In this article we will discuss about:- 1. Meaning of Plasmids 2. Preparation of Plasmids 3. Major Groups.
Meaning of Plasmids:
Plasmids are dispensable, extra pieces of DNA found mostly in bacteria, that exist separately from the main bacterial chromosome. They are simpler in organisation than viruses, have no protein coat, and cannot exist outside a cell.
They replicate independently within the host cell and are inherited in a regular manner when the cells multiply. First discovered about 30 years ago, they were later found to be of widespread occurrence in bacteria.
The presence of the plasmid appears to be not essential for any metabolic requirement of the host cell. As such, some cells of a plasmid-carrying strain may be devoid of a plasmid. The genes contained in plasmid DNA however, do provide accessory genetic information. The F factor for instance, is a plasmid that carries genetic information for conjugation or mating between bacteria.
Some plasmids cause diseases in plants and animals; others confer to their host cells, resistance to antibiotics, toxic compounds and UV radiation. They carry genes for traveller’s diarrhoea, staphylococcal impetigo, for fermentation of milk into cheese by lactic acid bacteria, for production of restriction and modification enzymes and for some metabolic functions of pseudomonads and soil bacteria.
One of the most important features of plasmids is that they replicate independently of the bacterial genome and segregate to the progeny when a bacterial cell divides. In this way they can be maintained indefinitely in a bacterial lineage. Most plasmids are circular DNA molecules, but some are linear. The number of copies of a plasmid in a cell is variable, depending on the mechanism by which replication is regulated.
As many as 50 copies of a plasmid could be present in a host bacterial cell (high copy number), or just 1 or 2 plasmids in a cell (low copy number). Plasmids vary in size from a few kilo-bases to several hundred kilo-bases per cell.
An E. coli cell could have small-sized plasmids up to 10 kb long, or large plasmids greater than 40 kb in length. In general, a typical E. coli cell contains 3 different small plasmids, each present in multiple copies per cell, and one large plasmid present in a single copy per cell.
Plasmids can be visualised in electron microscopic images, or by performing gel electrophoresis on DNA samples. Plasmids can also be detected through a phenotypic characteristic they confer on the host cell, such as antibiotic resistance. For example, if a plasmid is carrying a gene for resistance to tetracycline (tet-r gene), the host bacterial cell will be able to form colonies on a medium containing tetracycline.
Although plasmids replicate independently of host cell DNA, they depend on the DNA replication enzyme of the host cell for their reproduction. But the initiation of replication is controlled by plasmid genes. In bacterial cells having high copy number plasmids, replication of plasmids is initiated multiple times during replication of host genome.
But in low copy number plasmids, replication is initiated only once per round of replication of host genome. During division of host bacterial cell which takes place by fission, the segregation of plasmids to both daughter cells is promoted by sequences contained in the plasmid.
Preparation of Plasmids:
Restriction enzymes are valuable genetic tools in that they can recognise a specific short sequence of bases and cleave DNA at a particular site within that sequence; wherever that sequence occurs in the DNA molecule it will be cut. In a pure sample of plasmid DNA therefore, a particular endonuclease will cut each DNA molecule at precisely the same site so that a large number of identical molecular fragments will be obtained.
These fragments can be separated from each other on the basis of their length by gel electrophoresis. For this the mixture of fragments is placed in a rectangular slot at one end of gel. When an electric current is passed, the DNA molecules move through the gel toward the positive electrode.
Their rate of movement is inversely proportional to the logarithm of their molecular length; consequently, fragments of identical length form a narrow horizontal band. The positions of the bands are determined by first staining the gel with a fluorescent dye (ethidium bromide which binds to DNA) and photographing it under UV radiation.
The Major Groups of Plasmids:
i. R Factors:
In the 1960s Watanabe in Japan discovered that patients with bacterial dysentery did not respond to treatment with certain antibiotics. The bacterium causing this dysentery Shigella dysenteriae was found to contain genes that made it resistant to several antibiotics.
Moreover, the resistance genes were transferred to other intestinal bacteria in the same way as the F factor. They were called ‘R’ or resistance factors. The R factors were later found to be present in Staphylococcus and some enteric bacteria. Roth and Novick have found that the Staphylococcus plasmids carried genes for resistance to drugs like penicillin, chloramphenicol, tetracycline and to toxic metallic compounds of mercury and cadmium.
The R factors consist of two parts, the basic resistance transfer factor (RTF), and a variable genetic determinant for antibiotic resistance (r determinant), which contains the genes for drug resistance (R genes). The genetic determinants (r) cannot be transferred unless they fuse with the RTF.
ii. Col Factors:
Similar to the R factors are the bacteriocins which are toxic proteins produced by certain bacteria that can kill closely related strains of bacteria. Bacteriocins which are produced by E. coli or related enteric bacteria are known as colicins. The genes for production of colicins are present in plasmids called Col (Colicinogenic factors).
Depending upon the colicins they produce, Col factors have been designated Col B, Col E1, Col E2, Col I and Col V. Two of the Col factors produce sex pili and promote plasmid transfer. Colicins act by absorbing to specific receptors on the surfaces of sensitive bacteria causing modification or loss of the receptor sites.
iii. The Degradative or Metabolic Plasmids:
Some species of Pseudomonas inhabiting the soil have a unique characteristic. They are capable of breaking down organic compounds like hexane, phenol, xylene and camphor by special metabolic pathways.
It was found out by I. M. Chakrabarty and his colleagues that the enzymes required for each metabolic pathway are under the control of plasmid genes. These plasmids may be transferred by conjugation, transduction or together with the F factor, and the recipient cell acquires the ability to control an entire metabolic pathway.
In the last few years, plasmids have been more extensively studied and manipulated with the idea of introducing non-bacterial genes into bacteria by the techniques of molecular cloning. This technique has revolutionised genetic analysis and given birth to the much publicized and equally criticised science of genetic engineering.