hoffman Estates, IL
60179, USA
Figure 4a. Pulsed-field gel electrophoresis (PFGE) of 9. Precision Scientific, 3737 West Cortland Street,
Streptococcus pneumoniae whole cell DNA digested with Chicago, IL 60647-9987, USA
SmaI. The PFGE patterns are highly related. 'A size 10. Fisher Scientific, 711 Forbes Avenue, Pittsburgh, PA
standard is shown at right. 15219, USA
11. BioRad Laboratories, 2000 Alfred Nobel Drive,
Hercules, CA 94547, USA
5 7
-.
1 2 3 4 6 8 9 kb 12. Kimberly Clark Corporation, Roswell, GA 30076-
... . . .= --
2199, USA
. .... .
1430 Waukegan Road, McGaw Park, IL 60085, USA
,---
-291 14. Fotodyne, Inc., 950 Walnut Ridge Drive, Hartland, WI
.
-194 53029-9910, USA
-
.~.-. iii ,..... '. .. 15. Sigma Chemical Company, PO Box 14508, St. Louis,
~ ~ • ;: ~ ~. I j iii
No.9, Oakdale, IA 52319, USA
-48.5
18. USA Scientific Plastics, PO Box 3565, Ocala, FL
34478, USA
19. Sarstedt, Inc., Newton, NC 28658-0468, USA
20. WWR Scientific Products, West Chester, PA 19380,
Figure 4b. Pulsed-field gel electrophoresis (PFGE) of USA
Streptococcus pneumoniae whole cell DNA digested with 21. Becton Dickinson & Company, Cockeysville, MD
SmaI. The PFGE patterns in Lanes 2, 3, and 4 represent 21030, USA
related strains, whereas those in Lanes 1,5, 6, 7, 8, and 9 22. Difco Laboratories, Detroit, MI 48232-7058, USA
were produced from DNA from distinct strains. 'A size 23. ICN Biomedicals, Inc., 1263 Chillicothe Road,
standard is shown at right. Aurora, OH 44202, USA
239
24. EM Science Industries, Inc., 400 South Democrat faecium during its emergence in a city in southern
Road, Gibbstown, NJ, USA Texas. Clin Infect Dis 21: 1234-1237.
25. Boehringer Mannheim, 9115 Hague Road, PO Box 10. Munoz R, Coffey TJ, Daniels M, et al. (1991).
50414, Indianapolis, IN, USA Intercontinental spread of a multiresistant clone of
serotype 23F Streptococcus pneumoniae. J Infect Dis
164: 302-306.
References 11. Murray BE (1990). The life and times of the entero-
coccus. Clin Microbiol Rev 3: 46-65.
1. Barnes DM, Whittier S, Gilligan PH, Soares S, 12. Murray BE, Singh KV, Heath JD, Sharma BR,
Tomasz A, Henderson FW (1995). Transmission of Weinstock GM (1990). Comparison of genomic DNAs
multidrug-resistant serotype 23F Streptococcus pneu- of different enterococcal isolates using restriction
moniae in group day care: Evidence suggesting endonucleases with infrequent recognition sites. J Clin
capsular transformation of the resistant strain in vivo. Microbiol 28: 2059-2063.
J Infect Dis 171: 890-896. 13. Patterson JE, Sweeney AH, Simms M, Carley N,
2. Blumberg HM, Stephens DS, Modansky M, Erwin M, Mangi R, Sabetta J, Lyons RW (1995). An analysis of
Elliot J, Facklam RR, Schuchat A, Baughman W, 11 0 serious enterococcal infections: Epidemiology,
Farley MM (1996). Invasive group B streptococcal antibiotic susceptibility, and outcome. Medicine 74:
disease: The emergence of serotype V. J Infect Dis 191-200.
173: 365-373. 14. Ramage L, Green K, Pyskir D, Simor AE (1996). An
3. Coffey TJ, Dowson CG, Daniels M (1991). Horizontal outbreak of fatal nosocomial infections due to group
transfer of multiple penicillin-binding protein genes, A streptococcus on a medical ward. Infection Control
and capsular biosynthetic genes in natural populations Hosp Epidemiol 17: 429-431.
of Streptococcus pneumoniae. Mol Microbiol 5: 15. Stevens DL, et al. (1989). Invasive Group A strepto-
2255-2260. coccal infections associated with a toxic shock-like
4. Harrison LH, Ali A, Dwyer DM, Libonati JP, Reeves syndrome and scarlet fever toxin A. New Engl J Med
MW, et al. (1995). Relapsing invasive Group B strep- 321: 1-7.
tococcal infection in adults. Annals Int Med 123: 16. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA,
421-427. Murray BE, Persing DH, Swaminathan B (1995).
5. Hellstein J, Vawter-Hugart H, Fotos P, Schmid J, SoIl Interpreting chromosomal DNA restriction patterns
DR (1993). Genetic similarity and phenotypic diver- produced by pulsed-field gel electrophoresis: Criteria
sity of commensal and pathogenic strains of Candida bacterial strain typing. J Clin Microbiol 33:
albicans isolates from the oral cavity. J Clin Microbiol 2233-2239.
31: 3190-3199. 17. Tenover FC, Arbeit RD, Goering RV, and the
6. Holt JG (editor-in-chief) (1984). Bergey's manual of Molecular Typing Working Group of the Society for
systematic bacteriology, Volumes 1 and 2. Baltimore: Healthcare Epidemiology of America (1997). How to
Williams and Wilkins. select and interpret molecular strain typing methods
7. Lai E, Birren BW, Clark SM, Simon MI, Hood L for epidemiological studies of bacterial infections: A
(1989). Pulsed field gel electrophoresis. review for healthcare epidemiologists. Infection
BioTechniques 7: 34-42. Control Hosp Epidemiol 18: 426-439.
8. Moreno F, Crisp C, Jorgensen JH, Patterson JE
(1995). The clinical and molecular epidemiology of
bacteremias at a university hospital caused by Address for correspondence: Jan E. Patterson, MD,
pneumococci not susceptible to penicillin. J Infect Dis Department of Medicine/lD, University of Texas Health
172: 427-432. Science Center at San Antonio, 7703 Floyd Curl Dr., San
9. Moreno F, Grota P, Crisp C, Magnon K, Melcher GP, Antonio, TX 78284-7881, USA
Jorgensen JH, Patterson JE. Clinical and molecular Phone: (210) 358-2927; Fax: (210) 358-4702
epidemiology of vancomycin-resistant Enterococcus E-mail: pattersonj@uthscsa.edu
Methods in Cell Science 20: 241-249 (1998).
© 1998 Kluwer Academic Publishers.
Abstract. Surface proteins provide a multitude of the surface adhesin protein Fap 1 were produced
functions for the bacterial cell. Antibodies to these using phage display. The immune repertoire of a
proteins can provide tools for tagging bacteria and mouse injected with purified Fapl was cloned into
characterizing protein function. Phage display the phagemid vector pCOMB3, and a combinatorial
technology has emerged as a powerful method for Fab library was expressed in E. coli. A cell-based
producing monoclonal Fabs in Escherichia coli. In panning method using whole S. parasanguis cells
an effort to study the adhesion mechanisms of was developed and has been shown to be a means
Streptococcus parasanguis FW213, Fabs specific for for enriching for Fabs specific for the Fapl protein.
- Sephadex G-50 DNA Grade F Cat. #17-0573- C. RNA extraction and cDNA synthesis
02 Pharmacia Biotech. Extract RNA from the spleen cells with
- Spin Columns Cat. #C1281. TriReagent according to the manufacturer's
- Dye Terminator Cycle Sequencing Ready instructions [for explanation see 8]. Briefly,
Reaction Kit Part #402079 Perkin E1mer/ homogenize spleen cells in 5ml TriReagent and
Applied Biosystems.1 4 centrifuge to remove cell debris. Extract the
D. Supplies and equipment resulting supernatant with 0.2ml chloroform/ml
- Centric on 100 Cat. #4212 Amicon. 15 supernatant. Save the upper aqueous phase and
- Gene Pulser Apparatus Cat. #165-2105 Bio- use a Centricon 100 to concentrate while
Rad. 16 exchanging buffer for nuclease free water.
- Rapidcycler Idaho Technologies. Reverse transcribe the first strand of cDNA
- 96 well tissue culture treated plates Cat. with Superscript II. Heat 5 J..lg of total RNA and
#25860 Costar. I? 1 J..lg of oligo(dT) (I5-mer) primer to 70°C for
- E.I.A.lR.I.A. Plate AI2 96 well microtiter 10 minutes and then quickly coolon wet ice. Add
plates Cat. #3690 Costar. manufacturer's buffer to a final concentration of
- Nunc-Immuno Wash 12 Cat. #62409-160 VWR Ix along with 500 units of Superscript II. Incubate
Scientific Products. IS the reaction at 40°C for 1-1.5 hours and
- Automated Microplate Reader EL311sBio-Tek inactivate the enzyme by incubating at 70 °c for
Instruments, Inc. 19 10 minutes. Concentrate the reaction mix and
exchange the buffer for water with a Centricon
100.
