Biology 310: Prokaryotic and Viral Genetics

Plasmids

I. What is a plasmid?
Any DNA molecule in cells that replicates independently of the chromosome and
regulates its own replication so that the number of copies of the DNA molecule
remains relatively constant. Can be infectious (self-transmissible via conjuga-
tion) and sometimes can integrate into the main chromosome, in which case it is
known as an episome.

A. Naming plasmids

1. Originally, naturally occurring plasmids were named for phenotypes

they conferred (F for fertility, R for antibiotic resistance, etc.).

Plasmid Phenotypic trait Original source

F Fertility Escherichia coli1

RK2 Resistance to ampicillin, tetracycline, and Klebsiella aerogenes
kanamycin
ColE1 Produces a bacteriocin which kills E. coli Escherichia coli
Tol Degradation of toluene and benzoic acid Pseudomonas putida
Ti Tumor initiation in plants Agrobacterium tumefaciens
pJP4 2,4-D (dichlorophenoxyacetic acid) degrada- Alcaligenes eutrophicus
tion
pSym Nodulation on roots of legume plants Rhizobium meliloti
SCP1 Antibiotic methylenomycin biosynthesis Streptomyces coelicolor

2. Artificially constructed plasmids are usually given the designation

“p” for plasmid, “XY” the initials of the person or team that
constructed the plasmid, and “nnn,” its serial number. Thus
pBR322 was the serial number 322 plasmid constructed by Bolivar
and Rodriquez.

B. Functions encoded by plasmids

1. Plasmids do not usually encode routine housekeeping functions

1 Found in 28% of human E. coli isolates (MULEC, J., M. STARCIC, & D. ZGUR-BERTOK. F-like plasmid
sequences in enteric bacteria of diverse origin, with implication of horizontal transfer and plasmid host
range. Current Microbiology 44(4): 231–235, April 2002.)

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2. Rather they tend to confer metabolic adaptations to specialized
environments

a) Growth in the presence of an antibiotic

b) Competitive growth advantage through targeted killing of

competitors

c) Growth on specialized carbon sources

d) Growth inside eukaryotic hosts

(1) Pathogenicity is often plasmid-mediated

(2) Many exotoxins are encoded by plasmid genes

(a) E. coli heat-stable and heat-labile enterotoxins

(b) Staphylococcus aureus enterotoxin

(c) Tetanus toxin of Clostridium tetani

(3) Yersinia pestis, the causative agent of bubonic plague,

must have at least three plasmids for pathogenicity

(a) One encodes outer membrane proteins (Yops)

(b) One encodes a toxin and an antiphagocytic protein

(c) One encodes a protease

(d) A fourth plasmid encodes an iron scavenging

system

3. Thus plasmids essentially increase the genetic diversity of bacteria

without overburdening the main chromosome with seldom-used
genes.

4. However, they always act as free agents. They are not out there
for the sake of any particular set of host cells.

C. Plasmid structure: negatively supercoiled covalently closed circular

dsDNA, usually

1. Some plasmids in Streptomyces or Borrelia burgdorferi are linear,

with telomere-like ends

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LECTURE 04: PLASMIDS

II. Properties of plasmids

A. Replication

1. DNA molecules that can replicate “autonomously” in the cell are

called replicons

a) This actually would include all viable plasmids, plus the main
chromosome.

b) Sometimes authors will state that their artificially constructed

plasmids are based on the so-and-so replicon, for example
the RK2 replicon. This seems to mean that the construct
has on it all of the sequence of RK2 necessary for replica-
tion, plus other interesting features not found on the naturally
occurring plasmid. Most of the sequence absolutely neces-
sary for replication seems to map around the ori site.

c) “Autonomously” is in quotes, because many, if not most, of

the proteins necessary for plasmid replication are encoded in
the host chromosome, and this then necessarily limits the
host range of the plasmid.

2. All replicons have an ori site, the origin of replication.

3. Two general mechanisms of replication

a) Theta (θ) replication, with either one or two replicating

systems beginning at ori and either replicating the whole
plasmid (if single), or replicating half the plasmid (if double).

b) Rolling circle replication, with a nick at ori, followed by the 3’

end serving as the primer for replication. A single-stranded
replicate is made, and then its complementary strand must
be synthesized from RNA primers.

B. Functions of the ori region

1. Host range: because of the intimate interactions of the ori region

with host-derived products, the ori region pretty much determines
the broadness of the host range

a) ColE1 and its derivatives pBR322, pET, and pUC all have
host ranges narrowly confined to Escherichia coli, Salmo-
nella, and Klebsiella.

