Appl Microbiol Biotechnol (2011) 89:1061–1073

DOI 10.1007/s00253-010-2907-6

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Functional characterization of a new holin-like antibacterial

protein coding gene tmp1 from goat skin surface metagenome
Thangamani Rajesh & Thangamani Anthony &
Subramani Saranya & Paul Lavanya Pushpam &
Paramasamy Gunasekaran

Received: 30 April 2010 / Revised: 31 August 2010 / Accepted: 1 September 2010 / Published online: 7 October 2010
# Springer-Verlag 2010

Abstract We have identified a holin-like gene from a goat
skin surface metagenome. The ORF designated tmp1
coding for 34 amino acids shared sequence similarity with
putative holin-like toxin genes. To analyze the antibacterial
activity of tmp1 encoded protein, this ORF was cloned and
expressed in Escherichia coli BL21(DE3). The expressed
gene product Tmp1 exhibited antibacterial activity against
Gram-positive bacteria but not to Gram-negative bacteria.
A single transmembrane domain (TMD) was identified
within Tmp1 and deletion analysis of the N-terminal region
and TMD indicated TMD to be responsible for antibacterial
activity. The TMD-dependent antibacterial activity was
validated using a synthetic peptide with the amino acid
sequence of TMD. Besides antibacterial activity, Tmp1 also
complemented the function of holin in a lysis-defective
bacteriophage lambda. To broaden the spectrum of antibac-
terial activity, a mutant library of tmp1 was generated by
random mutagenesis. Four mutants with amino acid
substitutions at the N-terminus of Tmp1 exhibited increased
antibacterial activity against Gram-positive and Gram-
negative bacteria and were not hemolytic. An improved
activity of these mutant proteins is attributed to their
increased hydrophobicity.
Keywords Metagenome . Holin-like protein . Antibacterial
activity . Transmembrane domain . Site-directed
mutagenesis . Hemolytic activity
Introduction

Microorganisms in any environment employ various de-

Electronic supplementary material The online version of this article fense mechanisms for their survival. Antagonistic organ-
(doi:10.1007/s00253-010-2907-6) contains supplementary material,
which is available to authorized users. isms generally produce antimicrobial substances to
suppress/inhibit growth of related and/or unrelated compet-
T. Rajesh : T. Anthony : S. Saranya : P. L. Pushpam :
ing microorganisms (Handelsman et al. 1998). The surface
P. Gunasekaran (*)
Department of Genetics, Centre for Excellence in Genomic of animal skin and hide is expected to have representatives
Sciences, School of Biological Sciences, of microorganisms in every environmental niche to which
Madurai Kamaraj University, the animal is exposed. Microbes from soil, water, gastro-
Madurai 625 021, India
intestinal tracts, and air are expected to be present in
e-mail: gunagenomics@gmail.com
conjunction with normal skin surface commensals. Hairs on
Present Address: hides of cattle have been reported to carry a microbial load
T. Anthony of 103–105 CFU/g (colony forming units) (Madden 1994).
P. G. Department of Botany, The H. H. Rajah’s College,
Fecal and soil bacteria associated with hoofs, hides of cattle
Pudukottai 622 001, India
and slaughterhouse sediments that exceed 109 CFU/cm2 are
Present Address: considered to be a major food safety hazard (Kang and
S. Saranya Frank 1990). In a previous study, we reported that on an
Structural and Computational Biology Group, International Centre
average, 45% of bacterial strains isolated from slaughter
for Genetic Engineering and Biotechnology,
Aruna Asaf Ali Marg, house sewage samples exhibited antibacterial activity
New Delhi 110 067, India (Anthony et al. 2009).
1062
Analysis of such a competitive microbial community
in a metagenomic scale is desirable for identifying
novel antibacterial proteins. Metagenomic studies em-
ploy culture-independent methods for exploring unculti-
vable microbial diversity in any environment to identify
new molecules for therapeutic and biotechnological
applications (Schloss and Handelsman 2005). Functional
and sequence-based screening strategies are used to
identify genes or proteins from the metagenome. In
functional-based screening, clones expressing expected
traits are obtained by phenotypic screening of a metage-
nomic library. In sequence-based screening, consensus
DNA sequences for the target gene in the metagenomic
library are identified by hybridization or by PCR-based
screening methods. Conversely, short DNA fragments
containing partial gene sequences are subjected to improved
inverse PCR methods to identify the complete coding genes
from metagenomes (Uchiyama and Watanabe 2006).
Function-based screening of metagenomic DNA library
for antimicrobial compound producing clones has
experimental limitations due to the occurrence of
functional clones in a low frequency that represent
antibiotic pathway genes (Courtois et al. 2003). However,
the sequence-based screening method has been success-
fully employed for identification of new polyketide
synthase genes and genes involved in the biosynthesis of
erythromycin, epothilone, and rifamycin (Gillespie et al.
2002; Seow et al. 1997).
Double-stranded DNA bacteriophages code hydropho-
bic proteins namely holins that are involved in perme-
ablization of membrane in host cells. Endolysins with
muralytic activity gain access to the cell wall through a
holin-dependent membrane permeabilization or in some
cases, by holin independent transport mechanisms. As a
result of cytoplasmic membrane permeablization, endo-
lysins with muralytic activity are released and they
degrade murein of host cells leading to bacterial lysis
(Sao-Jose et al. 2000; Xu et al. 2004; Young 1992). Due to
the rapid action of endolysins (also called virolysins) on
bacterial cell walls leading to cell lysis, they have been
widely applied as prophylactic agents (Courchesne et al.
2009). Several holin coding genes of bacteriophages of
Gram-positive and Gram-negative bacteria have been
reported (Wang et al. 2000). Recently, holins have been
studied as therapeutics in experimental model systems
(Agu et al. 2006, 2007). Here, we report the characteristics
of a novel gene encoding a holin-like protein Tmp1 from
goat skin surface metagenome. By deletion cloning and
random mutagenesis, several mutant proteins with im-
proved antibacterial activity were derived. This study also
indicates that the spectrum of antibacterial activity could
be extended by varying the average hydrophobicity of
holin-like proteins.
Appl Microbiol Biotechnol (2011) 89:1061–1073
Materials and methods
Bacterial strains and culture conditions
The strains of Escherichia coli, plasmids, and primers
used in this study are listed in Tables 1 and 2, respectively.
Indicator bacterial strains Bacillus subtilis B4219, Lacto-
coccus lactis B1821, Staphylococcus epidermidis B4268,
Bacillus smithii B173, Lactobacillus fermentum B1840,
Pediococcus acidilactici B14958, Micrococcus luteus
B287, Listeria innocula B33016, E. coli B409, Pseudo-
monas putida B8, and Pseudomonas stutzeri B775 were
obtained from the ARS culture collection, Northern
Regional Research Laboratories, Illinois, USA. Kurthia
gibsonii GCS6, a skin-putrefying bacterial isolate was
from our laboratory stock. Cultures were grown at 37°C in
Luria–Bertani medium under vigorous agitation (250 to
300 rpm). Appropriate concentrations of ampicillin
(100 μg/ml) or kanamycin (40 μg/ml) (Sigma-Aldrich
St. Louis, MO, USA) was added to the culture media
whenever required.
Metagenomic DNA isolation
Fresh goat skin samples were obtained from slaughter
house sources in and around Madurai, India. Metagenomic
DNA from goat skin samples was isolated according to
Yeates et al. (1998) with the following modifications.
Initially, skin samples were cut to a size of 5×5 cm,
suspended in 25 ml 0.85% saline and incubated for 30 min
at 37°C with constant shaking at 200 rpm. After removal of
skin, the saline suspension was centrifuged at 1,000×g for
3 min to remove larger size debris particles. Subsequently,
bacterial cells were harvested by centrifugation at 12,200×g
for 15 min and processed further. Isolated metagenomic
DNA was purified using DNeasy genomic DNA isolation
kit (Qiagen, Hilden, Germany) and resuspended in 1 ml TE
(10 mM Tris, 0.1 mM EDTA, pH 8.0) buffer.
Molecular biology techniques
Molecular cloning techniques were performed essentially
following standard procedures (Sambrook et al. 1989).
Restriction enzymes, Taq DNA polymerase, buffers, deoxy-
ribonucleotide triphosphates, and T4 DNA ligase were from
Fermentas (MBI-Fermentas, St. Leon-Rot, Germany) and
used as specified by the manufacturer. Conventional and
degenerate PCR protocols were used to amplify specific
gene sequences from metagenomic DNA. Degenerate
primers BACF2 and BACR2 were designed based on the
conserved regions of reported antibacterial proteins from
cultivable bacteria (Drider et al. 2006; Kayalvizhi et al.
2008), and they were used to isolate similar sequences from
Appl Microbiol Biotechnol (2011) 89:1061–1073 1063