3. Procedure D. PCR amplification of heavy and light chain
sequences
A. Immunization Use the following primers to PCR amplify heavy
Mix antigen (purified Fap1, 10-20 J..lg per injec- chain Fd (CHI + V H) DNA fragments: 5' primers
tion) with RIBI adjuvant as per manufacturer's - HeI-8 agg tcc a(g/a)c t(g/t)c tcg agt c(t/a)g and
instruction and inject into two BALB-c mice. Hc9 agg tii aic tic tcg agt c(a/t)g g; 3' class
Give each mouse two 100 J..ll injections, one under specific primers - IgG I agg ctt act agt aca atc cct
each arm, and boost twice at three week intervals ggg cac aat, IgG 2A ggt ctg act agt ggg cac tct ggg
with the same injections. Three days after the final ctc, IgG 3 ggg ggt act agt ctt ggg tat tct agg ctc.
boost, euthanize the mice by asphyxiation, remove Use the following primers to PCR amplify light
their spleens and place immediately in TriReagent chain genes: 5' primers - LeI cca gtt ccg agc tcg
for RNA extraction. Divide spleen/TriReagent in ttg tga ctc agg aat ct, Lc2 cca gtt ccg agc tcg tgt
half, place in two microfuge tubes, and homoge- tga cgc agc cgc cc, Lc3 cca gtt ccg agc tcg tgc
nize with a plunger centrifuge at 10,000 rpm for tca ccc agt ctc ca, Lc4 cca gtt ccg agc tcc aga tga
10 minutes in a microfuge to isolate serum. ccc agt ctc ca, Lc5 cca gat gtg agc tcg tga tga ccc
B. Immune response aga ctc ca, Lc6 cca gat gtg agc tcg tca tga ccc agt
Assess the general serum response of each mouse ctc ca, Lc7 cca gtt ccg agc tcg tga tga cac agt ctc
to antigen (purified Fap1 protein) by ELISA ca; 3' primer - kappa gcg ccg tct aga att aac act
assay. Suspend antigen in phosphate buffered cat tcc tgt tag a. Heavy chain extension primers
saline (PBS) consisting of 0.14 M NaCl, 1 mM are: VHI Ext gag aga gag aga gag aga agg tcc arc
KH2P0 4 , 20 mM Na2HP04 , and 3 mM KCl, pH7.4 tkc tcg a, VH9 Ext gag aga gag aga aga agg tii aic
[21] and immobilize on a Costar AI2 micro titer tic tcg a, IG I Ext gag aga gag aga gag aga agg ctt
plate (0.24 J..lg Fap1 protein/well) by incubating at act agt, IG 2 Ext gag aga gag aga gag aga gtt ctg
37°C for one hour. Block non-specific binding act agt, and IG 3 Ext gag aga gag aga gag aga ggg
sites with PBS/gelatin (0.5%). Add a 1:1000 ggt act agt. Light chain extension primers are: VL
dilution of mouse sera in PBS and detect antibody Ext gag aga gag aga gag aga ccacwt cyt agc tcg
binding with 1:5000 dilution of HRP (horse radish and CL Ext gag aga gag aga gag aga gcg ccg tct
peroxidase) conjugated anti-mouse IgG secondary aga [3].
antibody. Incubate each addition for one hour at All PCR was carried out with an Idaho
37°C. Before each new addition, wash three times Technologies Rapidcycler that works by air con-
with PBS. After incubating with secondary, wash vection and requires significantly less time than
twice with PBS and once with TPBS (PBS + 0.5% traditional thermocyclers. Specified concentra-
Tween 20). Perform all washes using a Nunc- tions of each reaction component in a Ix reaction
Immuno Wash 12. Detect a positive immune are as follows: 200 J..lM dNTPs, 0.5 J..lM each
response with an OPD (o-phenylenediamine) primer, 0.4 units/J..lI Amplitaq polymerase in
based substrate or a similar substrate that enzyme diluent composed of 10 mM Tris pH8.3,
produces a color reaction that is detectable by a 2.5 mg/ml BSA (bovine serum albumin), and a
spectrophotometer. reaction buffer containing 50 mM Tris, 250 J..lg/ml
244
BSA, 2% sucrose, 0.1 mM Cresol Red and either Blue with 1-2 fll of the ligation reaction by
2 mM, 3 mM or 4 mM Mg2+. PCR amplify heavy electroporation. Plate dilutions on Superbroth
chains using a combination of the 5' primers (SB) agar plates containing carbenicillin (50
(Hcl-8, 9) plus each 3' primer (IgG l , IgG 2a, or flg/ml) and tetracycline (10 flg/ml) to assess
IgG 3 ) in three separate reactions. Amplify light library size. Use 250 ng of insert plus 1 flg of
chains with each 5' primer (Lcl-7) plus the 3' vector in preparative library ligations. Construct
primer (kappa) in seven separate reactions. Carry redundant libraries by ligating heavy chains first
out PCR from the cDNA (1:10 dilution) in 10 fll followed by light chain PCR products and vice
reactions with the following PCR conditions: versa. Ligate the first PCR product, and transform
initial denaturation cycle of 95°C for 5 s E. coli XLI-Blue by electroporation with the
(seconds) followed by 37-40 rounds of 94°C for entire ligation reaction. Isolate plasmid the
o s, 52-54°C for 5 sand 72 °c for 35 s. Complete following day. Insert the second PCR product, and
with a final extension step of 72 °c for 60 s. again, transform E. coli XLI-Blue by electro-
E. Vector preparation and restriction digests poration with the entire ligation reaction.
Gel purify PCR products from 2.0% agarose gels Synthesize a high titer phage library in the
using the following method. Resuspend cut get following manner. Combine electroporations in
pieces in water (approximately 2x gel volume) 100 ml of SB containing 10 flg/ml tetracycline
and homogenize in a microfuge tube with a 1 ml that selects for the F factor in XLI-Blue cells.
syringe plunger. Centrifuge at 13,000 rpm in a Harboring the F factor. Incubate at 37°C for
microfuge to remove agarose, remove supernatant 30 minutes followed by addition of carbencillin
and repeat extraction. Concentrate supernatants to a concentration of 20 flg/ml. After an additional
containing DNA in a Centric on 100 while hour of incubation at 37°C, add carbenicillin to
exchanging buffer for distilled water. Combine a final concentration of 50 flg/ml. Carbenicillin
light chain cDNA PCR products while retaining selects for cells harboring the pCOMB3 plasmid.
heavy chains in three separate reactions for exten- Incubate culture for another hour at 37°C and add
sion PCR. Carry out preparative extension PCR helper phage VCSM13 (10 12 pfu) which carried a
in 50 fll reactions to total 250-500 fll all together. gene for kanamycin resistance. Continue to
Follow an initial denaturation step at 96°C for incubate for two more hours at 37°C before
10 s with 25 cycles at 94°C for 0 s, 60°C for adding kanamycin to a final concentration of
5-10 s, and 72 °c for 50 s. Complete the reaction 50 flg/ml to select for helper phage infection. At
with a final extension at 72 °c for 60 s. Gel purify this point, move the culture to 30°C and incubate
products as described above. Combine heavy overnight. Filamentous phage are secreted into the
chain products to result in a single population of medium as the culture grows. In the morning,
heavy chain PCR products and a single popula- centrifuge culture to remove cells and precipitate
tion of light chain products overall. the phage from supernatant (100 ml) with the
Isolate vector pCOMB3 (carbenicillin resis- addition of 30 ml of 20% PEG 8000/2.5 M NaCl.
tance) from E. coli XLI-Blue (recA- recAI, lac-, Recover phage by centrifugation at 10,000 rpm in
endAl, gyrA96, thi, hsdR17, supE44, relAl, {F' a Sorvall GSA rotor for 40 minutes and resuspend
proAB, lacI q, lacilMl5, TnlO}) using Wizard in 1.5 ml PBS/gelatin (high titer Fab library).
plasmid kits according to the manufacturer's G. Panning
instructions. Carry out restriction digests with the Perform solid-based panning by immobilizing
following units (u) of enzyme/flg DNA: digest protein on two wells of a Costar AI2 microtiter
1.7 flg of heavy chain PCR products with Spel plate and incubating at 37°C for 1 hour (Fap
(17 u/flg) and Xhol (70 u/flg), digest 2 flg of light 1-100 ng/well). Normal procedure calls for 1 flg
chain PCR products with Sac! (35 u/flg) and XbaI protein/well, but less was used here due to limited
(70 u/flg), digest 10 flg of pCOMB3 vector with supplies of purified Fap 1. Block non-specific
SpeI (3 u/flg) and Xhol (10 u/flg) and digest binding sites with PBS/gelatin before adding
10 flg of pCOMB3 with Sac! (5 u/flg) and Xbal 50 fll (-1 x 10 12/ml) of the high titer Fab library.
