Plasmids

It is often desirable to maintain two different plasmids in a single cell. What are
two important considerations when choosing the plasmids to use?
ANSWER: The plasmids should be compatible so they can stably co-exist in
the same cell, and the plasmids should have distinguishable phenotypes so
you can insure that both plasmids are present (ideally a phenotype that is
selectable).

Replication of plasmid ColE1 is catalyzed by the host encoded DNA polymerase

I protein (Pol I). In wild-type cells, Pol I is present in large excess over that
needed for plasmid replication. Initiation of plasmid replication is down
regulated by the plasmid encoded Rop protein which is present in limiting
amounts. If the protein synthesis inhibitor chloramphenicol was added to
sensitive cells that carry a ColE1 plasmid, what would happen to the plasmid
copy number and why?
ANSWER: Addition of chloramphenicol will prevent synthesis of new
proteins, including both Pol I and Rop. Pol I is present in excess, so even in
the absence of protein synthesis there is sufficient Pol I in the cells for many
rounds of plasmid replication. In contrast, Rop is limiting, so in the absence
of new protein synthesis there is insufficient Rop to inhibit plasmid
replication. This results in multiple rounds of plasmid replication and the
accumulation of plasmids to a copy number much greater than normally
found (up to about 1000 copies of the plasmid per cell), a process called
plasmid amplification. This can be used as a trick to facilitate purification of
plasmid DNA from cells.

Copy number mutants of ColE1 type plasmids have been isolated with "runaway" replication at 42 C (resulting in high copy number) but normal replication
(and thus copy number) at 30 C. What type of mutation in which gene might
cause this phenotype? [Explain your answer.]
ANSWER: The most likely mutation is in the rop gene because rop mutants
have increased plasmid copy number, and temperature sensitive mutations

are most often due to changes in protein structure. An alternative

explanation is that the mutation destablizes a stem-loop structure in the
RNA which plays a role in regulation. (It is possible to obtain temperature
sensitive mutations that decrease the stability of nucleic acid hybrids but,
since most nucleic acid hybrids involve a considerable number of base pairs,
a single nucleotide substitution is unlikely to have a substantial effect on the
Tm of the hybrid. Furthermore, in this case the hybrid is between
complementary strands so a nucleotide substitition would result in a
different base pair at that position, not mispairing at that position.)

Mutations in the "RNA I - primer RNA" overlap region of plasmid ColE1 can
result in a a very high copy number. Other mutations in the "RNA I-primer RNA"
overlap region decrease the plasmid copy number.
a. Based upon what you know about the role of these RNAs, propose an
explanation for the high copy number phenotype and the low copy number
phenotype [i.e., what do the mutations do in each case?].
ANSWER: Mutations that destabilize the RNA I - primer RNA would
result in a higher copy number (for example, mutations that result in
decreased base-pairing between the two RNAs). Mutations that
stabilize the RNA I-primer RNA hybrid would result in a lower copy
number (for example, mutations that result in stronger base-pairing
between the two RNAs).
b. Second-site mutations that map on the E. coli chromosome can suppress
the low copy number phenotype described above. What enzyme might be
affected in these mutants (and why)?
ANSWER: Anything that stabilizes the RNA I-primer RNA hybrid
would result in a lower copy number phenotype. A mutation that
increases a host protease with specificity for the plasmid Rop protein
could decrease the intracellular concentration of Rop protein and thus
decrease the stability of the RNA hybrid. Alternatively, an increase in
the concentration or specificity of RNase H could result in enhanced
cleavage of the primer RNA and thus increasing initiation of plasmid
replication and the plasmid copy number.

It is possible to obtain mutations in plasmid encoded genes by mutagenizing the

purified plasmid DNA in vitro (for example, with hydroxylamine) then
transforming the plasmid DNA into recipient cells. Given a plasmid that encodes
ampicillin resistance (AmpR), chloramphenicol resistance (CamR), and Bgalactosidase (lacZ), how would you use this approach to isolate single mutations
in the lacZ gene? Be sure to describe any genetic selections and/or screens used.
[Hint: you will need a way of measuring the relative amount of mutagenesis to
ensure a reasonable probability that the lacZ mutants are due to a single
mutation.]
ANSWER:
Mutagenize the purified plasmid DNA in vitro with hydroxylamine.
Move the plasmid into a lacI- lacZ+ lacY+ host (by transformation or
electroporation of the plasmid DNA), selecting for chloramphenicol
resistance.
Screen the cells for (i) AmpR and (ii) repression of lacZ (for example,
by screening for X-gal- in the absence of an inducer such as lactose or
IPTG).
In several independent experiments, quantitate the extent of
mutagenesis by plotting the number of Amps mutants and the number
of LacI- mutants vs time of exposure to the mutagen. The results will
indicate the number of cells that have a mutation in both genes (i.e. at
least two hits) compared to the number of cells that have a mutation in
only one gene (i.e. at least one hit). These results can be plugged into
the Poisson distribution to determine the probability of 0, 1, and >1 hit
per gene for each time point. Choose colonies from time points where
the probability of >1 hit per gene is rare.

