Plasmids
Devlina Sengupta
Department of Microbiology
Kanchrapara College
Bacterial
Plasmids
The term plasmid was first
introduced by
the American molecular
biologist Joshua Lederberg in
1952.
A plasmid is a short, usually
circular, and double-
stranded segment of DNA that is
found in the cytoplasm separate
from the main bacterial
chromosome.
The bacterial chromosome and bacterial plasmids, as shown
in the electron microscope. The plasmids(arrow) are the
circular structures, much smaller than the main
chromosomal DNA.
Plasmid
features
Plasmid sizes vary from 1 to over
1,000 kbp.
They usually contain 5 to 100 genes
and usually carry genes that are
useful but not essential to survival:
e.g. genes which make bacteria
resistant to antibiotics.
As long as the bacterium is thriving in a low-
stress environment, removing all the
plasmids would not affect the ability of the
bacterium to survive.
Classification Of
Plasmids.
[Link] on their ability to transfer to other
bacteria.
a) Conjugative plasmids - contain tra
genes, which perform the complex
process
of conjugation, the transfer of plasmids
to another bacterium.
e.g., F plasmid, many R plasmid &
some Col plasmid.
b) Non-conjugative plasmids - incapable
of initiating conjugation, hence they
can be transferred only with the
assistance of conjugative plasmids.
e. g., many R plasmid & most Col
plsmid.
c) Mobilisable plasmid - An intermediate
class of plasmid. They carry only a
subset of the genes required for
transfer. They can parasitize another
plasmid, transferring at high
frequency in the presence of a
conjugative plasmid.
Process of plasmid transfer
• The process by which non-conjugative plasmid is
effectively transferred into a recipient via the
contact provided by a conjugative plasmid is
called donation.
• The process by which self transmissible plasmid
via recombination carries along with it the non-
mobilizable plasmid to a recipient is called
conduction.
• Plasmid mobilization begins when a plasmid
encoded protein, which is a nicking protein of the
relaxation complex, makes a single strand break
in a unique base sequence called the transfer
origin or oriT. This nicking protein is attached at
5΄ end.
• This nick initiates rolling circle replication & the
linear branch of the rolling circle is transferred.
• DNA synthesis occurs both in donor (called
donor conjugal DNA synthesis that serves to
replace the single strand that is transferred) & in
recipient cells (called recipient conjugal DNA
synthesis that converts transferred single strand to
tra genes of F
• Transfer of R plasmids immediately after
receiving into a recipient cell is inhibited at
many times due to a multisubunit repressor
encoded by two fin genes which acts on an
operator & prevents transcription of the
genes required for transfer (tra genes). This
phenomenon is called fertility inhibition.
• Transfer of F plasmids is constitutive
because they have repressor sequence
disrupted by insertion of an IS3 sequence in
finO.
• Thus R plasmid transfer is initiated into a
recipient from a recent recipient, even before
the repressor protein synthesis has started
inside it.
• Most of the tra genes are controlled in an
operon which is involved in pili synthesis.
[Link] on function.
a) Degradative plasmids – They are able
to digest unusual substances like
toluene and salicylic acid.
e.g.,TOL plasmid of Psedomonas putida.
b) Virulence plasmids –contains vir
genes which turn the bacterium into
a pathogen.
e. g., Ti & Ri plasmids
c) Fertility (F)-plasmids - contain tra
genes. They are capable of
conjugation and result in the
d) Resistance (R)plasmids –
contain genes that provide
resistance against antibiotics
or poisons.
Historically known as R-factors,
before the nature of plasmids was
understood.
e. g., pRP4 of Pseudomonas sp.
e) Col plasmids - contain genes that
code for bacteriocins & toxins that
can kill other bacteria.
e. g., ColE1
III. Based on copy number.
a) Stringent Plasmid – It replicates only
along with the main bacterial
chromosome & is present as a single
copy, or at most several copies, per cell.
a) Relaxed Plasmid – It replicates within a
cell independently of the chromosomal
DNA replication. Thus multiple copies
of plasmids are present.
Plasmid copy number
• The average number of a particular plasmid maintained
inside a cell or the number of copies of plasmids in a
newborn cell immediately after cell division.
• All plasmids must regulate their copy number, otherwise
they would fill up the cell & become too great a burden for
the host or their replication would not keep up with the cell
replication & they would progressively be lost during cell
division
• Plasmids with high copy number have different mechanism
in copy number regulation as compared to plasmids with low
copy number.
Control of plasmid copy number
• Regulation of copy number is by a repressor protein encoded by
the plasmid itself, that negatively regulates the initiation of
replication.