(10 u/flg). Carry out SpellXhoI and Sac!lXbaI Wash with distilled water (3-4x) between each
double digests at 37°C overnight in manufac- step and incubate at 37°C for 1 hour after each
turer's buffers H and A, respectively. Digested addition. After incubating with phage, wash Ix
vector preparations were gel purified on 0.5% with TPBS and 4x with water to remove non-
agarose gels as described above. specific binders. Elute phage with 50 fll 0.1 M
F. Library construction glycine HCI (pH2.2), agitating and scraping sides
Perform all ligations with T4 DNA ligase in the of well with pipetman intermittently for 10-15
manufacturer's buffer at room temperature minutes. Add phage to 2 ml freshly grown XLI-
overnight. For test ligations, use 50 ng of insert Blue and dilute with 8 ml of SB containing
(heavy or light chain PCR products) plus 250 ng tetracycline (10 flg/ml). Plate dilutions (usually
of vector (pCOMB3). Transform E. coli XLI- > 1: 10,000) of culture onto tetracycline (10
24S
Ilg/ml) and carbenicillin (SO Ilg/ml) SB agar Analysis Software Version 2.1.1. Perform dideoxy
plates to assess phage yields and to provide sequencing using a dye terminator cycle
colonies to test individual clones. Add carbeni- sequencing ready reaction kit, and purify products
cillin (20 Ilg/ml) to the 10 ml culture after a half using spin columns packed with G-SO fine grade
hour of incubation. After an additional hour of sephadex. Use the following DNA sequencing
incubation, add 10 ml culture to 100 mls of SB primers: SeqT3 ata acc cct cac taa ag or Cy ggc
containing SO Ilg/ml carbenicillin and grow up to cag tgg ata gac aga for heavy chains and KEF gaa
synthesize phage as described above. Use the ttc taa act agc tag tcg or CK cac tgg atg gtg gga
resulting high titer phage stock from the first aga tg for light chains.
panning in a second round of panning and so on. Generate soluble Fabs by first purifying
Monitor enrichment by testing individual clones pCOMB3 DNA from individual clones using the
from each panning as described below. Wizard mini prep kit. Then digest 2 Ilg pCOMB3
Perform cell-based panning against whole S. DNA with Nhel (9 u/Ilg) and SpeJ (3 u/Ilg) to
parasanguis FW213 cells by centrifuging 1.2ml remove the gene III DNA that fuses the heavy
of overnight culture (grown at 37°C in S% CO 2 ) chain to the pIlI capsid protein. Digestion with
in a microfuge tube at 3000 rpm for 2-3 minutes. these enzymes produces compatible cohesive
Resuspend cells in 200 Ilg PBS/gelatin and ends. Gel purify digested, linear vector and ligate
incubate for 20-30 minutes to block non-specific cohesive vector ends together using ligation
binding sites on the plastic tube before adding conditions as describe above. Transform E. coli
SO III (-1 x 1012/ml) of the high titer Fab library. XLI-Blue with I III of the ligation by electro-
Incubate cells with phage for 2-3 hours at room portation and plate dilutions to obtain single
temperature and centrifuge at 3000 rpm for colonies. Inoculate 10 ml SB plus carbenicillin
1 minute to trap Fabs that are bound to cells. Wash (SO Ilg/ml) with single colonies and incubate at
cells repeatedly by resuspending in 1 ml of PBS 37°C for six hours. Induce by adding 300 IlM
followed by centrifugation (approximately ten IPTG and moving to 30°C to incubate overnight.
times). Resuspend the final pellet in 200 III PBS Harvest soluble Fab by concentrating with
and add phage-bound S. parasanguis cells to 2 ml ammonium sulfate (SO%) directly from the growth
freshly grown E. coli XLI-Blue. At this point, the medium or release Fabs from cells by suspending
phage are competed off of S. parasanguis by in PBS and freezing at -70°C followed by
infection sites on the E. coli. Culture the E. coli thawing at 37°C three times.
to produce phage as described above for solid- FW213 and VT120S (Fapl- 1 kanamycin inser-
based panning. tion mutant) growth culture medium was precip-
H. Clone characterization and soluble Fab produc- itated with 4S% ammonium sulfate and dialyzed
tion in O.S x PBS to provide samples for Western blots.
Pick individual colonies from plates resulting Precipitates from FW213 supernatants contain the
from each round of panning and synthesize phage 200 kDa Fapl protein whereas VT120S contains
as described above. Test individual clones for the all other proteins with the exception of Fap 1. Run
ability to bind wildtype FW213 cells versus an samples (2S Ilg/well) in SDS loading buffer
insertion mutant, VTI20S, that does not express containing beta-mercaptoethanol on SDS poly-
the Fapl protein using a whole cell ELISA assay acrylamide gels (S%). Transfer proteins to nitro-
(BactELISA) [9]. Briefly, dry cells (FW213 and cellulose and after blocking with S% non-fat dry
VT120S) suspended in SO mM carbonate buffer milk/PBS, probe blots with Fabs. Detect Fab
(16 mM sodium carbonate/34 mM sodium bicar- binding with the HRP conjugated anti-mouse IgG
bonate) onto 96 well tissue culture treated plates. as described above (1 :SOOO dilution). React blots
Carry out ELISA in the same general fashion as with a chemi-illuminescent substrate and expose
described above. Add a SO Ill/SO III mixture of to x-ray film to visualize protein bands. Two
culture supernatant containing approximately 109 clones that were isolated by phage display were
phage and PBS/gelatin to the well. Detect Fab used in Western blots (PhS and PhI2). An
binding with a mixture of HRP conjugated anti- ammonium sulfate concentrated stock of soluble
mouse IgG and HRP conjugated anti-M13 Fab was used for clone PhS, while the phage
secondary antibodies (l :SOOO dilution). Although bound form of Ph12 was used directly from
it is not necessary to use two secondary anti- culture supernatants. Since affinity purified phage
bodies, enhanced signals can be achieved in this were not used, functional Fab concentrations were
way because the anti-mouse IgG reacts with the determined using ELISA assays (data not shown).
antibody and the anti-Ml3 reacts with the Fab
bearing phage. Select positive clones for sequence
analysis. Heavy and light chain genes from
pCOMB3 DNA were sequenced on an ABI 373
Stretch Sequencer and analyzed with Sequence
246
4. Results and discussion enough library some of the original heavy and light
chains will be recombined [12].
The serum immune response to antigen was tested by Panning is the process by which antigen specific
ELISA against purified Fap 1 protein compared to the Fabs are selectively sorted from phage libraries.
reaction with blocking agent alone (PBS/gelatin). Multiple rounds of panning are often necessary to
There was no apparent immune response in mouse bring enrichment to a detectable level [2], Initially,
#1. However, a ten-fold immune response over back- solid-based panning against purified Fap 1 immobi-
ground in mouse #2 indicated the immune repertoire lized on a microtiter plate was used, but five rounds
of this mouse contained antibodies specific for the of panning did not yield any positive clones. This
Fapl protein (Figure 1). may have been the result of using small amounts of
Total RNA was isolated from the spleen of mouse the Fap 1 protein, which was in limited supply.
