- Bor Shin Chee
Plasmids are extrachromosomal DNA elements which retain important proportion of overall genome and gene that necessary for the growth of the cell. Plasmids usually are circular but some are linear, they have supercoiling DNA structure which undergoes the process of under-winding of DNA strands in order to compact DNA molecules. Moreover, plasmids can also be classified into two groups: low copy number plasmids and high copy number plasmids. For example, F plasmid aka fertility plasmid is a low copy number plasmid, it presents as a single copy in a cell, active partitioning rather than randomly distribution occurred with the purpose of stabilizing the plasmid in cell division. After the cell division of F plasmid, it ended up to have two copies for each cycle. Col E1 is a high copy number plasmid, in the cell division, random distribution between two daughter cells which results at least one copy of the plasmid contain in each cell. It is said to be correct partitioning at the cell division is very important as incorrect partitioning leads to plasmid segregation.
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When comparing plasmid with chromosomal DNA, plasmid is smaller than chromosomal DNA, even the largest plasmid is also smaller than chromosomal DNA. Thus, when running an agarose gel, the plasmid will run faster and be found ahead of the chromosomal. Although there is one case that plasmid will be larger than a Chromosomal DNA is the chromosomal DNA is broken into short linear fragments while the plasmid still stayed as an intact circular molecule. Plasmids usually are small molecules which have a few kilobase in length, but some of the plasmids exclusively genus Pseudomonas have several hundred kilobase long. ColE1 which is the prototype of E.coli, a small plasmid with the size of 6.4 kb is commonly used as cloning vector to partially control the plasmid replication. As mentioned above, ColE1 is a high copy number plasmid, so the cell contains this plasmid is usually small. High plasmid copy number produces multiple copies within the cell that increases the amount of cellular energy required for replication and expression of plasmids, causing metabolic load and impair normal cellular functioning. The consequences of the increase of metabolic load is the loss of recombinant plasmid or recombinant gene from a plasmid. ColE1 is non conjugative, it is not able to transfer itself from one cell to another because the plasmid is small.
F plasmid is a large plasmid with approximately 100 kb long, so conjugation occurred, 30 kb out of 100kb is transferred from one to another. This plasmid has a nine repeats of a 17bp sequences called interons which RepA protein binds to it to promote application.
Some bacterial characteristics can be determined by plasmids such as antibiotic resistance, colicins and bacteriocins, virulence determinants, plasmids in plant-associated bacteria and metabolic activities. Antibiotics resistance is the most relevant way to characterize bacterial. A bacteria becomes resistant to antibiotics by taken up a plasmid which encodes with an antibiotics gene from one bacteria to another. On the other ways, bacteria which has the ability to resist to some drugs such as nalidoc acid and rifampcin is because of the mutation of the gene that codes for the target protein. Some bacteria are not only resistance to one antibiotics but resistance to several antibiotics at the same time either possess several independent plasmids in a cell or possess single plasmid with many resistance determinants on it. For example, when bacteria which have plasmids encoded for kanr gene aka kanamycin resistance gene are pipetted into a medium contained kanamycin antibiotics, as the bacteria resist to kanamycin, after a certain incubation period, they survived and colonies are observed in the medium.
Plasmids have the ability to produce a protein such as colicins and bacteriocins which has an antimicrobial action against closely related organism. ColE1 carried a gene called colicinogenic for the synthesis of colicins E1 and another genes to confer immunity to the action of colicins in order to protect the cell against lethal effects of its own product. Colicins which released into the environment are toxic to some strains of Escherichia coli and it can reduce competition from other E.coli strains.
Plasmids can carry types of toxic genes that are essential for virulence. Plasmid of E.coli has a gene called labile toxin gene or LT gene which produces labile toxin that closely related to the cholera toxin. Thus, plasmids contained LT genes have the ability to resemble cholera and caused a disease. Another example is the 70 kb virulence plasmid of Yersinia species especially Yersinia pestis which is the causative microorganism of plague. These plasmids synthesis proteins and attack the cells immunity response in order to disrupt cells communication or kill the cells in the human body. Besides that, Bacillus anthracis, the microorganism which causes anthrax has two significant large plasmids known as pOX1 and pOX2. The pOX1 plasmid encodes the genes that synthesis anthrax toxin and pOX2 encodes the genes that synthesis capsule to protect the bacterium from the immune response. The absence of pOX1 and remaining the pOX2 in the cells result to loss of virulence; the presence of pOX1 and lack of pOX2 such as Sterne strain can become medical use as a live attenuated vaccine for cattle. Bacillus species such as Bacillus cereus and Bacillus thuringiensis do not have anthrax strains, thus these bacteria which have pOX1 and pOX2 virulence plasmids can only cause anthrax-like illness such as mild, food borne illness.
Another characteristic of bacteria is the pathogenicity. Pathogenicity is associated with the transfer of a specific plasmid DNA from Ti plasmids of Agrobacterium tumefaciens into the plant cells, these particular Ti plasmid induces tumour-like growth called crown gall in some plants. The species of genus Rhizobium have the relationship of symbiotic with the plants, they fix nitrogen into a useable source of reduced nitrogen for the plant by forming nodules on the roots of leguminous plants.