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LECTURE 04: PLASMIDS
b) RK2, on the other hand, has a very broad host range, and
will replicate in almost any Gram-negative organism. It
seems to encode more of its own proteins, rather than rely-
ing on host cell functions.

2. Regulation of copy number

Plasmid Approximate copy number

F 1
P1 prophage 1
RK2 4–7 in E. coli
pBR322 16
pUC18 30–50
pIJ101 40–300

a) Relaxed plasmids inhibit the initiation of plasmid replication

when copy numbers get too high.

b) Stringent plasmids like F, P1, RK2, and ColE1 and its deriva-
tives have more elaborate mechanisms.

(1) Regulation via antisense RNA. In the ColE1 family of

plasmids, replication is dependent on the availability of
RNA II, which serves as the primer. RNA I, transcribed
from the strand opposite that from which RNA II is tran-
scribed, is (necessarily) complementary (antisense) to
RNA II, forms a double helix with it, and gums up its ability
to serve as a primer. Apparently RNA I levels increase
with copy number; enough is made from a copy number of
16 that replication is shut down nearly completely.

(2) Regulation via both RepA protein and antisense RNA.

In the R1 family of plasmids, the RepA protein is necessary
for initiation of replication. repA is transcribed from both
the pcopB and prepA promoters. prepA only produces a repA
transcript, but pcopB produces a transcript with both the
upstream copB and repA open reading frames. The CopB
protein is a repressor of transcription at prepA, so after a few
minutes in a new cell, transcription at prepA shuts down.
Further, transcription of an antisense RNA called copA
from promoter pcopA, which is in the middle of the repA
gene and oriented backwards, inactivates the copB repA
transcript, totally shutting down production of RepA.

(3) Regulation via RepA protein handcuffing iterated

sequences at ori. Plasmids pSC101, F, R6K, P1, RK2,
and RP4 all use some variation of the system seen at its

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 BIOLOGY 310: PROKARYOTIC AND VIRAL GENETICS
LECTURE 04: PLASMIDS
simplest in pSC101. Once again, the RepA protein is
necessary for initiation of replication. First, RepA serves
as its own repressor protein by binding to its promoter
sequence and shutting down its own transcription.
Second, RepA interacts with repeated iteron sequences,
R1, R2, and R3. Apparently RepA can mediate the asso-
ciation of the R1, R2, and R3 sequences of one copy of
plasmid with the same three sequences in another, gum-
ming up replication.

C. Mechanisms to prevent curing of plasmids

1. Resolution of multimeric plasmids

a) Sometimes plasmids will accidentally form catenanes, inter-

locking circles like links on a chain. A catenane will then
partition as a unit, and raise the effective copy number, while
increasing the possibility of a cure.

b) Sometimes plasmids will accidentally form concatemeric

structures, which are linear repeats of the entire sequence,
which also raise the effective copy number, while increasing
the possibility of a cure.

c) This problem is usually addressed with site-specific recombi-

nases, which open certain sites so frequently that catamers
and concatamers cannot form.

d) The most famous plasmid site-specific recombinase is the

Cre protein, which acts so rapidly on the loxP site of the
phage P1 genome that it has a linear genetic map, in spite of
having a circular DNA genome.

2. Partitioning

a) If n plasmids partitioned randomly, without any mechanism

promoting an equal distribution, the probability that all plas-
mids will end up in one daughter cell is
F 1I
p = 2×G J
2n

H 2K (the multiplicand of 2 indicates that it doesn’t

matter which daughter gets none of the plasmids, and a cell

with n plasmids will have 2n plasmids at the time of parti-
tioning).

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LECTURE 04: PLASMIDS
b) Thus if n = 16, with 32 at the time of partitioning, the prob-
ability of a cure is 2.3 x 10-10, but if n = 1, the probability of a
cure is 0.5. Low copy number plasmids have a problem.

c) Partitioning mechanisms of low-copy-number plasmids are

only now being characterized.
Accurate partitioning of the F plasmid seems to depend on
three “stability of plasmid” loci, sopA, sopB, and sopC.2
sopC is a series of 11 or 12 43-bp repeats. SopA and SopB
are proteins that bind to the repeat region and bring the
plasmids to the membrane at the midpoint of the cell.