#2 using the method developed by Chomczynski and Alternatively, high affinity binders may not have
Sacchi [8]. cDNA was reverse transcribed from eluted efficiently.
mRNA encoding all the antibodies expressed within Fapl is associated with fimbriae on the surface of
mouse #2. Light chain genes and the Fd fragments S. parasanguis and is important for the adhesion of
of the heavy chain genes that encode V H+ CHI were these bacteria to the tooth surface [26]. As an
amplified from the cDNA by polymerase chain alternative, a cell-based panning method using whole
reaction (PCR). A second round of extension PCR S. parasanguis FW213 cells was developed. This
provided unique restriction sites for cloning products technique has several advantages over traditional
into the phagemid vector pCOMB3. Libraries were solid-based methods. Whole cells provide ample
constructed in a redundant fashion resulting in two supplies of the Fap 1 antigen. Additionally, native
identical libraries differing only in the order in which proteins are presented on cells preventing epitopes
the light or heavy chain PCR products were inserted. from being concealed or denatured by binding to
This method prevents loss of clones due to restric- plastic microtiter plates [10]. Thus, whole cells
tion of the first cloned PCR product with the enzymes increase the chance of isolating antibodies that will
used to clone the second PCR product. Ligations be more relevant in analyzing a protein's biologically
were transformed into E. coli XLI-Blue by electro- active state. Using whole cells also facilitated the
poration and upon the addition of helper phage, a elution of phage after panning. S. parasanguis cells
combinatorial Fab library was synthesized. Insertion with Fabs bound to their surface were added directly
of heavy chain PCR products first resulted in poor to the E. coli culture, which allowed for the phage
electroporation efficiencies. However, insertion of to be competed from the S. parasanguis by infection
light chains first gave good efficiencies and upon sites on the E. coli. Because of the greater numbers
cloning heavy chain PCR products, a library on the of E. coli present in the mixture and the longer
order of 3 x 106 clones resulted. Each clone within elution time allowed by this method, even Fabs with
this library has the potential to possess a different a high affinity for the Fapl protein would be
combination of heavy and light chains encoding a expected to elute. Traditional elution methods may
unique Fab. However, it is expected that with a large not elute the highest affinity Fabs in the given time
frame. Because S. parasanguis does not grow in the
absence of carbon dioxide and it does not have a
2.5 natural resistance to the antibiotics used to culture
the E. coli, there is little chance of S. parasanguis
contamination. With another bacterium where this
might not be the case, it would be feasible to use heat
-= 1.5 +------------- killed cells [5].
~ Two populations of phage were used in cell-based
panning against S. parasanguis cells: the original
0.5
phage library (PL I) and the library that resulted from
two rounds of solid-based panning against purified
o Fapl (PL2). Selective enrichment can be monitored
2 by following the percentage yield of phage from each
Mouse Number
panning if input phage levels and panning surfaces
are approximately equivalent for each round of
panning [2]. Five rounds of panning surfaces are
Figure 1. Serum immune response of mouse #1 and
mouse #2 as tested by ELISA. Mouse sera was diluted
approximately equivalent for each round of panning
1: 1000 in PBS and added to blocked wells on a microtiter [2]. Five rounds of panning were carried out and
plate containing 0.24 Jlg Papl protein/well or PBS/gelatin enrichment was evident with increasing phage yields
alone. Binding of antibodies from the serum was detected in each round of panning. Enrichment was also
with an HRP conjugated anti-mouse IgG secondary quantitated by screening ten random clones from
antibody. Gelatin 0; Pap 1 protein ~. each PL I panning one through four. Clones were
247
initially screened for their ability to bind to FW213 were searched for unique restriction sites to use as a
and not to VTI205, a Fapi insertion mutant that does means of eliminating this clone from the library, but
not express the 200 kDa Fap I protein [26]. A none were found. An attempt was also made to block
hybridoma monoclonal antibody specific for the Fap 1 enrichment of Ph5 by coating FW213 cells with
protein (MAb F51) was used as a positive control. soluble Ph5 Fab prior to panning. However, Ph5
An increase in the proportion of positive clones can clones were still isolated from these pannings and
be seen with each successive panning. The first no additional clones were identified. In fact, the
panning contained no positive three and the fourth continuous reappearance of this clone in independent
panning contained nine positive clones (Figure 2). pannings not only demonstrated the strength of this
A combined total of forty-nine clones from the fifth clone, but was also affirmation of the reproducibility
PL 1 and PL 2 pannings were screened and forty eight of the cell-based panning method.
of these were positive (data not shown). Results of Western blot analyses with Ph5 and
Six clones from each of the fifth PL 1 and PL 2 PhI2 demonstrated the specificity of these Fabs for
pannings were sequenced to differentiate clones. the Fapl protein (Figure 3). MAb F5I was used as
Heavy and light chain genes were sequenced from a positive control and has been shown to react with
pCOMB3 DNA isolated from each clone. All six a 200 kDa protein identified as Fap I as well as a
clones from PL 1 pannings proved to be identical and slightly smaller protein of approximately I80kDa
this Fab was designated Ph5. Three of the clones [26]. Presumably this is a modification, degradation
from PL 2 pannings were also identical to Ph5. product or precursor of the 200 kDa Fap I protein.
However, the remaining three clones had identical PhI2 reacted with the same two bands corresponding
sequences that were distinct from Ph5. This clone to Fap I as F5I. While Ph5 also reacted with these
was specified Ph12. Isolation of a distinct clone from same bands, it also reacted with an uncharacterized
PL 2 supports our hypothesis that this library was lower molecular weight protein that may represent a
biased as compared to the PL 1 library, which had not breakdown product of Fapl. Neither Ph5 nor PhI2
been panned on micro titer plates. Although no reacted with the Fapl- mutant, VT1205. Additionally,
positive clones were isolated from solid-based Ph5 reacted weakly in immunoblots as compared to
pannings alone, this library was probably enriched to Ph12. Although Ph5 and Phl2 Fab stocks used in
some degree. Western blots reacted comparably in ELISA assays
Sequencing clones from the second, third and (data not shown), Ph5 was only minimally detectable
fourth PL 1 pannings revealed Ph5 was present after
two rounds of panning and in every panning there-
MW Ph12 FSl MW PhS
after. Due to the prevailing presence of Ph5, it FW Fap- FW Fap-
FW Fap-
became apparent that further sequencing would not
be a productive means of identifying new clones. The
genes encoding the Ph5 Fd fragment and light chain
10 , - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - ,
9 +----- ·-------------------- 200
8 ~·----------------------------
0:>
:::: 7 - - - - - - .---------
fl
§ 6 +----------------------------~
-;;:; 5 - -- - - - - -- - - - - - - • --
.~
.":: 4 -
1 ----------------------------
&.
'It
3 1- - - - - - - - - - - - - - - - - 68
2 1- - - - - -
43
o \------------
4 Exposure Time:
Panning Number 10 second 2 minute
Figure 2. Selective enrichment in each round of panning Figure 3. Western blot analyses with Ph5 and Ph12.
quantitated by whole cell ELISA assay. Ten clones from Ammonium sulfate precipitated samples from wildtype
each round of panning 1-4 were tested for their ability to FW213 (FW) and VTl205 Jap1 mutant (Fap-) culture
bind to S. parasanguis FW2l3 and VTl205, aJap 1 inser- supernatants were probed in Western blots with a con-
tion mutant. Binding of phage bound Fabs directly from centrated ammonium sulfate precipitated stock of soluble
culture supernatants was detected with a mixture of HRP Fab clone Ph5, a stock of clone Ph12 in phage bound form
conjugated anti-mouse IgG and HRP conjugated anti-M13 directly from culture supernatants or MAb F51 (positive
secondary antibodies. Positive clones were scored as those control) antibodies. Binding was detected with HRP-
with the ability to bind specifically to FW213 and not to conjugated anti-mouse IgG and a sensitive chemi-illumi-
VTl205. nescent substrate.
248
6. Casson LP, Manser T (1995). Random mutagenesis of 18. McCafferty J, Griffths AD, Winter G, Chiswell DJ
two complementarity determining region amino acids (1990). Phage antibodies: filamentous phage dis-
yields an unexpectedly high frequency of antibodies playing antibody variable domains. Nature 348(6301):
with increased affinity for both cognate antigen and 552-554.
autoantigen. J Exp Med 182: 743-750. 19. Pereira S, Maruyama H, Siegel D, Van Belle P, Elder
7. Chiswell DJ, McCafferty J (1992). Phage antibodies: D, Curtis P, Herlyn D (1997). A model system for
Will new 'coliclonal' antibodies replace monoclonal detection and isolation of a tumor cell surface antigen
antibodies? Trends Biotechnol Mar lO: 80-84. using antibody phage display. J Immunol Methods
8. Chomczynski P, Sacchi N (1987). Single-step method 203: 11-24.
of RNA isolation by acid guanidinium thiocyanate- 20. Ruff-Jamison S, Glenney JR Jr (1993). Molecular
phenol-chloroform extraction. Anal Biochem 162: modeling and site-directed mutagenesis of an anti-
156-159. phosphotyrosine antibody predicts the combining site
9. Elder BL, Boraker DK, Fives-Taylor PM (1982). and allows the detection of higher affinity interactions.
Whole-bacterial cell enzyme-linked immunosorbent Protein Eng 6: 661-668.
assay for Streptococcus sanguis fimbrial antigens. J 21. Sambrook J, Maniatis T, Fritsch EF (1989). Molecular
Clin Microbiol 16: 141-144. cloning: a laboratory manual. New York: Cold Spring
10. Friguet B, Chaffotte AF, DJavadi-Ohaniance L, Harbor Laboratory Press.
Goldberg ME (1985). Measurements of the true 22. Smith GP (1985). Filamentous fusion phage: Novel
affinity constant in solution of antigen-antibody com- expression vectors that display cloned antigens on the
plexes by enzyme-linked immunosorbent assay. J virion surface. Science 228(4705): 1315-1317.