Table 1 List of bacterial strains, phages, and plasmids used in this study

Strains, phages, Genotype, phenotype, or description Reference

and plasmids

Bacteria
E. coli DH5α F− endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ Sambrook et al. (1989)
(lacZYA-argF)U169, hsdR17(rK− mK+), λ–
E. coli HB101 supE44, hsdS20(rB− mB−), recA13, ara-14, proA2, lacY1, galK2, rpsL20, xyl-5, Stratagene, CA, USA
mtl-1, leuB6, thi-1
E. coli LE392 sdR514(rk−, mk+), glnV(supE44), tryT (supF58), lacY1, galK2, galT22, metB1, Stratagene CA, USA
trpR55
E. coli BL21(DE3) F−, ompT, gal, dcm, lon, hsdSB, (rB− mB−), λ (DE3 [lacI lacUV5-T7 gene 1 ind1 Novogen Inc, CA, USA
sam7 nin5])
Bacteriophages
λgt11 chIts857; Sam100; lac promoter for expression of cloned genes Promega, WI, USA
λgt11::tmp1 tmp1 inserted into the EcoRI site of λgt11 This study
Plasmids
pTZ57R 2.6-kb cloning vector; lac promoter; Ampr Fermentas, Germany
pET30b 5.4-kb cloning and expression vector; T7 promoter; Kanr Novogene Inc, CA, USA
pM2 250-bp PCR product (amplified conserved domain) inserted by T-A cloning in to This study
pTZ57R
pTZTMP1 105-bp PCR product (encoding Tmp1) inserted into pTZ57R by T-A cloning This study
pTMP1 105-bp PCR product (encoding Tmp1) inserted into NdeI and XhoI site of pET30b This study
pTMP1A1T pTMP1 with modified NdeI site (ATG→TTG) This study
pNDEL pTMP1 with 30 bp deletion from 3′ end of tmp1 ORF This study
pTMDEL pTMP1 with 87 bp deletion from 3′ end of tmp1 ORF This study
pT1RM1 GTG (Met 1)→CTG (Leu) in ORF tmp1 This study
pT1RM2 GTG (Met 1)→GTT (Val); GAT (Asp5)→GTT (Val) in ORF tmp1 This study
pT1RM3 GTG (Met 1)→GAG (Glu); GAT (Asp 5)→GTT (Val) in ORF tmp1 This study
pT1RM4 GTG (Met 1)→CTA (Leu); GTT (Val 3)→CTT (Leu) in ORF tmp1 This study
pT1SDM1 GTG (Met 1)→GAG (Glu) in ORF tmp1 of pT1RM2 This study
pT1SDM2 GTG (Met 1)→GAG (Glu); GTT (Val 3)→Gln (CAA); in ORF tmp1 of pT1RM2 This study
pT1SDM3 GTG (Met 1)→Glu (GAG); GTT (Val 3)→Gln (CAA); GAT (Asp 5)→AAT (Asn) This study
in ORF tmp1 of pT1RM2
pRM1A1T ATG (Met 1)→Leu (CTG) in ORF in pT1RM1 This study
pRM2A1T ATG (Met 1)→Leu (CTG) in ORF in pT1RM2 This study
pRM3A1T ATG (Met 1)→Leu (CTG) in ORF in pT1RM3 This study
pRM4A1T ATG (Met 1)→Leu (CTG) in ORF in pT1RM4 This study