• Activity of this repressor is dependent on its own concentration. As
the cell grows, volume increases so the repressor concentration
drops & replication is not inhibited. Plasmid DNA molecules
doubles.
• At this point the initial number of repressor genes doubles as well,
causing larger amount of repressor protein synthesis & replication
to stop.
• For high copy number plasmids complete repression requires a
higher concentration of repressor than is required for low copy
number plasmids.
• Each plasmid controls its own copy number.
• Some high copy number exhibit a phenomenon called Plasmid
amplification whereby bacterial chromosomal DNA synthesis is
inhibited by presence of a replication protein synthesis inhibitor &
[Link] on compatibility
• It is possible for plasmids of different types to coexist in a single
cell. Several different plasmids have been found in E. coli. Eg:
Plasmid ColE1 & plasmid F can coexist together because they fall
under compatibility group/different incompatibility group i.e.
repressor of one plasmid does not impact the replication of other
plasmid.
• However, related plasmids are often incompatible, in the sense
that only one of them survives in the cell line, due to the
regulation of vital plasmid functions. Thus, plasmids can be
assigned into incompatibility groups based on the fact that
repressor protein of one plasmid influences the replication of
other plasmid & either of the plasmid is selected randomly for
replication.
• In such cases as a single cell containing two compatible plasmids
divides, the percentage of the progeny population containing only
plasmid type will increase with each generation.
Plasmid replication
• Plasmids rely on host replication proteins for their replication.
• Some plasmids use host DNA pol III(like F) or some use host DNA pol I(like
COLE1).
• Some replicate unidirectionally some bidirectionally (theta mode of
replication).
• Plasmids replicating bi-directionally terminate replication in either of two
ways: a) termination occurs when growing replication forks collide, b)
termination occurs when one of the two replication fork reaches a fixed
termination site.
• Plasmids have a separate mode of replication called “Butterfly or rabbit’s
ear mode” whereby replicated molecule contains untwisted replicated
portions & a supercoiled unreplicated portion.
• When replication cycle is completed, one of the circles is cleaved to separate
the daughters in which newly replicated molecule is sealed & supercoiled
later.
In image a) unidirectional replication is shown where origin region is
designated by oriV. Replication terminates when the replication fork gets
back to origin. In image b) bidirectional replication is shown where
replication terminates when the replication forks meet at a point
opposite oriV
Rolling circle replication where a nick is made at the double stranded origin (DSO) by
the plasmid encoded Rep protein which remain bound to the 5’p at the nick. The free
3’OH serves as primer for the DNA pol III which replicates around the circle displacing
the old strand as a ssDNA. The Rep protein makes another nick, releasing the single
stranded circle. DNA ligase then joins the end to form ds & SS circular DNAs. The host
RNA polymerase then makes a primer on SSDNA origin & then DNA pol III replicates
SSDNA to make another ds circle. DNA Pol I removes the primer, replacing it with
DNA & ligase joins the end to make ds circular DNA.
Plasmid curing
• Plasmid curing is the loss of plasmid during cell division.
• In general a plasmid with copy number n, the frequency of
curing is 2(1/2)2n considered when the sorting of the plasmid is
completely random. ( 2 is multiplied because at the time of cell
division the number of plasmid is twice the copy number).
• Several mechanisms exists to prevent plasmid curing like
plasmid addiction system, site specific recombinases that
resolve multimers or partitioning systems.
Plasmid Addiction
Plasmid partitioning
• Partitioning system ensures that a plasmid is not lost during cell
division & atleast one copy of plasmid is segregated in each
daughter cell.
• This is maintained by par function system of the cell. It consists
of a cis acting site called ParS & two trans acting genes that
encode proteins called ParA & ParB.
• ParS site is the site at which two copies of the plasmid are
pulled apart during partitioning & parA & parB proteins act at
this site.
• Although the mechanism of this action is still under research,
but it is known that many copies of parB binds at around parS
site blocking or silencing genes around this site.
• The parA protein next binds this complex of parBparS
hydrolyzing ATP to ADP.
Two models for par site functions: Model a) plasmid binds to the unique
site on the bacterial membrane & two copies of the plasmids are pulled
apart as the site on the membrane divides. In model b) two copies of the
plasmid bind to each other before they bind to a site on membrane. One
then associates with each site when the site of the membrane divides.
Plasmid host range
• The host range of a plasmid includes all types of bacteria in
which the plasmid can replicate & the host range is usually
determined by the ori region.