Immunol Methods 77: 305-319. 23. Watters JM, Telleman P, Junghans RP (1997). An
11. Hill HR, Stockley PG (1996). Phage presentation. Mol optimized method for cell-based phage display
Microbiol May 20: 685-692. panning. Immunotechnology 3: 21-29.
12. Huse WD, Sastry L, Iverson SA, Kang AS, Alting- 24. Webster DM, Pedersen J, Staunton D, Jones A, Rees
Mees M, Burton DR, Benkovic SJ, Lerner RA (1989). AR (1994). Antibody-combining sites. Extending the
Generation of a large combinatorial library of the natural limits. Appl Biochem Biotechnol 47: 119-
immunoglobulin repertoire in phage lambda. Science 132.
246(4935): 1275-1281. 25. Winter G, Griffiths AD, Hawkins RE, Hoogenboom
13. Iba Y, Kurosawa Y (1997). Comparison of strategies HR (1994). Making antibodies by phage display
for the construction of libraries of artificial antibodies. technology. Annu Rev Immunol 12: 433-455.
Immunol Cell BioI 75: 217-221. 26. Wu H, Mintz KP, Ladha M, Fives-Taylor P (1997).
14. Jacobsson K, Frykberg L (1996). Phage display shot- Isolation and characterization of Fapl, a fimbriae
gun cloning of ligand-binding domains of prokary- associated adhesin of Streptococcus parasanguis
otic receptors approaches lOO% correct clones. FW213. Mol Micro (submitted).
Biotechniques 20: lO70-1076.
15. Kohler G, Milstein C (1975). Continuous cultures of
fused cells secreting antibody of predefined speci-
ficity. Nature 256: 495-497. Address for correspondence: Paula Fives-Taylor,
16. Marks JD, Hoogenboom HR, Griffiths AD, Winter G Department of Microbiology and Molecular Genetics,
(1992). Molecular evolution of proteins on filamen- College of Medicine and College of Agriculture and Life
tous phage. Mimicking the strategy of the immune Sciences, Stafford Hall, University of Vermont,
system. J BioI Chern 267: 16007-160lO. Burlington, Vermont 05405, USA
17. Martin AC, Cheetham JC, Rees AR (1991). Molecular Phone: (802) 656-1121; Fax: (802) 656-8749
modeling of antibody combining sites. Methods E-mail: pfivesta@zoo.uvm.edu
Enzymol 203: 121-153.
Index, Vol. 20/1-4
16S rRNA genes, 223 antibiotic resistance, 233, 238 bacterial traits, 71 cell adhesion, 209
acapsular phenotype, 14 antibiotic resistance-encoding bacterial viability, 107 cell aggregation (Clu) pheno-
acceptor sequences, 133 genes, 59 bacteriocin-like inhibitory sub- type, 71
acid production, 119 antibiotic resistance gene, 51, stance, 1 cell constituents, 181
acid tolerance, 1 59 bacteriological techniques, 107 cell density, 65
acid-sensitive (AS) phenotype, antibiotic resistance maker, 46, bacteriophage attacks, 119 cell including protection, 241
1 53 bacteriophage contamination, cell lysis, 144
acmA gene, 47 antibiotic resistance plasmids, 119 cell membrane proteins, 199
Actinobacillus pleuropneumo- vii bacteriophage TI2, 51 cell surface, viii, 79, 95, 127
niae, 157 antibiotic susceptibility, vii, bacteriophage-protection sys- cell surface proteins, 103
Actinomyces, 230 107 tern, 119 cell wall anchor sequence, 79
Actinomyces odontolyticus, antibiotic treatment regimes, BamHI,48 cell wall peptidoglycan, 209,
224 203 batch cultication, 189 213
Actinomyces viscosus, 224 antibiotics, 3, 21, 29, 184 Bell sites, 116 cell walls, 109
actin polymerization, 214 antibodies, viii, 241 ~-galactosidase activity, 48, cell wall structure, 83
acute necrotizing ulcerative antibody/antigen interaction, 156 cell-based panning, 242
gingivitis, 230 241 ~-hemolytic species, vii cell-cell communication, viii
adaptation, 165 antigen recognition, 241 ~-Iactamases, 209 cell-surface associated pro-
adenine biosynthesis, 8 antigens 95 BHI agar plates, 133 teins, 209
adherence, vii, viii, 143, 191 antigen-encoding genes, 29, Bifidobacterium breve, 224 cellular functions, 85
adherence assays, 107 95, 103, 105 Bifidobacterium dentium, 224 cellular invasion, 107
adherence-associated genes, antimicrobioal agents, 95 binding capacity, 241 centrifugation, 198
150 antisera, 103 binding substance mutant, 83 chain length, 79
adherent populations, 181 aortic valve, 203 biochemical capabilities, 181 characterization, 28, 59
adhesion, 79, 143, 241 aphA3, I biochemical characterization, checkerboard hybridization
adhesion molecule, 79 architecture of the cell wall, 10 methodology, 223
adhesion properties, 214 83 biofilm reactor, 187 chemically-defined medium
aerobic growth, 150 arginine biosynthesis, 8 biofilms, viii, 181 (FMC),217
agarose, 143 ATCC A549 cells, 196 bioprocessing, 125 chemifluorescence (Storm sys-
agarose gel electrophoresis, 91, ATPase, 165 Bio-Rad,53 tem) procedures, 224
226, 233 ATPase ~-subunits, 171 biotechnology, 125 chemi-illuminiscent substrate,
agarose gels, 149 ATP-binding cassette (ABC), biotinylated PCR fragment, 248
age-dependent phenomena, 141 160, 209 116 chemilumenescent substrate,
aggregation substance (AS), 79 AT-rich DNA, 95 BLAST,103 116
aliquots 82, 107 autolysin, 30, 105 BLAST program, 156 chemiluminescence (X-ray
alleles, 67 auxotrophic (AX) strains, 2 BLIS, 1 film) procedures, 224
allelic exchange, 1, 14, 62 auxotrophy (AX), 9 bloodstream, 143, 203 chimeric shuttle plasmid, 135
allelic replacement, 29, 51, 82, Axospirillum brasilense, 157 blot assays, 191 chloramphenicol, 13, 35
85 baceriophage, 121 blotting membrane, 197 chloramphenicol acetyltrans-
allelic replacement vector, 15 baceterial cell-cell contact, 79 blue colonies, 45, 158 ferase (cat), 137, 186
Alul,47 bacilli, 209 blue/white screning, 46, 54 chloramphenicol acetyl trans-
amino acids, 160 Bacillus circulans, 132 Bordetella pertussis, 160 ferase gene, 174
amino acid sequences, 48, 172, Bacillus licheniformis, 213 Bradford assay, 189 chloramphenicol resistance
176 Bacillus megaterium, 158, 173 Burkholderia cepacia, 109 gene (Cm R), 63
aminoglycosides, 95 Bacillus methanolicus, 157 C-terminal sequence, 209 chloramphenicol resistance
amnion, 191 Bacillus stearothermophilus, calcium phosphate, 139 maker, 82
amnionic fluid, 191 158 Campbell-like mechanism, 156 chorioamnion, 191
ampicillin, 2 Bacillus subtilis, 35, 104, 125, Campbell-like recombination, chorioamnionitis, 191
amplification, 171, 242 144, 157, 160 59, 153 chorion, 191
anaerobic incubation, 6 background radioactivity, 198 capping, 121 chromosomal conjugative ele-
anchor plasmid, 128, 130 bacteremia, 191 capsular polysaccharide, 191 ments,59
anchorage of polypeptides, 209 bacteria, viii, 9, 18, 217, 230, capsule expression, 13, 18 chromosomal DNA, 18, 86,
anesthesia, 203 241 capsule gene sequences, 14 101, 233
animal environment, 137 bacterial adherence, 107 capsule synthesis, 13 chromosomal promotors, 35
animal infections, 139 bacterial blots, 197 carbohydrate limitation, 183 chromosomal walking, 59
animal~ age, 141 bacterial chromosomal genes, carbohydrate metabolism, 1 chromatography, 217
annealing temperature, 173 35 carbohydrates, 165, 181 CITase, 132
anti-microbials, 209 bacterial counts, 223 carbon sources, 143 Citrobacter freundii, 157
anti-phagocytic M protein, 209 bacterial genetics, vii cartogenicity, 128, 153 clidamycin, 238
antibiodies, 127 bacterial invasion, 107 cat fusion, 137 clinical samples, 223
antibiotic killing, 107 bacterial plaque, 224 cat gene, 13, 161, 185 cloning, 13,21,37,59,72,89,