the metagenomic DNA. The complete tmp1 ORF was
amplified using primers BACF3 and BACR3 designed based
on the reported gene sequence at the GenBank database
(accession number: CP000414.1). The PCR product was
directly cloned into pTZ57R, and the ligated product
(pTZTMP1) was used to transform E. coli DH5α. Plasmid
DNA was isolated from transformants, and the insert DNA
was sequenced. For expression analysis, tmp1 ORF was
amplified with primers BACF4; BACR4 and pTZTMP1 as
template, and the PCR product after NdeI and XhoI digestion
was cloned at the NdeI and XhoI sites into expression vector
pET30b (Novagen Inc, CA, USA). The resulting recombinant
plasmid pTMP1 was used to transform E. coli BL21(DE3).
Deletions of N-terminal and the transmembrane domain in
Tmp1 were made by PCR with appropriate primers DEL F,
NDEL R, and TMDEL R, pTMP1 as DNA template and
high-fidelity platinum Taq polymerase (Invitrogen, CA,
USA) according to manufacturer's instructions. The PCR
protocol followed was: initial denaturation step of 94°C for
2 min, followed by 30 cycles of denaturing for 30 s at 94°C,
annealing for 30 s at 55°C, 11 min extension at 68°C, and a
final extension step of 68°C for 10 min. The cloned DNA
fragments were sequenced at Macrogen, Seoul, South Korea.
Protein expression and zymogram analysis
E. coli BL21(DE3) harboring recombinant plasmid was
grown in LB broth at 37°C at 200 rpm overnight. A fresh
medium (25 ml) was inoculated with an aliquot of
overnight culture and grown for 2 to 2.5 h until it reaches
1064 Appl Microbiol Biotechnol (2011) 89:1061–1073

Table 2 List of primers used in this study

Primer Sequence (5′ to 3′) Purpose

BACF2 TT(AG)TAA(AG)TGGTT(CT)AG(CT)GG(AGCT)GG To amplify conserved region in immunity proteins of class IIa

BACR2 AT(AG)CC(TGCA)AT(AG)TC(CT)TA(AGT)TT(AG)TT(AG)AA bacteriocins
BACF3 GTGTCCGTTTCGGATGCT To amplify tmp1 ORF
BACR3 TCATTTTTTATTGCTGTT
BACF4 AAGGCATATGGTGTCCGTTTCGGATGCT To amplify tmp1 ORF for cloning in pET30b
BACR4 ACACTCGAGTCATTTTTTATTGCTGTT
DEL F TACATATAGAGGAAGTTTCAATTTG To make N-terminal and TMD deletion constructs of tmp1 in
NDEL R TACAATCCGAAACCTTGAAAAC pTMP1
TMDEL R TTTTTGTCGTTATTTTTTACTCTAGGC
TMP1L F ATTAGAATTCGTGTCCGTTTCGGATGCTG To amplify tmp1 for cloning in λgt11
TMP1L R GCGCGAATTCTTATTTTTTATTGCTGTTTTTTATC
RM F GTTTAACTTTAAGAAGAAGATATACAT To generate random mutant library
RM R TGGTGGTGGTGGTGCTCGAGTCA
RMV2E F ATATACATATGGAGTCCGTTTCGGA To create specific mutation in tmp1 and generate pT1SDM1
RMV2E R TCCGAAACGGACTCCATATGTATAT
RMV4Q F ATATGGAGTCCCAATCGGATGCTGT To create specific mutation in tmp1 and generate pT1SDM2
RMV4Q R ACAGCATCCGATTGGGACTCCATAT
RMD6N F GAGTCCCAATCGAATGCTGTGCAAC To create specific mutation in tmp1 and generate pT1SDM3
RMD6N R GTTGCACAGATTCGATTGGGACTC
T1A F CTTTAAGAAGAAGATATACATTTG To create specific mutation in tmp1 and generate pTMP1A1T,
T1A R CAAATGTATATCTTCTTCTTAAAG pRM1A1T, pRM2A1T, pRM3A1T, and pRM4A1T