It is possible to "cure" a strain of the plasmid pLAFR which encodes resistance to

tetracycline (TetR) by mating in a second plasmid pPH1JI which encodes
resistance to gentamycin (GenR).
a. What does this suggest about the properties of these two plasmids?
[Explain your answer.]

ANSWER: The two plasmids are probably incompatible. This means

that only one of the plasmids can be stably maintained in the cell.
Hence, selection for the second plasmid results in loss in the first
plasmid.
b. Would this trick work if the only selectable marker on pPH1JI was Tet R?
[Explain why or why not.]
ANSWER: No. Because the recipient cell is already TetR, there is no
selection for inheritance of the second plasmid. Hence, very few cells
would probably be transformed and even if they were you would not
be able to distinguish them from the untransformed cells.

Replication of plasmids with a pR6K origin (oriR6K) requires a protein called pi

(encoded by the pir gene).
a. What would happen if a plasmid with oriR6K that encodes ampicillin
resistance (AmpR) and carries the putA+ gene was introduced into a
recipient that is pir+ putA(Am)? [Would the plasmid replicate? Would the
plasmid integrate into the chromosome? Would the cell be Put + or Put- and
why?]
ANSWER: See figure below.

b. What would happen if a plasmid with oriR6K that encodes ampicillin

resistance (Ampr) and carries the putA+ gene was introduced into a
recipient that is pir putA(Am)? [Would the plasmid replicate? Would the
plasmid integrate into the chromosome? Would the cell be Put +or Put- and
why?]
ANSWER: See figure below.

Normally multicopy plasmids do not integrate into the chromosome even when
they contain considerable DNA sequence homology for recombination. Gutterson

and Koshland (1983. Proc. Natl. Acad. Sci. USA 80: 4894-4898) showed that
multicopy plasmids (such as pBR322) readily integrate into the chromosome
of polA mutants. Suggest an explanation for both of these results.
ANSWER: The polA gene encodes DNA polymerase I, which is required by
the cell in tiny amounts for survival but is needed in large quantities by
certain plasmid origins to initiate DNA replication. Thus, a polA(Am)
mutation, which is slightly leaky, provides enough DNA PolI for the cell to
grow but not enough for plasmids such as pBR322 to replicate autonomously
in the cytoplasm. Under this condition the plasmid can be stably inherited
only if it integrates into the chromosome via a single cross-over event using
regions homologous to the chromosome. In a polA<sup+< sup=""> cell the
plasmid can autonomously replicate in the cytoplasm, but the homology is
still there to allow integration into the chromosome, and this will happen at
the same frequency as in the polA mutant. However, the plasmid origin is
still functional and expressed at a high level to increase the copy number of
the plasmid. There is a lethal effect because of having a functional plasmid
origin disrupting the function of the chromosomal origin and increased copy
number could lead to overexpression of chromosomal genes located adjacent
to the integrated plasmid, which may also be lethal. So the take home point
is that integration still occurs in the polA+ strain, but cells with the
integrated plasmid are not recovered because of the lethal
consequences.</sup+<>

ColE1 plasmids replicate in enteric bacteria but cannot replicate

in Halobacterium salinarium. The bop gene from H. salinarium was cloned into
a ColE1 plasmid, and an insertion mutation constructed that disrupted the
plasmid encoded bop gene (indicated by bop'-'bop in the figure below). This
plasmid was then transformed into H. salinarium with selection for resistance to
the antibiotic mevinolin (MevR).

a. The plasmid does not have any functional replication origin and, except
for the bop gene, the plasmid lacks any homology with the H.
salinarium chromosome. How do the MevR transformants arise? [Show a
diagram and briefly describe your answer.]

ANSWER: The plasmid cannot replicate, so the MevR colonies must

arise by integration of the plasmid into the chromosome by
homologous recombination between the bop genes.

b. When the resulting transformants were subsequently grown for many

generations without mevinolin (Mev), some Mev s colonies were obtained.
About half of these Mevs colonies were Bop+ and half were Bop-. How do
the Mevs Bop- colonies arise? [Show a diagram and briefly describe your
answer.]
ANSWER: Segregration of the chromosomal bop duplication will
yield Mevs colonies. Sometimes the recombination will occur on the
same side of the bop disruption as the initial integration event and
these will result in bop+ colonies [crossover (i) in the figure below].
Sometimes the recombination will occur on the other side of the bop
disruption relative to the initial integration event and these will result
in bop- colonies [crossover (ii) in the figure below].