Part of the metabolic activities are manipulated by plasmids, for example, a plasmid which has the genes of fermentation of lactose introduced into a non-fermenting strain, will have the ability to utilize lactose. The Salmonella genus lack of lactose fermenting plasmid causes the delay of infection, due to the needs of lactose fermenting plasmid for the causative agents of Salmonella. Plasmids not only have the genes for the fermentation of lactose but also have the genes for the fermentation of other sugars such as sucrose, hydrolysis of urea, production of hydrogen sulphide, the ability to degrade potentially toxic chemicals and other metabolic activities. The pWWO plasmid from Pseudomonas putida is a plasmid which mediates metabolic activity for degradation of toxic chemical. In the upper pathway, pWWO has a series of enzymes that convert cyclic hydrocarbons such as toluene and xylene to benzoate and the degradation of benzoate to catechol, while in the lower pathway, catechol intermediate is breakdown into metabolic intermediate for energy production and biosynthesis via ring cleavage of catechol. The enzymes of the upper pathway are specialized, different plasmids have different specificities, the other plasmid mediated degradation include naphthalene, dichlorophenoxyacetic acid, camphor and chlorinated aromatic compounds such as 3-chlorobenzoate. Bioremediation is a waste treatment management technique which uses naturally occurring microorganisms to clean up the polluted sites. The organisms that have cyclic hydrocarbons as their sole carbon source are used to degrade organic contaminants in soil, groundwater, sludge and solids.
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Plasmids are often get loss during the mutation at a high rate from a population, the occurring plasmid is generally stable and easier to find for isolating a specify strain while the artificially constructed plasmid is usually unstable and harder to find. The unstable plasmids are an expensive and annoying problem in the industrial use as they can be easily lost within the manufacturing process. The instability of the plasmids are obviously related to plasmid integrity, differential growth rates and partitioning at cell division.
Plasmid integrity refers to retain the structure of the plasmid, the occurring plasmids have recombination hotspots resulting the plasmid have the possibilities to become unstable and losing genes. The recombination between the repeated sequences such as transposons or insertion sequences can causes deletion or inversion of genes. In order to identify the deletion of gene in a plasmid, agarose gel electrophoresis can be used to determine the size of the plasmid or pipetting the plasmids into specific antibiotics contained medium to test the plasmids resistance to which antibiotics. The identification of plasmids that undergoes inversion are more challenging than the deletion in the plasmid because the plasmids do not change their sizes and perhaps remain the original phenotypic characteristics, so there is one way to identify the presence of inversion, it is by sequencing the plasmid.
There is a difference in the growth rate between the cells that make the plasmid unstable or resulted elimination of the plasmid. With the high growth rate, the metabolic load produce from replication of plasmids and expression of genes will increase and the problem of instability become more critical.
Correct partitioning is significant because failure of partitioning will caused the increase of the proportion of plasmid free cells or the elimination of the cells. High copy number plasmids choose to have random partitioning while low copy number plasmids choose to have active partitioning, the plasmid copy number are controlled by counting the number of replication region rather than the number of molecules. A high copy number plasmid tends to have multimeric structures or recombination between monomer for the replication process because multimers posse more than one origin of replication. It is said to be the plasmids as multimers are not too effective to maintain each daughter cell can receive a copy of the plasmid than as monomers, dimers or trimers during cell division. In order to stabilize the plasmid, the site-specific recombination was used, for example, ColE1 has a site of cer which is a target for the action of the host proteins called XerC and XerD. If a plasmid dimer which has two copies of the cer sequence needs to resolve into two monomers, the XerC and XerD will causes site specific recombination between them, crosses them and breaks both molecules, rejoins them. Resolution of dimeric plasmids or multimeric plasmids into monomeric plasmid stabilize the plasmids in cell division.
The post segregation killing process is the process of killing any cells that have lost the plasmid and this killing requires two plasmid genes, one specifying a stable, long lived toxic agents and another one specifying an unstable antidotes to prevent the lethal effect action of the toxin. The toxin usually is a protein while the antidote is an antisense RNA that inhibits the translation of toxin protein. For example, the F plasmid has an operon which encodes two genes, there are CcdA which acts as an antidote and CcdB which acts a toxin. The CcdB protein obstruct the DNA replication via the interference on DNA gyrase mediated supercoiling while the CcdA neutralizes the toxicity of CcB, once all the unstable CcdA decays or destroys , the more stable CcdB are no longer neutralized and finally the cell is killed. For another example, the plasmid R1 consists of two genes: hok and sok. The toxic agent is hok which interferes the cell membrane while the sok, an antisense RNA molecule for suppression of killing by binding to the same region of hok but transcribed in the opposite direction. Once the antisense sok RNA binds to hok RNA, the translation prevented, thus if the R1 plasmid is lost, the unstable sok RNA molecule decays rapidly, in the plasmid free segregant, the stable hok RNA molecule is translated and leads to the death of the cell. There is advantage and disadvantage of the post segregational killing, the disadvantage is the death of the cells and the advantage is if the cells are under starvation condition, the death of some of the cells can help to promote the survival of the remaining cells.
Jeremy W. D. & Simon F. P. (2010) Molecular Genetics of Bacteria. 5th edn. , America: John Wiley & Sons, Ltd.
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