the optical density of 0.6 at 600 nm. The culture was
induced by the addition of isopropyl β-D-1-thiogalactopyr-
anoside (IPTG) (5 mM), grown for 30 min and harvested
by centrifugation at 5,000×g for 10 min. The cell pellet was
resuspended in Tris–HCl buffer (50 mM, pH 8.0) and lysed
by sonication for three times, 15 s each with an interval of
15 s (Vibra Cell™ Sonics Scientific, Inc). The cell lysates
were clarified by centrifugation at 12,200×g at 4°C for
20 min. Antibacterial activity of recombinant protein in
clarified lysates was determined by zymogram analysis and
lysoplate assay. In zymogram analysis, an autoclaved
overnight-grown bacterial cell suspension of K. gibsonii
GCS6 (0.2% w/v) was added to the SDS-polyacrylamide
gel (15% w/v) before polymerization. After electrophoresis
(3 h, 100 V, 22°C), the gels were equilibrated in sterile
water for 60 min at 4°C, then transferred to potassium
phosphate buffer (25 mM, pH 5.2) containing 0.1% Triton
X-100 and incubated for 12 h at 37°C. To visualize the
translucent lytic band, the gels were stained with 250 ml of
0.01% (w/v) KOH containing 0.1% (v/v) methylene blue,
followed by destaining with distilled water.
Peptide synthesis and antibacterial assay
Peptide TMDSP (LGFGTFVLMLLGLVVELIK) was syn-
thesized employing a solid-phase method (Genescript,
Piscatway, NJ, USA). One milligram of peptide was
dissolved in 1 ml TFE (trifluoroethanol, 0.02% v/v), and
the appropriately diluted sample was used to determine
antibacterial activity in a lysoplate assay. In the lysoplate
assay, bacterial lawn on LB agar was made by plating
100 μl of overnight-grown indicator bacterial culture. Wells
(5 mm) made on the lawn were filled with 50 μl of clarified
cell lysate or reconstituted peptide, incubated overnight,
and examined for growth inhibition. Lytic activity on cell
walls by TMDSP was determined with pure cell wall
preparations of Streptococcus faecalis STF-3 (Sigma-Aldrich
St. Louis, MO, USA) reconstituted in 50 mM MES (2-(N-
morpholino) ethanesulfonic acid) buffer containing 1 mM
MgCl2 as substrate. Mutanolysin, a cell wall degrading
enzyme [100 U in 50 mM TES buffer (50 mM Tris–HCl,
1 mM EDTA, 25% sucrose, pH 8.0) containing 1 mM
MgCl2] (Sigma-Aldrich St. Louis, MO, USA) was prepared
as stock solution. Reaction with mutanolysin (10 U) or
TMDSP (20 μM) and cell wall suspension was carried out in
a final volume of 3 ml and decrease in the optical density at
600 nm was measured at intervals.
Cloning of tmp1 in λgt11
The tmp1 ORF was PCR amplified with primers TMP1L F
and TMP1L R, cloned at the EcoRI site of λgt11 Sam100
(Stratagene CA, USA), and then packaged in vitro using
Packagene® Lambda DNA packaging system (Promega,
Appl Microbiol Biotechnol (2011) 89:1061–1073
WI, USA). λgt11 Sam100 was used as experimental
control. The packaged mixture was plated on E. coli
HB101 and E. coli LE392 to determine the plating
efficiency. To confirm a cloned gene in recombinant
phages, plaques were picked, eluted in 0.1 ml phage buffer
(20 mM Tris–HCl, 100 mM NaCl, 10 mM MgSO4,
pH 7.4), and PCR was then performed with gene-specific
primers BACF3 and BACR3.
Construction and screening of a random mutant library
Random mutagenesis of tmp1 ORF was carried out using
Genemorph® random mutagenesis kit (Stratagene, CA,
USA). The primers RM F and RM R were used to amplify
the template pTMP1 and the PCR-amplified mutant
plasmids were used to transform E. coli BL21(DE3) and
selected on LB kanamycin plates. Screening for antibacte-
rial activity was conducted in sterile 96-well microtiter
plates. Individual wells containing 100 μl of LB supple-
mented with kanamycin were inoculated with E. coli BL21
(DE3) cells bearing mutant plasmid and allowed for growth
at 37°C overnight. The next day, 10 μl of culture from the
wells were transferred to another microtiter plate containing
90 μl of LB supplemented with kanamycin and allowed for
growth until the optical density of 0.6 at 600 nm
(Spectromax M2e Elisa plate reader, Molecular Device
Corporation, CA, USA) was reached, and then cells were
induced by the addition of 1 mM IPTG. After 3 h of
induction, cells were lysed by freeze-thaw cycles (−70°C
for 1 h followed by 50°C for 1 h; this cycle was repeated
three times). Supernatants collected (90 μl) from wells
were transferred to another sterile microtiter plate con-
taining a kanamycin-resistant derivative of E. coli B409
(2×105 CFU/ml). The plates were incubated at 37°C
overnight and observed for growth inhibition. To eliminate
false positives, the screening procedure was repeated as
described above but with an increased titer of indicator
bacterial cells (5×105 CFU/ml).
Site-directed mutagenesis
Site-directed mutagenesis was carried out using
QuikChange® II Site-Directed Mutagenesis kit (Stratagene,
CA, USA) as per manufacturer's protocol, and sequencing
was done at Macrogen, Seoul, South Korea. To introduce
amino acid substitutions in Tmp1, PCR was performed with
plasmid pT1RM2 (20–50 ng) as DNA template and different
mutagenic primer sets (RMV2E F and RMV2E R; RMV4Q
F and RMV4Q R; RMD6N F and RMD6N R) (Table 2). To
introduce point mutations upstream the coding region of
tmp1, PCR was performed with pT1RM1, pT1RM2,
pT1RM3, and pT1RM4 as templates and mutagenic primers
T1A F and T1A R (Table 2).
1065
Assay of bactericidal and hemolytic activity
To exponentially growing cultures of E. coli B409
(5×107 CFU/ml) and P. putida B8 (6×107 CFU/ml),
50 μl of clarified cell lysate from induced cultures was
added and incubated at 37°C. At intervals, samples were
withdrawn, and suitable dilutions were plated on LB agar
plates to score residual viable cells. A culture without the
addition of cell lysate was used as control. The plates were
incubated at 37°C for overnight and then viable cell count
was determined. Sheep blood erythrocytes (SBE) freshly
collected in sterile tubes containing EDTA (0.1 mg/ml
blood) from slaughter house sources were used in hemo-
