Plasmid is a genetic structure in a cell that can replicate
independently of the
chromosomes, typically a small circular DNA strand in the
cytoplasm of a bacterium or protozoan
.
They are most commonly found in bacteria as small circular, double-stranded DNA
molecules; however, plasmids are sometimes present in archaea and eukaryotic
organisms. In nature, plasmids often carry genes that may benefit the survival
of the organism, for example antibiotic
resistance.
While
the chromosomes are big and contain all the essential genetic information for
living under normal conditions, plasmids usually are very small and contain
only additional genes that may be useful to the organism under certain
situations or particular conditions. Artificial plasmids are widely used as vectors in molecular
cloning, serving to drive the replication of recombinant
DNA sequences within host
organisms.
Plasmids
are considered replicons,
a unit of DNA capable of replicating autonomously within a suitable host.
However, plasmids, like viruses,
are not generally classified as life. Plasmids
can be transmitted from one bacterium to another (even of another species) via
three main mechanisms: transformation, transduction, and conjugation. This host-to-host
transfer of genetic material is called horizontal
gene transfer, and plasmids can be considered part of the mobilome. Unlike viruses (which encase
their genetic material in a protective protein coat called a capsid), plasmids are
"naked" DNA and do not encode genes necessary to encase the genetic
material for transfer to a new host. However, some classes of plasmids encode
the conjugative "sex"
pilus necessary for their own
transfer. The size of the plasmid varies from 1 to over 200 kbp, and the number of identical plasmids
in a single cell can range anywhere from one to
thousands under some circumstances.
Characteristics
of plasmid-
The American molecular biologist Joshua Lederberg first introduced the term plasmid in 1952 - originally to describe any
bacterial genetic material that exists in an extra-chromosomal state for at
least part of its replication cycle. Later
in 1968, it was decided that the term plasmid should be adopted as the term for
extra-chromosomal genetic element, and
to distinguish it from viruses, the definition was narrowed to genetic elements
that exist exclusively or predominantly outside of the chromosome and can
replicate autonomously.
In
order for plasmids to replicate independently within a cell, they must possess
a
stretch of DNA that can act as an
origin
of replication. The self-replicating unit, in this case the plasmid, is called
a
replicon. A typical bacterial
replicon may consist of a number of elements, such as the gene for
plasmid-specific replication initiation protein (Rep), repeating units called
iterons,
DnaA
boxes,
and an adjacent AT-rich region.
Smaller
plasmids make use of the host replicative enzymes to make copies of themselves,
while larger plasmids may carry genes specific for the replication of those
plasmids. A few types of plasmids can also insert into the host chromosome, and
these integrative plasmids are sometimes referred to as
episomes in prokaryotes.
Plasmids
are generally carrying at least one gene. Many of the genes carried by a
plasmid are beneficial for the host cells, for example: enabling the host cell
to survive in an environment that would otherwise be lethal or restrictive for
growth. Some of these genes encode traits for antibiotic resistance or resistance
to heavy metal, while others may produce virulence
factors that enable a bacterium
to colonize a host and overcome its defenses, or have specific metabolic
functions that allow the bacterium to utilize a particular nutrient, including
the ability to degrade recalcitrant or toxic organic compounds. Plasmids can also provide bacteria
with the ability to fix nitrogen.
Some plasmids, however, have no observable effect on the phenotype of the host
cell or its benefit to the host cells cannot be determined, and these plasmids
are called cryptic plasmids.
Naturally
occurring plasmids vary greatly in their physical properties. Their size can
range from very small mini-plasmids of less than a 1 kilobase pairs (Kbp), to
very large megaplasmids of several megabase pairs (Mbp). At the upper end,
little can differentiate between a megaplasmid and a minichromosome. Plasmids are generally
circular, however examples of linear plasmids are also known. These linear
plasmids require specialized mechanisms to replicate their ends.
Plasmids
may be present in an individual cell in varying number, ranging from one to
several hundreds. The normal number of copies of plasmid that may be found in a
single cell is called the copy
number, and is determined by how the replication initiation is regulated and
the size of the molecule. Larger plasmids tend to have lower copy numbers. Low-copy-number plasmids that exist
only as one or a few copies in each bacterium are, upon cell division, in danger of being lost
in one of the segregating bacteria. Such single-copy plasmids have systems that
attempt to actively distribute a copy to both daughter cells. These systems,
which include the parABS system and parMRC
system, are often referred to as the partition
system or partition function of a
plasmid.
Classification
and types
Plasmids may be
classified in a number of ways. Plasmids can be broadly classified into
conjugative plasmids and non-conjugative plasmids. Conjugative plasmids contain
a set of transfer or tra genes which promote sexual
conjugation between different cells. In the complex process
of conjugation, plasmid may be transferred from one bacterium to another
via sex pili encoded by some of the tra genes (see
figure). Non-conjugative plasmids are incapable of initiating conjugation;
hence they can be transferred only with the assistance of conjugative plasmids.
An intermediate class of plasmids is mobilizable, and carries only a subset of
the genes required for transfer. They can parasitize a conjugative plasmid,
transferring at high frequency only in its presence.