Two plasmids are shown below. The plasmid pUC19 was constructed from
pBR322 and the origin from pBR322 came from a pMB9 plasmid. The region
designated ORI on the two plasmids is identical, but pUC19 has greater than 100
copies per cell in E. coli while pBR322 has about 20 copies per cell.

a. Why is the copy number of these two plasmids different? [Briefly describe
the molecular mechanism.]
b. When multicopy plasmids are used for complementation analysis,
problems often arise. Describe an example where overexpression of a
protein on a multicopy plasmid could give you erroneous
complementation results.

Muro-Pastor and Maloy (1995. Biotechniques 18: 386-390) developed a method

to easily move mutations from a multicopy plasmid onto the chromosome or
from the chromosome onto a multicopy plasmid. [You will need to consult the
article to answer the following questions.]
a. What was the selection for integration of the plasmid onto the
chromosome? [Briefly describe how this selection works.]
ANSWER: The plasmid was transduced into a polA recipient strain,
selecting for AmpR KanR colonies. Because pBR derivatives require the
polA gene product to replicate, the antibiotic resistant transductants
must arise by integration of the plasmid into the chromosome by
homologous recombination. This integration event results in a tandem
duplication of the cloned putA gene with the plasmid genes between
the two copies of the gene.
b. What was the selection for moving a chromosomal mutation onto the
plasmid? [Briefly describe how this selection works.]
ANSWER: A P22 lysate was grown on the strain with the plasmid
integrated into the chromosome. This lysate was used to transduce the
plasmid into a polA+ recipient strain. The recombinants were selected
on plates with ampicillin and sucrose, then screened for KanS.
The linear DNA brought in on the transducing fragment can
recircularize by homologous recombination between the two copies of
the putA gene (the chromosomal copy and the cloned copy that is
disrupted with the kan and sac genes). Depending upon where the
recombination event occurs, this will re-form the original plasmid
(AmpR KanR SucroseS) or will leave the chromosomal putA gene on the
plasmid (AmpR KanS SucroseR).
Note that this selection scheme does not rely upon the PutA phenotype
because the phenotype of the putA mutant on the multicopy plasmid
was unknown.

Temperature sensitive mutations that prevent the initiation of replication of the E.

coli chromosome are lethal at 42 C. However, Hfr strains that are known to
contain such mutations survive at 42 C.
a. Why is the temperature sensitive phenotype not observed in Hfr strains?

ANSWER: When the chromosomal origin is inactive, replication can

be initiated at the integrated F-plasmid origin and proceed around the
chromosome. This process is called "integrative suppression".
b. How could you prove that a Hfr strain still contains the original
temperature sensitive mutation?
ANSWER: (i) Loss of the Hfr would restore the original replication
mutant phenotype. (For example, it is possible to select for loss of the
Hfr, by recombination with adjacent markers but such transductants
are rare due to the ccd plasmid addiction system of the F-plasmid.) (ii)
Transfer of the chromosome origin into a new strain would confer the
replication mutant phenotype. (For example, this could be done by
selecting for an adjacent marker in an Hfr cross.) (iii) If the Hfr was
integrated at a site distinguishable from the chromosomal replication
origin, you could determine where replication initiates using
molecular approaches. If replication initiates at the Hfr under
nonpermissive conditions but at the chromosomal origin under
premissive conditions, then the original replication mutation is present
in this strain.

The plasmid RSF1010 carries three genes required for its own replication: a
replication initiator protein, a DNA helicase, and a primase. Because RSF1010 is
independent of the corresponding host replication functions, it is a very broad
host range plasmid that can replicate in essentially any gram negative bacterium.
However, there is one notable exception -- RSF1010 is unable to replicate
in Bacteriodes.
a. Suggest a potential explanation why RSF1010 is unable to replicate
in Bacteriodes.
b. How could you test this hypothesis?

The lac operon is often used as a target for mutagenesis studies because it is so
easy to screen for the Lac phenotype on indicator plates. In many mutagenesis
studies the lac genes are present on an F'. To calculated the mutation rate, the
number of mutants are counted and the number of cell divisions are is estimated
based upon the number of cells in the population. The validity of using this
calculation to determine the mutation rate on the F' lac rests upon the assumption

that replication of the F' is coordinated with the chromosome. This is a valid
assumption when the F' replicates via bidirectional replication, but not when F'
replication occurs via rolling circle replication. To avoid this complication, how
could you prevent rolling circle replication of the F'?
ANSWER: The most effective way of preventing rolling circle replication
would be to remove oriT from the F-plasmid. This would prevent
conjugation but would not affect "vegetative" replication.

Mahan, Slauch, and Mekalanos (1993. Science 259: 686-688) developed a

method to identify genes that are turned on when Salmonella infects a eukaryotic
host. The plasmid they used for these studies is shown below. Briefly describe the
functions provided by mob and oriR6K on this plasmid.