antibiotic protection, 108 bacterial quantification, 107 CAT reporter gene, 175 104, 132, 153, 167, 246
antibiotic protection procedure, bacterial samples, 223 catheter insertion, 203 cloning vector, 52
107 bacterial strain identity, 238 cDNA, 47, 246 Clostridium, I
252
Clostridium acetobutylicum, degenetate oligonucleotides, enterococca1 phage, 79 fimA operon, 150
157 177 enterococca1 pyrimidine, 29 fimbriae, 246
clustal alignment, 171 deletion derivative, 89 enterococcal strains, 102 flanking region, 45
cluster investigations, 233 dental caries, 1, 85, 127, 143, enterococci, vii, 21, 79, 95, fluorogram, 213
codons,l72 153, 165 181, 233 fluorography, 212
coexistance, viii dental plaque, 143, 165,217, Enterococcus, vii, 1, 82, 229 flurorescence microscopy, 189
cognate Tra region DNA, 75 229 Enterococcus faecalis, vii, 21, FMC agarose plates, 217
CORl,18 dental root surfaces, 230 55, 79, 87, 95, 157, 234, food, 217
cointegrate plasmids, 71 detection, 137, 229 367 food production, 71
colonization, 127, 143, 191, dextranase, 127 Enterococcus faecium, vii, 21, foreign genes, 127
209,217 dextrin, 139 104, 234 formaldehyde, 143
colony forming units (CFU), dilution factor, 8 Enterococcus hirae, 21, 157 fragment antigen binding
107, 109, 195 diseases, vii Entoemeba histolytica, 157 (Fab), 241
comC gene, 67 disruption, 144 enumeration, 229 fragmentation, 47
cosmid,95 dissecting functional epitopes, enumeration of viable organ- frame shift mutation, 214
community-acquired patho- 241 isms, 107 free-living sources, 230
gens, 233 distilled water, 139 environment, 137, 143, 165, freezing, 82
competence, 65, 85 dizziness, 206 181,209,223 frozen cells, 66
competence factor, 66 DNA, vii, 9, 21, 36, 51, 54, 66, environmental conditions, 85 fruA genes, 158
competence factor inactivator 72, 82, 91, 102, 116, 128, enzymatic digestion, 144 fruA::lacZ, 160
(CFI),67 153, 167,233,241 enzyme assays, 128 fructosyltransferase (ftf), 137,
competence induction, 67 DNA biosynthesis, 8 enzyme system, 85 188
competence stimulating pep- DNA fragments, 124 enzymes, 127 ftf gene, 137, 158, 185
tide (CSP), 65 DNA hybridization, 55 epidemiologic analysis, viii fusion genes, 127
competent cells, 66 DNA probes, 223 epidemiologic typing, 233 fusions, 175
complete transformation me- DNA sequencing, 104 epidemiology, 233 Fv fragment, 242
dium (CTM), 65 DNA-DNA hybridization tech- epithelial cell lysate, 107 gel electrophoresis, 76, 148
complex polymers, 181 niques,223 epithelial cell membrane gel fixation, 177
computer algorithms, 171 DNase, 149 proteins, 191 gel loading buffer, 103
computers, 166 donor cells, 79 epihtelial cells, 191 Genbank database, 9
conditional replication, 35 donor culture, 77 Erm resistance gene, 21 gene cloning, 59
conjugal elements, 77 double cross-over, 14, 30, 82, erythrogenic toxin (speA), 56 gene disruption, 214
conjugation, vii, 31, 71, 86 91 erythromucin, 8, 35, 45 gene expression, viii, 18, 119,
conjugation experiments, 89 double cross-over integration, erythromycin resistance, 1, 52 127, 138, 143, 153, 165,
conjugative transfer, vii 130 Escherichia coli 1, 15, 24, 35, 181
continuous chemostat cui tic a- double crossover recombina- 51,54,59,72,82,86, 101, gene fusion, 135
tion, 181 tion,59 108, 121, 127, 153, 157, gene inactivation, 36
continuous flow bioreactors, double-stranded DNA, 238 175, 194,213,241 gene isertion, 51
181 downstream expression, 18 ESP buffer, 235 gene isolation, viii, 95, 107
contour-clamped homologous drinking water, 217 ethidium bromide staining gene mapping, viii, 113
electric fields (CHEF), drug-resistance determinant, pattern, 148 gene physiology, 181
233 153 etiologic agents, 85 gene replacement, 35
Coomassie blue, 177 ebs locus, 83 eucaryotic integrins, 79 gene segments, 165
cosmid clones, 23 ecological niches, 143 eukaryotes, vii gene transfer systems, 85
cosmid libraries, 95 electrocompetence, 53 eukaryotic cells, 107, 242 gene/phenotype relationship,
cosmid vectors, 101 electrodes, 82 eukaryotic cell membrane 13
coupler fragments, 131 electrophoresis, 149 proteins, 196 gene-specific mutants, 14
covalent linkage, 209 e1ectroporation, viii, 13, 15,21, evasion, 214 general export pathway, 209
covalent modification, 181 29, 39, 55, 77, 79, 80, 85, exopolysaccharide synthases, genes, 165
covalently-bound radiolabel, 135,246 188 genes back -cross, 5
212 electrotransformation, 39, 55, exponensial growth, 183 genetic fusions, 165
cpsB gene, 18 61,85 external forces, 181 genetic manipulation, 79, 85
cpsB mutagenesis, 14 ELISA, 246 extracellular competence fac- genetic maps, 35
cps genes, 14 Emr gene, 47, 130 tor, 65 genetic marker, 238
cRNA,1 enamel surfaces, 85 extracellular fluid, 214 genetic mutations, 165
crosslinked probes, 230 end-labeling, 117 extracellular matrix, 143 genetic screen, 2
cross-over integrations, 36 end-probing, 113 extracellular matrix proteins genetic transfer, viii, 71, 85
cross-streak matings, 74 endocarditis, 95, 203 (ECM),191 genetic transfer systems, 85
cryptic plasmid, 86 endocarditis infections (EfaA), extracellular polymers, 85 genomic copy, 51
cshA gene, 213 105 extra-chromosomal element, genomic fragments, 123
cshB gene, 213 endogenous signals, 105 91, 238 genomic libraries, 7, 95
cycloisomal-tooligosaccharide energy, 85 extraction, 209 genotype, 241
glucanotransferase (CITase), energy equivalents, 181 F factor-based vectors, 101 genus-specific probe, 223
128 Enterobacter aerogenes, 48 famine, 217 glassware, 220
cytoplasmic membrane, 209 Enterobacteriaceae, vii Fap1 protein, 242 glucan binding protein, 8
dairy organisms, vii enterococcal binding substance feast, 217 glucose permease, 160
dairy products, 119 (EBS),79 fermentation environments, glucosyltransferases (GTF),
dairy streptococci, vii enterococcal infections, 21 119 127, 187
ddH 20,220 enterococcal infectious endo- fermented foods, 71 glutaldehyde, 177
decant supernatant (LHB), 235 carditis, 95 fibronectin, 196 glutamate transporter, 8
253
glutamine, 183 host chromosome, 133 industrial phages, 125 Lactobacillus leichmannii, 104
glutaraldehyde, 177 host conditions, 143 inertion mutants, 21 Lactobacillus sake, 158
glycine, 31 host functions, viii infection, 119, 203, 217, 233 lactococcal matings, 76
glycosylating, vii host immune defences, 214 inflammatory periodontal lactococcal strain development,
Gram-negative microorgan- host immunodefenses, 206 disease, 143 71
isms, 217 host infection, 137 initial temperature elevation, lactococci, vii, 35, 71, 80, 119,
Gram-negative bacteria, I, 36 host inflammatory response, 110 181
Gram-positive bacteria, I, 36, Vlll inoculation, 108, 205 Lactococcus, viii, I, 71
79, 135, 144, 209 host proteins, 217 insertion-duplication mutagen- Lactococcus lactis, vii, I, 35,
Gram-positive cocci, vii, 183 host range, 35 esis, 30, 59 55, 119, 157
Gram-positive diplococci, 191 host surfaces, 241 insertion mutants, 29 Lactococcus lactis NCKI68,
Gram-positive organisms, 59, host-associated sources, 230 insertional inactivation, 74, 79 74
95, 113 host -encoded integration host insertional mutagenesis, 35, Lactococcus lactis supsp.