lytic assays. Briefly, SBE was washed three times with
sterile 0.85% (w/v) saline and centrifuged at 500×g for
10 min. The washed SBE were then diluted to threefold
with 0.85% (w/v) saline, and to this suspension, 50 μM of
TMDSP or 200 μl of clarified cell lysate (50–200 μg of
total proteins) was added. Triton X-100 (1% v/v), which
completely lysed the SBE, was used as a positive control.
After 1 h of incubation, the suspension was centrifuged to
remove intact erythrocytes. The supernatant was diluted,
and absorbance at 540 nm was measured.
Bioinformatic analyses
A putative ORF was identified using the ORF finder
program (http://www.ncbi.nlm.nih.gov), whereas similarity
searches at the level of nucleotide and amino acid
sequences were made using BlastX and BlastP programs,
respectively, at NCBI (Altschul et al. 1990). A transmem-
brane region in protein sequence was determined using
TMpred program (http://www.ch.embnet.org/software/
TMPRED_form.html).
Nucleotide sequence submission
The nucleotide sequence of tmp1 was submitted to
GenBank under accession number EU934232.
Results
PCR-based screening of goat skin surface metagenome
Based on the conserved regions of immunity proteins in
class IIa bacteriocin gene clusters, primers BAC F2 and
BAC R2 were designed (Supplementary material S1). To
identify antibacterial protein coding sequences, PCR was
performed with degenerate primers BAC F2 and BAC R2
and metagenomic DNA as template. The resulting PCR
product (250 bp) was cloned into pTZ57R, and the
sequence analysis of randomly selected 256 clones revealed
1066
Fig. 1 Sequence alignment of deduced amino acid sequence of Tmp1
with related sequences. Multiple sequence alignment was performed
with ClustalW. The GenBank accession numbers of similar putative
holin-like toxin protein sequences from L. mesenteroides ATCC 8293
(YP_818422.1; YP_818566.1), L. casei ATCC 334 (YP_805841.1),
one of the recombinant clones, pM2 to harbor a partial ORF
that shared sequence identity (100%) with a predicted
holin-like toxin gene of Leuconostoc mesenteroides ATCC
8293 (Supplementary material S2). However, the upstream
sequence of the ORF in pM2 did not indicate similarity
with any sequence available in databases. To obtain the
complete ORF from the metagenome, specific primers
BACF3 and BACR3 were designed based on the putative
ORF from the GenBank database (accession number:
CP000414). The PCR product (105 bp) was cloned in
pTZ57R, and sequence analysis revealed this ORF to have
a GTG as start codon. This ORF was designated tmp1, and
the corresponding protein Tmp1 with 34 amino acids had a
calculated molecular mass of 3.9 kDa. The amino acid
sequence of Tmp1 displayed an overall sequence similarity
with uncharacterized holin-like toxins of L. mesenteroides
ATCC 8293 (100%, 97%), Lactobacillus casei ATCC 334
(85%), and Staphylococcus saprophyticus ATCC 15305
(79%) (Fig. 1).
tmp1 encodes an antibacterial protein
To determine the functional property of the protein encoded
by tmp1, the putative ORF was amplified from pTZTMP1,
cloned in pET30b, and expressed in E. coli BL21(DE3). The
recombinant strain E. coli BL21(DE3) (pTMP1) at its early
log phase was induced with IPTG (5 mM) and subsequently
the growth and cell viability were monitored. It was found
that the overexpression of tmp1 strongly affected the growth
and viability of host cells (Fig. 2). Further, clarified cell
lysate from an induced culture of E. coli BL21(DE3)
(pTMP1) exhibited antibacterial activity against K. gibsonii
GCS6 (Fig. 3). These results suggested that Tmp1 functions
as an antibacterial protein. The tmp1 gene was inserted into
vector pET30b to obtain pTMP1, making use of the NdeI
sites in pET30b and in primer BACF4 (see Table 2) for
fusion of the 5′ end of the PCR-amplified gene into the
vector. As a result of this, an ATG codon appeared upstream
to the proposed GTG start codon in tmp1. This provided a
possibility of this ATG to act as initiation codon. This ATG
codon was modified to TTG by site-directed mutagenesis,
Appl Microbiol Biotechnol (2011) 89:1061–1073
and S. saprophyticus ATCC 15305 (YP_301697.1) are indicated.
Identical residues are indicated by an asterisk, whereas colons and
periods indicate conserved and semi-conserved amino acids. Methi-
onine in position 1 of Tmp1 is given under the presumption that GTG
is the start codon in tmp1 ORF
and the plasmid carrying the resultant mutant gene was
designated pTMP1A1T. The clarified cell lysate from E. coli
BL21(DE3) pTMP1A1T-bearing cells, however, still
exhibited antibacterial activity against K. gibsonii GCS6 in
a lysoplate assay similar to that of Tmp1, and this suggested
that even in the absence of an ATG, the GTG could function
as the tmp1 ORF start codon.
Tmp1 exhibits holin-like activity
To identify whether tmp1 encoded protein could function as
a holin, complementation of a holin-defective lambda
phage was examined. The tmp1 gene was cloned into
λgt11 carrying the Sam100 mutation in holin coding gene,
and its ability to complement the Sam100 mutation was
examined. Phages λgt11 Sam100 formed visible plaques
(PFU 1.5×106) (plaque forming units) on a permissive host
E. coli LE392, but not on a non-permissive host, E. coli
HB101. The recombinant phage carrying the cloned tmp1
Fig. 2 Effect of tmp1 expression on the growth of E. coli BL21(DE3)
(pTmp1). Growth kinetics of E. coli BL21(DE3) (pET30b) (empty
circle) and E. coli BL21(DE3) (pTMP1) uninduced (empty triangle)
compared to that of E. coli BL21(DE3) (pET30b) (filled circle) and E.
coli BL21(DE3) (pTMP1) (filled triangle) induced with IPTG
(5 mM). Samples (n=3) were drawn at an interval of 30 min and
OD600 was measured
Appl Microbiol Biotechnol (2011) 89:1061–1073
Fig. 3 Antibacterial activity of Tmp1. Cell lysates (50 μl adjusted to
50 μg of total protein) from uninduced cultures of E. coli BL21(DE3)
(pET30b) (A), E. coli BL21(DE3) (pTMP1) (B), and induced cultures
of E. coli BL21(DE3) (pET30b) (C) and E. coli BL21(DE3) (pTMP1)
