In this article we will discuss:- 1. Definition of Plasmids 2. Physical Nature and Copy Number of Plasmids 3. Properties 4. Incompatibility 5. Types 6. Replication 7. Plasmid Curing 8. Use of Plasmids as Coning Vectors.
Definition of Plasmids:
In addition to bacterial chromosome (nucleoid), bacterial cells normally contain genetic elements in their cytoplasm. These genetic elements exist and replicate separately from the chromosome and are called plasmids. The very existence of plasmids in bacterial cytoplasm was revealed by Lederberg in 1952 while working on conjugation process in bacteria.
Lederberg coined the term ‘plasmid’ to refer to the transmissible genetic elements that were transferred from one bacterial cell to another and determined the maleness in bacteria.
Literally, thousands of plasmids are now known; over 300 different naturally occurring plasmids have been isolated from strains of Escherichia coli alone. Besides naturally occurring plasmids, many artificially modified plasmids have been developed and used as vectors in the process of gene cloning (genetic engineering).
Physical Nature and Copy Number of Plasmids:
The physical nature of plasmids is quite simple. They are small double-stranded DNA molecules. Majority of the plasmids are circular, but many linear plasmids are also known.
Naturally occurring plasmids vary in size from approximately 1 kilobase to more than 1 megabase, and a typical plasmid DNA is considered to be less than 5% the size of the bacterial chromosome. Most of the plasmid DNA isolated from bacterial cells exist in the supercoil configuration, which is the most compact form for DNA to exist within the cell.
The copy number refers to the fact that different plasmids occur in cells in different numbers. Some plasmids are present in the cell in only 1-3 copies, whereas others may be present in over 100 copies. Copy number is controlled by genes on the plasmid and by interactions between the host and the plasmid.
Properties of Plasmids:
(i) They are specific to one or a few particular bacteria.
(ii) They replicate independently of the bacterial chromosome.
(iii) They code for their own transfer.
(iv) They act as episomes and reversibly integrate into bacterial chromosome.
(v) They may pick-up and transfer certain genes of bacterial chromosome,
(vi) They may affect certain characteristics of the bacterial cell,
(vii) Plasmids differ from viruses in following two ways.
(viii) They do not cause damage to cells and generally are beneficial.
(ix) They do not have extracellular forms and exist inside cells simply as free and typically circular DNA.
Incompatibility of Plasmids:
In some cases, a single bacterial cell contains several different types of plasmids. Borrelia burgdorferi that causes Lyme disease, for convenience, possesses 17 different circular and linear plasmids.
In a condition when a plasmid is transferred to a new bacterial cell that already possesses another plasmid, it is commonly observed that the second (transferred) plasmid is not accommodated and is lost during subsequent replication.
This condition is called plasmid incompatibility and the two plasmids are said to be incompatible. A number of incompatibility groups of plasmids have been recognised in bacteria. The plasmids of one incompatibility group exclude each other from replicating in the cell but generally coexist with plasmids from other groups.
Plasmids of an incompatibility group share a common mechanism of regulating their replication and are thus related’ to one another. Therefore, although a bacterial cell may possess various types of plasmids, each is genetically distinct.
Types of Plasmids:
Various types of plasmids naturally occur in bacterial cells, and the most favoured classification of such plasmids is based on their main functions encoded by their own genes.
Following are the main type of plasmids recognised on the basis of above mentioned characteristic feature:
1. F-plasmid (or F-factor):
F-plasmid or F-factor (“F” stands for fertility) is the very well characterised plasmid. It plays a major role in conjugation in bacteria E. coli and was the first to be described. It is this plasmid that confers ‘maleness’ on the bacterial cells; the term ‘sex-factor’ is also used to refer to F-plasmid because of its this property. F-plasmid is a circular dsDNA molecule of 99,159 base pairs.
The genetic map of the F-plasmid is shown in Fig. 5.31. One region of the plasmid contains genes involved in regulation of the DNA replication (rep genes), the other region contains transposable elements (IS3, Tn 1000, IS3 and IS2 genes) involved in its ability to function as an episome, and the third large region, the tra region, consists of tra genes and possesses ability to promote transfer of plasmids during conjugation. Example F-plasmid of E. coli.
R-plasmids are the most widespread and well-studied group of plasmids conferring resistance (hence called resistant plasmids) to antibiotics and various other growth inhibitors.
R- plasmids typically have genes that code for enzymes able to destroy and modify antibiotics. They are not usually integrated into the host chromosome. Some R-plasmids possess only a single resistant gene whereas others can have as many as eight.
Plasmid R 100, for example, is a 94.3 kilobase-pair plasmid (Fig. 5.32) that carries resistant genes for sulfonamides, streptomycin and spectinomycin, chloramphenicol, tetracyclin etc. It also carries genes conferring resistance to mercury.