ANSWER: The mob function allows mobilization of the plasmid when

transfer functions are provided in trans by a conjugative plasmid. The
oriR6K plasmid replication origin depends upon the pi protein (encoded by
the pir+ gene) which can also be provided in trans. In the absence of pi
protein, the plasmid cannot replicate but if it carries a fragment of
chromosomal DNA it can integrate into the host chromosome.

Integration of a cloned gene on a suicide plasmid is a useful trick for constructing

a tandem chromosomal duplication. Given the plasmid shown below and a
recipient with a pyrA(Am) mutation, how could you construct a chromosomal
duplication for complementation analysis? [Draw a diagram showing the
crossover and resulting chromosomal duplication]

Rob Edwards constructed Salmonella enteritis mutant with a KanR insertion in

the sefA gene (designated sefA::Kan). He then cloned thissefA::Kan gene onto a
derivative of plasmid R6K that lacks the pir gene. When the plasmid was
transformed into S. enteritis, a low number of transformants was obtained. The
plasmid was also transformed into two Salmonella typhimurium strains: strain A
carried a copy of the pir+gene cloned onto the chromosome and strain B did not

have a copy of the pir+ gene. Many KanR transformants were obtained in strain A
but none were obtained in strain B. Suggest a likely reason for these results. [For
help, check out the discussion of "Allele exchange" on the 316 supplement.]
ANSWER: See figures below.

It is possible to disrupt a chromosomal gene by integration of an internal

fragment from that gene cloned onto a suicide plasmid. How would integration of
the plasmid shown below disrupt the adh gene? [Note that 'adh' represents an
internal fragment of the adh gene that is missing DNA sequences from the
beginning and end of the gene.]

The plasmids pBR328 and pUC19 are shown below. Both have a pMB9 origin.
Because these plasmids share a common origin, they are incompatible. If an E.
coli cell is co-transformed with both plasmids, then grown for many generations
in medium with ampicillin, which of the plasmids would be most likely to be
maintained? Why?

Recombination can occur between multiple copies of a plasmid in a cell. Such

recombination results in plasmid dimers or multimers. Many plasmids encode a
site specific recombination system that rapidly monomerizes any plasmid dimers
formed.
a. Draw a homologous recombination event that would generate a plasmid
dimer, and the site-specific recombination event that would regenerate
monomers.
b. Why might a plasmid need an efficient site-specific recombination system
to prevent the accumulation of dimers?

The plasmid pBR322 can replicate in E. coli but not in Bacteriodes

thetaiodomicron, and many Bacteriodes plasmids can replicate in B.
thetaiodomicron but not in E. coli. What parts of each plasmid would be needed
to make a "shuttle plasmid" that could replicate in both hosts?
ANSWER: The shuttle plasmid would require the regions of DNA from both
plasmids which function as an origin in each host. This would include any
cis-acting sites as well as any trans-acting factors required from each
plasmid.

The CcdA and CcdB gene products from F-plasmid function as a "plasmid
addiction system". Given an F' that carries a gene for resistance to tetracycline:
a. What would be the phenotype of a ccdB mutant? Why?
ANSWER: It would be easier to detect segregrants of the F-plasmid
because any segregrants would survive in the absence of the CcdB
toxin.
b. What would be the phenotype of a ccdA mutant? Why?
ANSWER: These mutants would be lethal. In the absence of the CcdA
anti-toxin, the CcdB toxin would kill the cells.

Simons, Housman, and Kleckner (1987. Gene 53: 85-96) developed a very useful
trick for moving genes from a multicopy plasmid onto phage lambda by
homologous recombination. Because lambda can be integrated into the
chromosomal att site in a single copy, this trick makes it possible to easily do
complementation tests with cloned genes. [You should be able to answer the
following questions without referring to the published paper.]
Draw a diagram showing how you could use homologous recombination to move
a DNA fragment from a plasmid onto phage lambda.
ANSWER: You could use homologous recombination between a region on
the plasmid that has also been cloned into a nonessential region of phage
lambda. Any gene cloned onto the plasmid between the homologous flanking
regions could be recombined onto lambda as shown in the figure below.

How could you could select for the desired lambda derivatives?
ANSWER: If you placed an antibiotic resistance gene within the homologous
flanking gene, you would have a simple selection for the desired
recombinants. Simply grow the lambda lysate in a strain with the plasmid,
and then infect a recipient strain selecting for antibiotic resistant lysogens.

When phage P1 lysogenizes cells, instead of integrating into the chromosome it

remains as an extrochromosomal closed circular DNA. Nevertheless, P1 lysogens
are quite stable. What functions would be required to ensure that the P1 DNA is
not segregrated upon cell division?
ANSWER: Phage P1 requires partitioning (par) functions to insure that it
co-segregrates with the host chromosome at each cell division.