Plasmids can
also be classified into incompatibility groups. A microbe can harbour different
types of plasmids; however, different plasmids can only exist in a single
bacterial cell if they are compatible. If two plasmids are not compatible, one
or the other will be rapidly lost from the cell. Different plasmids may
therefore be assigned to different
incompatibility groups depending on whether
they can coexist together. Incompatible plasmids normally share the same replication
or partition mechanisms.
Another way to classify plasmids is
by function. There are five main classes:
·
Fertility F-plasmids,
which contain tra genes. They are capable
of conjugation and result in the expression of sex pili.
·
Resistance
(R) plasmids, which contain genes that provide resistance
against antibiotics or poisons.
Historically known as R-factors, before the nature of plasmids was understood.
·
Col
plasmids, which contain genes that code for bacteriocins, proteins that
can kill other bacteria.
·
Degradative
plasmids, which enable the digestion of unusual substances,
e.g. toluene and salicylic acid.
·
Virulence
plasmids, which turn the bacterium into a pathogen.
Plasmids can belong to more than one
of these functional groups.
Plasmid maintenance
Some plasmids or microbial hosts include an addiction system or postsegregational killing system
(PSK), such as the hok/sok (host killing/suppressor of killing)
system of plasmid R1 in Escherichia
coli. This variant produces
both a long-lived poison and a short-lived antidote. Several types of plasmid
addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were
described in the literature and
used in biotechnical (fermentation) or biomedical (vaccine therapy)
applications. Daughter cells that retain a copy of the plasmid survive, while a
daughter cell that fails to inherit the plasmid dies or suffers a reduced
growth-rate because of the lingering poison from the parent cell. Finally, the
overall productivity could be enhanced.
In contrast, virtually all biotechnologically used plasmids
(such as pUC18, pBR322 and derived vectors) do not contain toxin-antitoxin
addiction systems and thus need to be kept under antibiotic pressure to avoid
plasmid loss.
Yeast Plasmid-
Yeast
are organisms that naturally harbour plasmids. Notable plasmids are 2 µm
plasmids - small circular plasmids often used for genetic engineering of yeast, and
linear pGKL plasmids from Kluyveromyces lactis, that are responsible for killer phenotypes.
Other types of
plasmids are often related to yeast cloning vectors that include:
·
Yeast integrative plasmid (YIp), yeast vectors that
rely on integration into the host chromosome for survival and replication, and
are usually used when studying the functionality of a solo gene or when the
gene is toxic. Also connected with the gene URA3, that codes an enzyme related
to the biosynthesis of pyrimidine nucleotides (T, C);
·
Yeast Replicative Plasmid (YRp), which transport a
sequence of chromosomal DNA that includes an origin of replication. These
plasmids are less stable, as they can get lost during the
budding.
Plasmid
DNA extraction-
As alluded to above, plasmids are often used to purify a
specific sequence, since they can easily be purified away from the rest of the
genome. For their use as vectors and for molecular
cloning, plasmids often need to be isolated.
There
are several methods to isolate
plasmid DNA from bacteria, the
archetypes of which are the miniprep and the maxiprep/bulkprep. The former can be used to quickly find
out whether the plasmid is correct in any of several bacterial clones. The
yield is a small amount of impure plasmid DNA, which is sufficient for analysis
by restriction digest and for some cloning techniques.
In the latter, much larger volumes of bacterial suspension are
grown from which a maxi-prep can be performed. In essence, this is a scaled-up
miniprep followed by additional purification. This results in relatively large
amounts (several hundreds micrograms) of very pure plasmid DNA.
In recent times, many commercial kits have been created to
perform plasmid extraction at various scales, purity, and levels of automation.
Commercial services can prepare plasmid DNA at quoted prices below $300/mg in
milligram quantities and $15/mg in gram quantitie.
Conformations-
Plasmid
DNA may appear in one of five conformations, which (for a given size) run at
different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic
mobility (speed for a given applied voltage) from slowest to fastest:
·
Nicked open-circular DNA has one strand cut.
·
Relaxed circular DNA is fully intact with both strands uncut, but has been
enzymatically relaxed (supercoils removed). This can be
modeled by letting a twisted extension cord unwind and relax and then plugging
it into itself.
·
Linear DNA has free ends, either because both strands have been
cut or because the DNA was linear in vivo. This can be modeled with
an electrical extension cord that is not plugged into itself.
·
Supercoiled (or covalently closed-circular) DNA is fully
intact with both strands uncut, and with an integral twist, resulting in a
compact form. This can be modeled by twisting an extension cord and then
plugging it into itself.
·
Supercoiled denatured DNA is like supercoiled DNA, but has unpaired
regions that make it slightly less compact; this can result from excessive alkalinity
during plasmid preparation.
The
rate of migration for small linear fragments is directly proportional to the
voltage applied at low voltages. At higher voltages, larger fragments migrate
at continuously increasing yet different rates. Thus, the resolution of a gel
decreases with increased voltage.
At
a specified, low voltage, the migration rate of small linear DNA fragments is a
function of their length. Large linear fragments (over 20 kb or so) migrate at
a certain fixed rate regardless of length. This is because the molecules
'resperate', with the bulk of the molecule following the leading end through
the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These
enzymes specifically break the DNA at certain short sequences. The resulting
linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the
bands out of the gel and dissolving the gel to release the DNA fragments.
Because
of its tight conformation, supercoiled DNA migrates faster through a gel than
linear or open-circular DNA.