3. Plasmid “addiction”

a) Some plasmids ensure that cells that lose them die.

b) The parB locus of the R1 plasmid can be divided into hok

(host killing) and sok (suppressor of hok). hok encodes the
Hok protein that destroys the cellular membrane potential,
causing a loss of cellular energy and cell death. sok, on the
other hand, encodes an antisense RNA that inactivates hok
mRNA. As long as the cell has both hok and sok loci, it is
safe. However, if it loses the plasmid, sok RNA degrades
faster than hok mRNA, Hok protein is made, and the cell
dies.

c) Thus plasmids are capable of killing potential competitor

cells that have lost the plasmid. Scary.

D. Incompatibility

1. Not all types of plasmids can stably coexist in a given cell.

2. Two plasmids that cannot coexist in the same host cell for long are
members of the same incompatibility group. For example, RK2
and RP4 both belong to the IncP incompatibility group.

3. Incompatibility due to replication control: plasmids that share

replication control will reach only half the copy number they usually
would, increasing the probability of a cure.

2 NIKI, H. & S. HIRAGA. Subcellular distribution of actively partitioning F plasmid during the cell division
cycle in E. coli. Cell 90(5): 951–957, September 5, 1997.

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 BIOLOGY 310: PROKARYOTIC AND VIRAL GENETICS
LECTURE 04: PLASMIDS
4. Incompatibility due to partitioning: plasmids that share partition
mechanisms will compete for distribution into daughter cells,
increasing the probability of a cure.

III. Plasmid genetics

A. Methods for extracting and analyzing plasmids

1. The alkali lysis method is a popular method for the extraction of

plasmid DNA.

a) High salt and pH causes denaturation of DNA and precipita-

tion of chromosomal DNA.

b) Plasmid DNA remains relatively soluble, and renatures

easily.

2. The Qiagen® chromatographic columns are popular methods for

purifying extracted plasmid DNA, although centrifugation of plasmid
DNA in a cesium chloride density gradient in the presence of
ethidium bromide gives very good results also (supercoiled plasmid
DNA will take up less ethidium bromide than broken chromosomal
DNA will, and so remains at a higher density).

3. Analysis of DNA extracts is usually done by agarose gel electro-

phoresis. Uncut supercoiled plasmid DNA will usually run at a
position different from that of nicked plasmid DNA and from frag-
mented chromosomal DNA.

4. Pulsed-field gel electrophoresis is best for larger plasmids.

B. Determining the incompatibility group

An unknown plasmid with one antibiotic resistance marker can be intro-
duced into a cell with a plasmid of a known incompatibility group and a
different antibiotic resistance marker. Rates of curing under conditions of
no antibiotic selection, compared to rates of curing of each plasmid alone
under similar conditions gives an indication of compatibility.

C. Maintaining plasmids belonging to the same incompatibility group

Two plasmids can be maintained together if each has its own selectable
antibiotic resistance marker and cultures are maintained in the presence
of both antibiotics.

D. Determining the host range

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If the plasmid has a selectable marker, it can be introduced into various
species by transformation or conjugation, and its ability to be maintained
in the presence of selection ascertained.

E. Finding the plasmid ori region

Various fragments of the plasmid can be ligated to a selectable marker;
only those fragments that carry a complete ori region (an origin of replica-
tion plus any genes that encode necessary trans-acting factors) will repli-
cate in a host cell.

F. Studying the requirements for plasmid replication

1. Trans-acting factors can be identified by inactivating the putative

gene on one plasmid and introducing the wild type sequence on
another, compatible plasmid. Replication of the first plasmid will
only then occur in the presence of the second plasmid, if the puta-
tive gene on the first plasmid is indeed essential.

2. Cis-acting sequences in the ori region can only be studied on

plasmids with an additional, conditional, ori region. Constructs are
maintained under conditions permissive for the secondary ori
region, then switched to non-permissive conditions to test the
effects of manipulations at the primary ori region under study.

IV. Plasmid cloning vectors: autonomously replicating DNA into

which other DNA can be inserted

A. Desirable features of plasmid cloning vectors

1. It should be small, so that it can be easily isolated and introduced

into various bacteria.

2. It should have a relatively high copy number, so that it can be easily

purified in sufficient quantities.

3. It should carry an easily selectable trait that can be used to select

cells that contain the plasmid.

4. It should have one or more restriction enzyme sites, preferably in a

second selectable gene, into which DNA sequences can be
inserted.

5. Other features that are sometimes desirable

a) pac or cos sites recognized by phage packaging systems

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b) Expression systems so that cloned genes can be expressed
in bacteria

c) Mobilization (mob) sites, to facilitate conjugation into other

cells

d) Broad-host-range ori regions

e) Shuttle vectors will have two ori regions, one say for bacte-
ria, and the other for yeast

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