• Some plasmids such as those with ori regions of the colE1
plasmid type including PBR322, pUC, pET are narrow host
range plasmids as they replicate only in [Link] & some other
closely related bacteria like Salmonella & Klebsiella species.
• Plasmids with broad host range includes RK2 & RSF1010
which will replicate in most gram negative bacteria & even in
some gram positive bacterias.
• Some gram positive plasmids like pUB110 are also broad
range plasmids as they would replicate in most gram positive
bacteria.
F plasmids
• F plasmids apart from providing mobilizable function also acts
in “integrative suppression”
• F plasmids have the ability to integrate at random sites(unlike λ
phage’s site specific integration) into bacterial chromosome to
generate an Hfr cell.
• F plasmids utilizes many proteins of host cells to replicate itself
& excludes some other proteins involved in host chromosomal
replication. One such protein is dnaA
• Bacterial chromosome mutant in replication protein called dnaA
cannot replicate its own chromosomal DNA at high temperature
(42 ̊C) but F plasmid with the help of other host replicative
protein can continuously replicate itself at this temperature.
• In Hfr cells, however mutated dnaA protein called dnaA(Ts) is
compensated by F integration as bacterial chromosome is able to
replicate using integrated F origin of replication (oriV).
• This way Ts mutation in dnaA of chromosomal DNA is
suppressed by F integration.
Drug resistant plasmids
• Most R plasmids have two contiguous segments of DNA: a)
Resistance transfer factor(RTF) b) R determinant
• RTF carries genes regulating DNA replication & copy number,
transfer genes & sometimes the genes for antibiotic resistance.
• In some strains & with some plasmids an RTF-R composite
plasmid could not be stably maintained & are continually
segregated.
• Plasmid amplification happens when both segments coexist
together in large number of plasmids. This increases the size of
plasmid & if any antibiotic is added to the medium of growth,
this increase is observed prominently. Reversion to original size
of plasmid occurs when antibiotic concentration is withdrawn.
• An interesting group of R plasmids are F-like R plasmids
including R1, R6 & R100. Genetic analyses showed that F-like
R plasmids consists of a large segments of F linked to a typical
R determinant
Colicinogenic plasmid
• Plasmids which produce proteins called colicins that prevent growth of
susceptible competing bacteria, are called Colicinogenic plasmids.
• Colicins interact with sensitive bacteria & inhibit one or more essential
processes such as DNA replication,transcription, translation, or energy
metabolism.
• Colicin production is detected by an assay similar to that for detecting phage
where colicin producing cell if placed on a lawn of sensitive cells inhibit growth
of nearby bacteria producing a clear area in the turbid layer known as a lacuna.
• Colicins are of two types: true colicins & defective phage particles (gene
products from remnants of ancient defective prophages) that resemble phage tail
structure.
• Some colicins are inactive against a cell that contains a related col plasmid. This
phenomenon is called immunity. Immunity is conferred by a small protein that
binds larger colicin protein & also kills Col- cells.
• ColE1 is a colicinogenic plasmid that depends on compatible F plasmid for
its conjugative mobilization.
Linear plasmids
• It is still a common belief that plasmids are circular. However,
linear plasmids have been reported to exist more than a decade
ago.
• Two types of linear plasmids are known. One type contains
covalently closed ends and are commonly found in Borrelia, the
causative agent of lyme disease. The other type is characterized
by the covalent attachment of proteins at the 5' ends and exists in
a number of bacterial genera including Streptomyces,
Rhodococcus, Mycobacterium and Planobispora.
• Such plasmids like linear chromosomes face a primer problem
whereby the extreme 5’end of lagging strand cannot be replicated
by DNA polymerase as no primers exists upstream. This is not a
problem of circular plasmids as upstream primer DNA exists.
• Such problems are thought to be solved in Borrelia linear
plasmids by having telomeric sequences on their ends. Also CC
ends have same inverted sequences, which allow the plasmids to
be replicated till the end without losing DNA from the ends.
Uses of plasmids
Plasmids serve as important tools in
genetics and biotechnology labs, where they
are commonly used to multiply (make many
copies of) or express particular genes.
Disease Models - Plasmids were historically
used to genetically engineer the embryonic
stem cells of rats in order to create rat
genetic disease models.
Gene therapy- plasmid vectors are
used for the insertion of therapeutic
genes at pre- selected chromosomal
target sites within the human genome.
Another major use of plasmids is to
make large amounts of proteins. In this
case, researchers grow bacteria
containing a plasmid harboring the gene
of interest.
eg: insulin & antibiotics.