were tested for antibacterial activity by a lysoplate assay against K.
gibsonii GCS6
gene (λgt11::tmp1) produced visible plaques on both
permissive and non-permissive hosts E. coli LE392 (PFU
1.3×106) and E. coli HB101 (PFU 1.6×106), respectively.
These results suggested that Tmp1 functions as a holin-like
protein in E. coli, besides exhibiting antibacterial activity.
Fig. 4 a Deletion constructs of tmp1 gene and their antibacterial
activity. Antibacterial protein coding gene tmp1 (pTMP1) and their
deletion constructs namely N-terminal deletion—NΔTMP1
(pNΔTMP1) and N-terminal and TMD deletion—TMΔTMP1
(pTMΔTMP1) were expressed in E. coli. Highlighted sequences in
TMP1 (11–29) represent the TM domain. Positive and negative signs
indicate the presence and absence of antibacterial activity, respectively.
1067
TMD in Tmp1 is required for antibacterial activity
A single transmembrane domain (TMD) spanning 11 to 29
amino acid residues in Tmp1 was identified using TMpred
program (Hofmann and Stoffel 1993). The N-terminal and
TMD deletion mutants of Tmp1 were constructed as shown
in Fig. 4a, and the ability of mutant proteins to inhibit
growth of bacterial cells was examined. The N-terminal
deletion mutant of Tmp1, NΔTmp1, retained antibacterial
activity comparable to Tmp1, whereas the N-terminal and
TMD deletion mutant, TMΔTmp1, had completely lost
antibacterial activity (Fig. 4b, wells 1–3). At the same time,
a synthetic peptide TMDSP (0.17 μM) with amino acid
sequence identical to TMD exhibited strong antibacterial
activity (Fig. 4b, well 4). These results suggested that TMD
in Tmp1 is essential for antibacterial activity. Further, the
lytic activity of TMDSP was studied with a Gram-positive
cell wall preparation as substrate. The TMDSP peptide
exhibited cell wall hydrolytic activity comparable to that of
a known muralytic enzyme, mutanolysin (Yokogawa et al.
1974) (Fig. 5). Cell wall lytic activity of Tmp1 was also
confirmed by zymogram analysis with cellular proteins of
E. coli BL21(DE3) (pTMP1) (Fig. 6). In zymogram
analysis with K. gibsonii GCS6 as the substrate, the
appearance of a single clearance zone was observed, and
this result suggests that Tmp1 could act on cell membranes
of Gram-positive bacteria and lyse the cell membranes.
b Antibacterial activity of deletion constructs of Tmp1 in a lysoplate
assay. Antibacterial activity of Tmp1, N-terminal, and transmembrane
domain deletion mutants and synthetic peptide. The clarified cell lysate
(50 μl) from an induced culture of (1) E. coli BL21(DE3) (pNΔTMP1),
(2) E. coli BL21(DE3) (pTMΔTMP1), (3) E. coli BL21(DE3)
(pTMP1), and (4) synthetic peptide TMDSP (0.17 μM in 0.02% v/v
TFE) were assayed for antibacterial activity on K. gibsonii GCS6
1068
Fig. 5 TMDSP exhibiting lysis of bacterial cell walls. Pure cell wall
preparation of S. faecalis STF3 was incubated with TMDSP (filled
triangle) (20 μM) and the decrease in absorbance at 600 nm was
measured. Mutanolysin (10 U) (empty triangle) was used as positive
control. Reconstituted cell wall in 0.02% TFE was employed as
negative control (empty circle)
Antibacterial spectrum of Tmp1
In a lysoplate assay for antibacterial activity, the clarified
cell lysates from an induced culture of E. coli BL21(DE3)
(pTMP1) and synthetic peptide TMDSP (20 μM) were
used. Antibacterial activity of recombinant protein Tmp1
and synthetic peptide TMDSP against various indicator
bacterial strains was examined. Both the proteins strongly
inhibited the growth of K. gibsonii GCS6, B. subtilis
a b
M 1 2 3 4 5
kDa kDa
43
29
14
6.5
3 3
Fig. 6 SDS-PAGE and zymogram analysis of Tmp1 for the lysis of
bacterial cell wall. a SDS-PAGE indicating the protein profile of E. coli
BL21(DE3) harboring various plasmids. Lane 1 E. coli BL21(DE3)
induced, lane 2 E. coli BL21(DE3)/pET30b uninduced, lane 3 E. coli
BL21(DE3)/pET30b induced, lane 4 E. coli BL21(DE3)/pTMP1
uninduced, lane 5 E. coli BL21(DE3)/pTMP1 induced for 30 min. b
Appl Microbiol Biotechnol (2011) 89:1061–1073
B4219, L. lactis B1821, S. epidermidis B4268, B. smithii
B173, L. fermentum B1840, P. acidilactici B14958, M.
luteus B287, and L. innocula B33016. However, these
proteins failed to inhibit the growth of Gram-negative
bacteria such as E. coli B409, P. putida B8, and P. stutzeri
B775 (data not shown).
Mutants of Tmp1 with enhanced target specificity
To obtain variants of Tmp1 with an increased spectrum of
antibacterial activity, error-prone PCR was employed to
introduce random mutations in tmp1. A mutant library with
750 clones was screened for antibacterial activity in a
microtiter plate assay as described in the “Materials and
methods”. Four mutants of Tmp1 designated T1RM1,
T1RM2, T1RM3, and T1RM4 inhibited both Gram-
positive (K. gibsonii GCS6) and Gram-negative bacteria
(E. coli B409). Sequence analysis of their corresponding
coding region revealed nucleotide substitutions causing
either a single or two amino acid substitutions (Table 3).
These four mutants were further examined for their ability to
inhibit the growth of Gram-negative bacteria. The killing
curve data revealed a significant reduction in number of
bacteria within 2 h of addition of mutant proteins to the
cultures, and no viable bacteria were detected after 6 h
(Fig. 7). Interestingly, in all these four mutants, the first
codon, GTG, was altered and other substitutions were
confined within the N-terminal region of Tmp1. In our earlier
experiments, GTG was shown to function as the start codon in
the absence of an ATG codon for the expression of Tmp1. But
in these mutants of tmp1, GTG was found to be modified.
M C T
43
29
14
6.5
Clarified cell lysate (50 μl) from induced (T) and uninduced (C) cultures
of E. coli BL21(DE3) (pTMP1) were resolved on a polyacrylamide gel
containing an autoclaved cell suspension of K. gibsonii GCS6. M
molecular weight protein marker. The arrow indicates a zone of
clearance due to the lysis of bacterial cell walls
Appl Microbiol Biotechnol (2011) 89:1061–1073 1069