Many R-plasmids are conjugative and possess drug- resistant genes as transposable elements, they play an important role in medical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections.
Virulence-plasmids confer pathogenesity on the host bacterium. They make the bacterium more pathogenic as the bacterium is better able to resist host defence or to produce toxins.
For example, Ti-plasmids of Agrobacterium tumefaciens induce crown gall disease of angiospermic plants; entertoxigenic strains of E. coli cause traveller’s diarrhoea because of a plasmid that codes for an enterotoxin which induces extensive secretion of water and salts into the bowel.
Col-plasmids carry genes that confer ability to the host bacterium to kill other bacteria by secreting bacteriocins, a type of proteins. Bacteriocins often kill cells by creating channels in the plasma membrane thus increasing its permeability. They also may degrade DNA or RNA or attack peptidoglycan and weaken the cell-wall.
Bacteriocins act only against closely related strains. Col E1 plasmid of E. coli code for the synthesis of bacterioein called colicins which kill other susceptible strains of E. coli. Col plasmids of some E.coli code for the synthesis of bacteriocin, namely cloacins that kill Enterobacter species.
Lactic acid bacteria produce bacteriocin NisinA which strongly inhibits the growth of a wide variety of gram-positive bacteria and is used as a preservative in the food industry.
5. Metabolic plasmids:
Metabolic plasmids (also called degradative plasmids) possess genes to code enzymes that degrade unusual substances such as toluene (aromatic compounds), pesticides (2, 4-dichloro- phenoxyacetic acid), and sugars (lactose).
TOL (= pWWO) plasmid of Pseudomonas putida is an example. However, some metabolic plasmids occurring in certain strains of Rhizobium induce nodule formation in legumes and carry out fixation of atmospheric nitrogen.
A brief summary of important types of bacterial plasmids, their hosts, and properties is given in Table 5.2.
Replication of Plasmids:
Plasmids replicate autonomously because they have their own replication origins. The enzymes involved in plasmid replication are normal cell enzymes particularly in case of small plasmids. But, some large plasmids carry genes that code for enzymes that are specific for plasmid replication.
Plasmids possess relatively few genes, generally less than 30, and the genes are concerned primarily with control of the replication initiation process and with apportionment of the replicated plasmids between daughter cells; the genetic information carried in plasmid genes is not essential to the host because the bacteria that lack them usually function normally.
Since the plasmid DNA is of small size, the whole process of its replication takes place very quickly, perhaps in 1/10 or less of the total time of cell division cycle.
Most plasmids in gram-negative bacteria replicate in a manner similar to the replication of bacterial chromosome involving initiation at the replication origin site and bidirectional replication around the DNA circle giving a theta (Ө) intermediate.
However, some plasmids of gram-negative bacteria replicate by unidirectional method. Most plasmids of gram-positive bacteria replicate by a rolling circle mechanism similar to that used by phage φx174. Most linear plasmids replicate by means of a mechanism that involves a protein bound to the 5′-end of each DNA strand that is used in priming DNA synthesis.
Plasmids can be eliminated from bacterial cells, and this process is called curing. Curing may take place spontaneously or it may be induced by various treatments, which inhibit plasmid replication but do not affect bacterial chromosome replication and cell reproduction. The inhibited plasmids are slowly diluted out of the growing bacterial population.
Some commonly used curing treatment agents are acridine dyes, ultraviolet (UV) and ionizing radiation, thymine starvation and growth above optimal temperatures. These curing treatment agents interfere with plasmid replication than with bacterial chromosome replication.
Use of Plasmids as Cloning Vectors:
Significance of plasmids dramatically increased with the advent of recombinant DNA technology as they became the first cloning vectors, and even today they are the most widely used cloning vectors especially in gene cloning in bacteria.
They enjoy this status because they have very useful properties as cloning vectors that include:
(i) Small size, which makes the plasmid easy to isolate and manipulate;
(ii) Independent origin of replication, which allows plasmid replication in the cell to proceed independently from direct chromosomal control;
(iii) Multiple copy number, which makes them to be present in the cell in several copies so that amplification of the plasmid DNA becomes easy; and
(iv) Presence of selectable markers such as antibiotic resistance genes, which make detection and selection of plasmid-containing clones easier.
The plasmid vector is isolated from the bacterial cell and at one site by restriction enzyme. The cleavage converts the circular plasmid DNA into a linear DNA molecule.
Now the two open ends of linear plasmid are joined to the ends of the foreign DNA to be inserted with the help of enzyme DNA ligase. This regenerates a circular hybrid or chimeric plasmid, which is transferred to a bacterium wherein it replicates and perpetuates indefinitely.
One of the most widely used plasmids in gene cloning in bacteria is pBR322, which has both resistance genes for ampicillin and tetracycline and many restriction sites. When a foreign DNA is inserted into the ampicillin resistance gene of pBR322, the plasmid is no longer able to confer resistance to ampicillin.