Gram-positive signal peptide, factor (IHF), 55 51, 71 cremoris 157
79 host:parasite relationship, viii integrase protein (Int), 51 Lactococcus lactis subsp. lactis
Griffith's transforming prin- host-pathogen interactions, 200 integration, 35, 51, 54, 59 ML3,71
ciple, vii host-vector system, viii integration host factor (IHF), LacZ, 187
groep A streptococci (GAS), hppA gene, 212 51 LacZ activity, 186
51, 199,209,233 human dental caries, 217 integration plasmid, 130 LacZ gene, 45, 63, 153
groep B streptococci (GBS), human flora, viii integration vectors, viii LacZ promoter, 53
viii, 10, 13, 107, 113, 191, human host, 127 integration-mediated transfor- LacZY fusion, 137
233 human platelets, 108 mation system, 134 LamB,242
group A streptokinase gene human recto-vaginal tract, 191 integrational vector, 153 lantibiotic synthesis, 8
(ska), 54 human saliva, 220 integrative plasmids, 153 leader peptides, 209
Group D streptococci, vii hybrid genes, 165 intercellular compartment, 107 left terminal repeat (LTR), 117
Group N streptococci, vii hybridization, 28, 229 intracellular polymers, 85 Leuconostoc, 71
growth, 9, 65, 165,217 hybridization assays, 223 inter-generic matings, 77 library construction, 95
growth phase, 143 hybridization signals, 123 inter-species matings, 77 ligand blot analysis, 199
growth rate, 181 hybridoma methods, 241 interrupted genes, 1 ligation mixture, 63
growth temperature, 35 hydrolytic enzyme treatments, invasion, 241 light chain genes, 241
gtf gene, 188 209 invasion assay, 107 line blot hybridization, 223
gtjBIC, 156 hydroxylapatite, 187 investigator safety, 203 linear DNA, 13, 59
gtjBK, 153 hypothetical enzymes, 103 INY3000,82 linearized DNA, 133
guanosine-mono-phosphate hypoxia, 206 INY3039,82 linkage analysis, 59
(GMP), 121 identification of species, 223 iosine, 172 lipid-modification, 212
guanosine-tri-phosphate (GTP), identification, 153, 209 iron, 143, 217 lipid turnover, 213
121 immune defences, 209 IS946-mediated mutagenesis, lipoprotein processing, 213
guanyltransferase, 121 immune response, 246 71 lipoproteins, 209
HaeIl,89 immune sera, 95 IS elements, 35, 76 lipoteichoic acid synthesis, 79
Haemophilus inJluenzae, 109, immune system, ix isertion duplication, 61 Listeria monocytogenes, 214
157, 160 immunization, 248 isocitrate dehydrogenase, 8 Llal operon, 119
heart tissue, 143 immunoblot analysis, 17, 196, isogenic mutants, 85, 213 L-methionine, 139
heart valves, 203 213 isolates, 107 lung epithelial carcinoma cells,
heat-stock response, 46 immunoblots, 247 isolation, 241 199
heavy chain genes, 241 immunoblotting, 21 isolation of genes, 48 lung epithelial cell monolayers,
helper plasmid, 37 immunopositive cosmid clones, isolation of mutants, 9 109
heritable traits, 65 100, 103 ivasion fibronectin, 191 lysis buffer, 235
heterodimer plasmids, 130 immunopositive subclones, jack pots, 9 lysozyme, 144
heterodimer plasmid system, 103 JHI005, 7 lytic enzymes, 144
127 immunoprotecti ve strategies, kan gene, 89 Lytic enzyme solution, 148
heterologous gene, 135 203 kanamycin omega cassette, 14 lytic phages, 120
heterologous proteins, 127, 135 immunoscreening, 29, 102 kanamycin (KM) resistance, Macaca nememstrina primates,
heterologous streptococcal in vitro bacterial cultivation, 153 199
host, 63 223 kanamycin resistance gene, 1, macrolide-lincosamide-strep-
higher taxa, 229 in vitro juxtaposition, 173 24 togramin, 238
HindIII,89 in vivo evaluation, 206 ketamine, 206 macroscopic visible clumps, 79
HindIII fragment, 29, 54, 101, in vitro methods, 206 key specific parameters, 181 magnesium, 217
123 in-frame fusions, 175 Km resistant transconjugant, Mal mutants, 47
hockey sticking, 109 inactivated genes, 1 89 maltose-negative strain, 48
homologous DNA, 89 inactivated genetic loci, 2 Kock's postulates, viii mammalian host, 143, 181,217
homologous recombination, inactivation, 59 Krebsiella pneumoniae, 158 mammalian oral cavity, 137
13,21,35,45,51,59,76, indentification, 209 laboratory-derived mutants, manganese, 217
92, 127 independant replicon, 91 107 mapping, 74, 113, 119
homology, 82 inducible promotors, 123 lac promotor, 105 marker rescue, 1
hospital-acquired infections, inductively coupled agon lactic acid, 85 matings,82
21,95 plasma (ICAP) analysis, lactic acid bacteria (LAB), 71, melting temperature, 171
hospital-acquired pathogens, 219 119,181 membrane localization, 213
233 inductively coupled plasma lactic acid bacterial starter meningitis, 191
hospitalized patients, 233 mass spectroscopy (ICP- cultures, 71 metabolic enzymes, 103
host cell, 14, 191, 143 MS),220 Lactobacillus, 71 metabolic phenotype, 107
254
protein databases, 165 randomly amplified polymor- rough derivates, 68 site-specific mutations, 13
protein repertoire, 165 phic DNA (RAPD), 233 rRNA,148 skin, 136
protein secretion, 8 rat, 138, 203 Saccharomyces cerevisiae 121, SMS101, 137
proteins, 160, 241 rat nuclei, 121 157,176 sodium chloride, 139
proteolytic enzyme treatments, reagents, 223 Saccharopolyspora erythracea, sodium dodecyl sulphate
209 Rec+ lactococca1 strain, 76 158 (SDS),209
proteomic datadases, 175 Rec- 1actococcal strain, 76 salivarius group, 144 sodium lauroylsarcosinate
proteomic maps, 176 Rec- recipient strain, 76 salivary components, 143 (SLS),212
proteomics, 165 Rec-independant recombina- salivary glycoprotein pellicle soft sugar technique, 107
protocols for gene transfer, viii tion, 74 in vitro, 214 software, 166
proton-trans1ocating ATPase, receptor sequences, 248 Salmonella typhimurium 104, solid-based method, 242
172 recipient cells, 79 137, 157 sortase, 209
protoplast transformation, 80 recipient chromosome, 91 salt buffer, 82 Southern analysis, 9, 17
pRS01,71 recipient culture, 77 sampling error, 109 Southern blot analysis, 30, 46
Pseudomonas aeruginosa, 104, recombinant DNA, 86, 128 saturation level, 79 Southern blot hybridization, 63
157 recombinant DNA technology, Sau3A,48 Southern blots, 91, 156
pSF143, 60 vii, 71 scanning confocal laser Southern blotting, 116
pSF151, 60, 155 recombinant chromosome microscopy, 189 Southern hybridization, 9, 121
pSF152,60 structure, 17 Schizosaccharomyces pombe spc gene, 89
pTEX5176,101 Recombinant Phage Antibody 157 speciation of streptococci, 69
pTEX5235, 27 System, 248 scrA gene, 89, 153, 158 species identification, viii
pTEX5236, 27 recombinant technology, 214 scrA::lacZ, 160 species-specific isolation inser-
pTRK28, '71 recombination, 13 screening, 9, 107 tion media, viii
pTRK28::pRS01, 74 recombination efficacy, 17 screening procedures, 125 specific growth rate, 183
pTRK414H, 119 recombination frequencies, 46 SDS-PAGE gels, 217 spheroplasting buffer, 210
PTS system, 160 recovery vector, 6 SDS-PAGE patterns, 213 slice-overlap extension PCR,
pTS204, 131 refractory periodontal disease, secretion, 127, 133 175
pTV1-0K,1 230 selectable marker, 45, 52 Sp' gene, 130
pTVlOK,l13 regulatory elements, 173 sepsis, 191 SpRKms phenotype, 91
pTV21~TetM, 1 regulatory system, 79 sequence motifs, 79 SspI fragment, 123
pUC-based rescue technique, 7 Rep- strains, 37 sequence analysis, 213 stability over time, 233
pulse times, 237 repA gene, 37 sequencing, 120 standard plating methods, 107
pu1sed-field gel electrophos- RepN helper strain, 47 sequential integration, 133 staphylococci, 209
resis (PFGE), 233 repAts, 1 serine tRNA, 54 Staphylococcus, 1
purification, 209 replication, 1 serodiagnostic tools, 95 Staphylococcus aureus, 109,