Hemolytic activity
Therefore, the ATG that preceded GTG possibly functioned
as the start codon in these mutant genes. To confirm this, the
ATG codon in the mutants of tmp1was modified to TTG by
site-directed mutagenesis, and the resultant mutants were
−
designated RM1A1T, RM2A1T, RM3A1T, and RM4A1T.
+
−
−
−
−
−
−
−
Time-kill experiments with clarified cell lysates from the
Gram-negative

induced cultures of E. coli BL21(DE3) harboring pRM1A1T,

pRM2A1T, pRM3A1T, or pRM4A1T suggested that the
modification of ATG to TTG resulted in complete loss of
antibacterial activity against both Gram-positive and Gram-
−
−
+
+
+
+
−
−
−
Inhibitory spectrum

negative bacteria. These results confirmed that ATG acted as

the start codon in the four mutant genes coding T1RM1,
Gram-positive

T1RM2, T1RM3, and T1RM4.

Hydrophobic substitutions in Tmp1 enhance the antibacterial

+
+
+
+
+
+
+
+
+

activity
Net charge

Although the mutant proteins T1RM1, T1RM2, T1RM3,

and T1RM4 showed similar antibacterial activity, their
+1

+1
+2
+1
+1

+1
0

0
0

net charge and average hydrophobicity was found to be

different from that of Tmp1 (Table 3). The net charge of
Hydrophobicity

Tmp1 was found to be +1, indicating the cationic nature of

this protein. Despite the amino acid substitutions, the net
charge of the T1RM1, T1RM3, and T1RM4 remained +1.
0.639
1.005
0.669
0.712
0.659
0.683
0.602
0.561
0.566

However, the net charge of T1RM2 was increased from +1

to +2, and the hydrophobicity was increased from 0.639 to
Table 3 Amino acid sequence of proteins/peptides used in the study and their biological properties

0.712. Therefore, the average hydrophobicity could be

Peptide length

responsible for the broad spectrum of antibacterial activity.

To confirm this reason, valine residues at the second, fourth,
and sixth position in T1RM2 were substituted with glutamic
34
19
35
35
35
35
35
35
35

acid, glutamine, and aspargine, respectively, by site-directed

mutagenesis, and the mutant proteins were designated
MESQSDAVQLMLGFGTFVLMLLGLVVELIKNSNKK
MESQSNAVQLMLGFGTFVLMLLGLVVELIKNSNKK
MLSVSDAVQLMLGFGTFVLMLLGLVVELIKNSNKK

MESVSDAVQLMLGFGTFVLMLLGLVVELIKNSNKK
MLSLSDAVQLMLGFGTFVLMLLGLVVELIKNSNKK
MVSVSVAVQLMLGFGTFVLMLLGLVVELIKNSNKK
MESVSVAVQLMLGFGTFVLMLLGLVVELIKNSNKK

Amino acids in bold face indicate the modified residues in the mutant proteins
MSVSDAVQLMLGFGTFVLMLLGLVVELIKNSNKK

T1SDM1, T1SDM2, and T1SDM3. As these substitutions

resulted in both modification of net charge and overall
decrease in hydrophobicity, we believed that the broad
spectrum antibacterial activity could be affected. As expected,
these mutant proteins T1SDM1, T1SDM2, and T1SDM3 lost
their ability to inhibit Gram-negative bacteria (Fig. 8) while
retaining the antibacterial activity against Gram-positive
LGFGTFVLMLLGLVVELIK

bacteria (Table 3). Thus, the decrease in hydrophobicity,

independent of net charge of these proteins, could be
responsible for the loss of antibacterial activity against
Gram-negative bacteria.

Effect of antibacterial proteins on sheep blood erythrocytes

Sequencea

As the mutant proteins with an increased hydrophobicity

displayed broad spectrum activity, we evaluated the
hemolytic activity of these proteins (Table 3). The
Protein/peptide

antibacterial protein Tmp1 and its derivatives did not show

T1SDM1
T1SDM2
T1SDM3

hemolytic activity, while the synthetic peptide TMDSP

TMDSP
T1RM1
T1RM2
T1RM3
T1RM4

(50 μM) showed mild hemolytic activity (Supplementary

Tmp1

material S3).
a
1070 Appl Microbiol Biotechnol (2011) 89:1061–1073

a a 8
6

5
6

4
Log10 CFU

Log10 CFU
4
3

2
2
1

0 0
1 2 3 4 5 1 2 3 4 5

b b 8

6
6
Log10 CFU

Log10 CFU

4
4

2
2

0 0
1 2 3 4 5 1 2 3 4 5

Fig. 7 Antibacterial activity of Tmp1 and mutant derivatives. To the
exponentially growing cultures of E. coli B409 (a) and P. putida B8
(b), clarified cell lysates from induced E. coli BL21(DE3) containing
Tmp1 (1), T1RM1 (2), T1RM2 (3), T1RM3 (4), and T1RM4 (5) were
added. At intervals, samples (n=3) were withdrawn and colony
forming units (CFU) were determined. Vertical bars indicate residual
CFU recovered after 0, 2, 4, and 6 h, respectively
Fig. 8 Antibacterial activity of T1RM2 and their derivatives. To the
exponentially growing cultures E. coli B409 (a) and P. putida B8 (b),
the clarified cell lysate (50 μl) from induced E. coli BL21(DE3)
containing T1RM2 (1), Tmp1 (2), T1SDM1 (3), T1SDM2 (4), and
T1SDM3 (5) were added. At intervals, samples (n=3) were withdrawn
and CFU were determined. Vertical bars indicate residual CFU
recovered after 0, 2, 4, and 6 h respectively

