Plasmid


A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and archaea; however plasmids are sometimes present in eukaryotic organisms as well. Plasmids often carry useful genes, such as those involved in antibiotic resistance, virulence, secondary metabolism and bioremediation. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain additional genes for special circumstances.
Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the internet by various vendors using submitted sequences typically designed with software, if a design does not work the vendor may make additional edits from the submission.
Plasmids are considered replicons, units of DNA capable of replicating autonomously within a suitable host. However, plasmids, like viruses, are not generally classified as life. Plasmids are transmitted from one bacterium to another mostly through conjugation. This host-to-host transfer of genetic material is one mechanism of horizontal gene transfer, and plasmids are 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. Plasmids vary in size from 1 to over 400 kbp, and the number of identical plasmids in a single cell can range from one up to thousands.

History

The term plasmid was coined in 1952 by the American molecular biologist Joshua Lederberg to refer to "any extrachromosomal hereditary determinant." The term's early usage included any bacterial genetic material that exists extrachromosomally for at least part of its replication cycle, but because that description includes bacterial viruses, the notion of plasmid was refined over time to refer to genetic elements that reproduce autonomously.
Later in 1968, it was decided that the term plasmid should be adopted as the term for extrachromosomal genetic element, and to distinguish it from viruses, the definition was narrowed to genetic elements that exist exclusively or predominantly outside of the chromosome, can replicate autonomously, and contribute to transferring mobile elements between unrelated bacteria.

Properties and characteristics

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, 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 almost always carry 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 defences 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, called cryptic plasmids, don't appear to provide any clear advantage to its host, yet still persist in bacterial populations. However, recent studies show that they may play a role in antibiotic resistance by contributing to heteroresistance within bacterial populations.
Naturally occurring plasmids vary greatly in their physical properties. Their size can range from very small mini-plasmids of less than 1-kilobase pairs to very large megaplasmids of several megabase pairs. At the upper end, little differs between a megaplasmid and a minichromosome. Plasmids are generally circular, but 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 plasmid 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.
Plasmids of linear form are unknown among phytopathogens with one exception, Rhodococcus fascians.

Classifications 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 genes which promote sexual conjugation between different cells. In the complex process of conjugation, plasmids may be transferred from one bacterium to another via sex pili encoded by some of the transfer genes. 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 are mobilizable, and carry 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, but 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 and can thus not be kept together in a single cell. Incompatibility typing was traditionally achieved by genetic phenotyping methods, testing whether cells stably transmit plasmid pairs to their progeny. This has largely been superseded by genetic methods such as PCR, and more recently by whole-genome sequencing methods with bioinformatic tools such as PlasmidFinder.
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. F-plasmids are categorized as either or and contribute to the difference of being a donor or recipient during conjugation.
  • Resistance plasmids, which contain genes that provide resistance against antibiotics or antibacterial agents was first discovered in 1959. R-factors where seen as the contributing factor for the spread of multidrug resistance in bacteria, some R-plasmids assist in transmissibility of other specifically non- self transmissible R-factors. 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. e.g. Ti plasmid in Agrobacterium tumefaciens. Bacteria under selective pressure will keep plasmids containing virulence factors as it is a cost - benefit for survival, removal of the selective pressure can lead to the loss of a plasmid due to the expenditure of energy needed to keep it is no longer justified.
Plasmids can belong to more than one of these functional groups.

Sequence-based plasmid typing

With the wider availability of whole genome sequencing which is able to capture the genetic sequence of plasmids, methods have been developed to cluster or type plasmids based on their sequence content. Plasmid multi-locus sequence typing is based on chromosomal Multilocus sequence typing by matching the sequence of replication machinery genes to databases of previously classified sequences. If the sequence allele matches the database, this is used as the plasmid classification, and therefore has higher sensitivity than a simple presence or absence test of these genes.
A related method is to use average nucleotide identity between plasmids to find close genetic neighbours. Tools which use this approach include COPLA and MOB-cluster.
Creating typing classifications using unsupervised learning, that is without a pre-existing database or 'reference-free', has been shown to be useful in grouping plasmids in new datasets without biasing or being limited to representations in a pre-built database—tools to do this include mge-cluster. As plasmid frequently change their gene content and order, modelling genetic distances between them using methods designed for point mutations can lead to poor estimates of the true evolutionary distance between plasmids. Tools such as pling find homologous sequence regions between plasmids, and more accurately reconstruct the number of evolutionary events between each pair, then use unsupervised clustering approaches to group plasmids.