purine genes, 29 replication-conditional vector, serum, 65 158, 248
pVA380-1,86 1 serum components, 143 start codon, 48
pVA891,60 replicon fusions, 8 sessile, 181 starter cultures, 71, 119
PVE6007, 13, 40 reporter gene fragments, 173 sex pheromone, 79 starvation, 85
PvuII, 76, 116 reporter gene fusions, 181 shot-gun cloning, 1, 120 steady state conditions, 182
pWM130,53 reproducibility, 233 shuttle factor, 82 stem-loops, 175
pWM139,51 reproductive tract, 136 shuttle plasmids, 127, 135 step-down PCR, 173
pWM245,51 residant plasmid, 133 shuttle vectors, 86 sterile culture filtrates, 67
pWM401,82 resistance phenotypes, 91 shuttle-suicide plasmid vectors, Stomatococcus mucilaginosus,
pWV01,36 resolvase, 30 51 224
pyrC gene, 31 respiratory rate, 181 signal transduction, 241 stop codon, 48, 136
quantitative assays, 107 restriction activity, 119 signals, 153 stop-transfer signal, 209
quasi-growing organisms, 188 restriction endonuclease anal- silent gene replacement, 35 STRA-B498, 199
rabbit endocarditis, 82 ysis (REA), 233 silver-stained gels, 177 strain characteristics, 30
rabbit serum, 102 restriction endonuclease cas- single base changes, 175 strain differentiation, viii
radioactively-labeled fatty acid, sette (LlaIR+), 119 single copy transcriptional strain enumeration, 138
212 restriction endonuclease sites, fusions, 35 strain improvement regimes,
radioactively-labeled palmi- 89 single crossover, 14, 30, 51, 71
tate, 212 restriction enzyme digestions, 156 strain specificity, 65
radiolabeled GBS, 199 95 single crossover homologous strand-specific repair, 159
radiolabeling, 194 restriction enzyme mapping, recombination, 59 streptavidin-HRP conjugate,
ramp times, 173 113 single crossover recombina- 116
random chromosomal frag- restriction fragments, 82 tion, 153 Streptococcaceae, vii
ments,47 restriction mapping, 29, 123 single solid support membrane, streptococcal adherence, 108
random chromosomal muta- restriction pattern, 56 223 streptococcal origin (ori), 59
tions, 47 resuspension, 159 single stage chemos tat, 183 streptococcal replicon, 86
random chromosomal strains, reverse genetics, 248 single-copy plasmid integra- streptococci, 13, 51, 59, 65, 80,
37 RNA integrity, 146 tion system (SCIS), 130 143,181,203,209,217,233
random chromosomal tran- RNA isolation, 143 single-stranded replicative Streptococcus, vii, 59, 71, 223
scriptional fusions, 37 RNA markers, 149 intermediates, 89 Streptococcus agalactiae, vii,
random mutagenesis, 46, 82, RNA polymerase (RNAP), 121 site bias, 113 55,82,113,191,233
241 RNA transcripts, 123 site-directed mutagenesis, 241 Streptococcus anginosus, 66
random transposition, 35 RNase, 144 site-specific integration, 51 Streptococcus constellatus, 66
random transcriptional fusions, Rothia dentocariosa, 224 site-specific integration vec- Streptococcus crista, 66
48 Rototorque, 187 tors, 53 Streptococcus downei, 55
256
Streptococcus equisimitis, 55, sucrose catabolic enzymes, 85 TEP buffer, 210 Ts-helper plasmid, 48
104 sucrose metabolic enzyme test medium, 217 Ts-system, 37
Streptococcus equisimilis systems, 85 tetracycline, 238 tuberculosis, viii
(skc), 54 sucrose permease, 89 tetracycline resistance marker, tubing, 184
Streptococcus ferus, 86 sugar, 184 46 tumor cell lines, 191
Streptococcus gordonnii, 55, sugar alcohols, 161 throat, 136 two-dimensional gel elec-
63, 65, 87, 108, 127, 143, sugar metabolism, 153 THY-HS, 53 trophoresis, 175
199, 212 sugar regulation, 153 tissue culture techniques, 191 two-dimensional gel separa-
Streptococcus hygroscopicus, sugar uptake systems, 48 Tn4001, 1 tions, 176
158 sugars, 153, 160, 165 Tn5, 82 TX52,102
Streptococcus intermedius, 66 sugar-regulated phenotype, 156 Tn916, 1, 21, 82 typing of streptococci, 69
Streptococcus macacae, 55 sugar-responsive gene expres- Tn91ME,13 urease enzyme activity, 185
Streptococcus milleri, 66, 135, sion, 160 Tn917, 1, 13, 21 UV irradiation, 224
223 suicidal plasmids, 59 Tn917 insertions, 113 vaccines, 95, 209
Streptococcus mitis, 55, 66 suicide factors, 1 TnphoA,113 vaccinia virions, 121
Streptococcus mutans, vii, 1, suicide system, 119 Todd Hewitt broth horse serum vancomycin resistance, 95, 233
55, 63, 85, 127, 153, 157, suicide plasmid, 51 (THS),65 vegetable oil, 139
165, 184,206,212,217 suicide vectors, viii, 13 Todd-Hewitt agar, 109 vegetation formation, 203
Streptococcus mutans dexA, 63 surface colonies, 11 0 tooth enamel, 85 Veillonella parvula, 224
Streptococcus mutans rodD, 63 surface expression system, 135 tooth loss, 141 Veillonella dispar, 224
Streptococcus mutans se- surface plots, 176 tooth surface, 143 Vibrio cholerae, 137
quences cloned in pFP1, surface proteins, vii, 209, 241 touchdown approach, 173 vigorous vortexing, 144
157 surface structures, 79 toxins, viii virulence, viii, 65, 68, 79, 85,
Streptococcus mutans transfor- surface-associated proteins, TRa1,76 127, 165
mations, 159 209 Tra1 region insertions, 75 virulence determinants, 221
Streptococcus oratis, 55, 66, surface-growing organisms, Tra3,76 virulence factors, 1, 95, 103,
143, 158 188 transconjugants, 74, 89 143, 181, 191, 128, 203
Streptococcus parasanguis, vii, SurtZAP Vector system, 248 transcription, viii virulence genes, 21
67,143,212,241 surgery, 204 transcription-repair coupling, virulence properties, 209, 215
Streptococcus pneumoniae, vii, swab matings, 72 159 virulence-associated genes,
55, 63, 65, 66, 157, 160, Swagelock type fittings, 188 transcriptional fusion, 35, 153 137, 141, 148
214,233 synthetic competence simu- transcriptional regulation, 143 vitamimes, 139, 183
Streptococcus pyogenes, vii, lating peptides, 65 transfer frequencies, 74, 77, 82 washing, 82, 228
10,51,55,63,82,104,214, T7 Select Phage Display transfer phenotype, 76 water-insoluble glucan syn-
233 System, 248 transference, 5 thesis, 127
Streptococcus pyogenes redA, TAE,143 transformation, vii, 1, 15, 55, water-soluble glucan synthesis,
63 tailor transformation media, 68 61, 65, 82, 85, 127 127
Streptococcus rattus, 9 tandem intersertions, 56 transformation frequencies, 91 Watson-Crick base-paring
Streptococcus sativarius, 55, targered mutagenesis, 21 transformation system, 127 properties, 173
144, 184 targered mutations, 21 translation, viii Western blot analysis, 247
Streptococcus sanguis, 55, 65, target gene sequence, 167 translational regulation, 165 Western blots, 102, 213
82, 104, 109, 143, 212 target genes, 47 transport, 241 white colonies, 158
Streptococcus sobrines, 85, target vector, 113 transporters, 103 whole cell ELISA assay, 247
127, 132 TE buffer, 235 transposase, 30 wild types, 82, 206
Streptococcus thermophilus, 36 temperate bacteriophages, 51 transposition, 35 wild type strains, 68
Streptococcus typhimurium, temperature, 143 transposition frequency, 8 wild-type gene, 10, 91, 45
160 temperature shift, 47 transposon miniy~-200 (my~), wobble base positions, 172
structural genes, 82 temperature-sensitive derivate, 22 X-gal,45
Student's t-test, 177 1 transposon mutagenesis, 82 Yersinia entercolitica, 157
subacute bacterial endocarditis, temperature-sensitive plasmids, transposon mutants, 107 Zwittergent method, 103
143 13 transposons, viii, 1, 13,21,35, zymography, 133
subcloning, 30, 73, 100 temperature-sensitive factor, 1 83, 113, 153 Zymomonas mobilis, 157
substrate availability, 181 temperature-sensitive proper- tRNA,51
sucrose, 1, 139 ties, 35 trypsin, 215