Discussion cytoplasmic membrane to promote access of cell wall-

A degenerate primer-based PCR strategy was initially used
to amplify antibacterial protein-related gene sequences. The
organization of immunity genes involved in class IIa
bacteriocin production has been shown to be conserved,
and the identified culturable bacteria from goat skin surface
have also been shown to produce multiple bacteriocins
(Anthony et al. 2009; Drider et al. 2006; Kayalvizhi et al.
2008). Previously, immunity gene sequences in bacteriocin
gene clusters have been shown to be associated with a
holin-like protein coding gene bhlA of Bacillus lichen-
iformis AnBa9 (Anthony et al. 2010). BhlA was also found
to exhibit antibacterial activity against Gram-positive
bacteria. Holins are hydrophobic membrane proteins in-
volved in the formation of nonspecific pores or lesions in
hydrolyzing endolysins towards peptidoglycan and to
release of the tailed bacteriophages (Young 1992). Al-
though bacteriophage endolysins have been reported as
antibacterial and therapeutic agents (Borysowski et al.
2006; Courchesne et al. 2009), the property of holins or
holin-like proteins as antimicrobials remains to be studied.
In the present study, a DNA fragment carrying a partial
ORF, which had sequence homology with uncharacterized
holin-like toxin genes, was identified. Further, the complete
ORF tmp1 was amplified from a goat skin surface
metagenome. The expression of Tmp1 was toxic to host
cells comparable to bacteriophage PRD1 holin and NucE
from Serratia marcescens (Berkmen et al. 1997; Ziedaite et
al. 2005). Heterologous holins in general have an ability to
complement a defective S allele in bacteriophage lambda
Appl Microbiol Biotechnol (2011) 89:1061–1073
and thus facilitate the transport of lambda endolysin
through the E. coli cytoplasmic membrane (Damman et al.
2000; Delisle et al. 2006; Henrich et al. 1990; Loessner et
al. 1999). In the present study, we have shown that Tmp1
could complement an S allele defective bacteriophage,
suggesting that Tmp1 could function as a holin-like protein.
Since holin-like proteins with antibacterial activity against
Gram-positive bacteria have not been reported earlier, the
present observation of Tmp1 as a holin-like protein with
antibacterial activity seems to be novel.
The membrane-damaging property in the EJh holin of
the temperate pneumococcal EJ-1 phage is associated with
an N-terminal hydrophobic TMD (Haro et al. 2003).
Similarly, we observed that the lytic activity of Tmp1 is
dependent on the single TMD. Our result is also in
agreement with previous studies on S21 holin of phage 21
which has two TMDs, and a hole-forming property is
associated exclusively with TMD2 (Park et al. 2006). A
synthetic peptide TMDSP with the amino acid sequence of
TMD of the Tmp1 exhibited antibacterial activity against
Gram-positive bacteria only. Although a variety of antibac-
terial peptides with a cationic nature similar to Tmp1 has
been shown to interact with anionic phospholipids of
bacterial inner membranes or with hydrophilic lipopolysac-
charides of the outer cell membranes (Kourie and Shorthouse
2008), the mechanisms of action of these peptides on Gram-
positive bacteria is unknown. A common target for cationic
peptides in Gram-positive bacteria is the cytoplasmic
membrane, wherein a voltage or transmembrane potential-
dependent interaction results in membrane permeabilization,
thereby leading to cell lysis (Koo et al. 1996; Kordel et al.
1988). In addition to a cationic nature, the average
hydrophobicity of cationic antibacterial proteins play an
important role for their efficient interaction with bacterial
membranes (Friedrich et al. 2000; Wu and Hancock 1999).
Based on these results, we suggest that the cationic nature of
Tmp1 and its hydrophobic lytic domain favor a strong
interaction with cell membranes of Gram-positive bacteria,
leading to selective inhibition. In addition, we have
demonstrated the cell wall-hydrolyzing activity of Tmp1 by
zymogram analysis and of TMDSP by its ability to a
hydrolyzed cell wall preparation from S. faecalis STF3.
Without having significant level of similarity with conserved
domains of known peptidoglycan hydrolases (Croux et al.
1993), the cell wall-hydrolyzing activity of Tmp1 seems to
be a new property of this protein.
Four mutant derivatives of Tmp1 with increased average
hydrophobicity (>0.639) and change in net charge showed
a broad spectrum of activity (against Gram-positive and
Gram-negative bacteria). Several other mutants with de-
creased average hydrophobicity (<0.639) failed to exhibit
antibacterial activity against Gram-negative bacteria. Previ-
ously, increase in hydrophobicity among antibacterial
1071
peptides has been shown to exhibit a negative correlation
with peptide specificity (Chen et al. 2007; Wieprecht et al.
1997). Several studies with cyclic, α-helical, amphipathic,
and linear synthetic peptides revealed the importance of
hydrophobicity and net positive charge in determining
antibacterial activity. Magainins are cationic, amphipathic
α-helical peptides identified in the skin extracts of the frogs
Phyllomedusa sauvagei and Xenopus laevis (Zairi et al.
2009). Magainins exhibit antibacterial activity against
Gram-positive bacteria and to a certain extent against
Gram-negative bacteria. Their action on eukaryotic cells is
limited (Cuervo et al. 1990). In magainin, an optimal
balance between the α-helical structure, hydrophobicity,
and net positive charge needs to be maintained, and any
change in these properties results in loss of antibacterial
activity (Chen et al. 1988). By modulating hydrophobicity
and net positive charge independent of α-helical structure
in magainin, selective antibacterial activity has been
successfully explained (Oren et al. 1997). With hydropho-
bic substitutions in the cyclic peptide GS14K4 that resulted
in change of average hydrophobicity, modulation of
antibacterial activity has been explained (Kondejewski et
al. 2002). Similar studies on brevinin 1E amide and certain
synthetic amphiphilic α-helical peptides indicated that
increase in hydrophobicity results in altered target selection
(Kiyota et al. 1996; Kwon et al. 1998).
It is known that highly hydrophobic antibacterial
peptides exhibit cytotoxic activity against neutral mem-
branes of mammalian cells (Oren and Shai 1997; Shai et al.
1996). In histatin and magainin derivatives, the hemolytic
activity strongly correlates with mean hydrophobicity,
independent of amphipathicity or net peptide charge
(Helmerhorst et al. 1999). In our studies, mutant proteins
T1RM1, T1RM2, T1RM3, and T1RM4 with their increased
hydrophobicity exhibited broad spectrum antibacterial
activity but failed to exhibit hemolytic activity. In these
derivatives of Tmp1, the spectrum of antibacterial activity
was correlated with the amino acid substitutions at the N-
terminal region of Tmp1. This extended spectrum of
activity was abolished with modifications that resulted in
the decrease in average hydrophobicity. TMDSP with the
high level of hydrophobicity failed to show antibacterial
activity against Gram-negative bacteria due to the loss of
the N-terminal region of Tmp1. On the other hand, TMDSP
exhibited hemolytic activity. Therefore, we believe that the
substitutions at the N-terminal region of Tmp1 leading to
increase in the hydrophobicity facilitated the interaction of
these mutant proteins with the outer membrane of Gram-
negative bacteria.
In conclusion, a holin-like protein Tmp1 with antibacterial
property has been identified. Proteins with broad spectrum
antibacterial activity and without hemolytic activity were
obtained by altering the average hydrophobicity of Tmp1. The
1072
antibacterial property of Tmp1 together with its ability to
function as a holin-like protein suggests that holin-like
proteins could be explored as therapeutic proteins.
Acknowledgements The authors thank the Department of Biotech-
nology, New Delhi, India for financial support through research grant
(BT/PR-8346/BCE/08/489/2006). TR thanks UGC, India for providing a
meritorious student research fellowship in Biosciences (No. F. 4-1/2006
(BSR)/5-67/2007). The Centre for Advanced Studies in Functional
Genomics, The Centre for Excellence in Genomic Sciences and the
Networking Resource Centre in Biological Sciences are gratefully
acknowledged for support facilities.
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