PLASMIDS

PLASMIDS

Komposisi genetik sel bakteri secara keseluruhan mencakup bakteriofaga yang terintegrasi ke dalam kromosom  (profaga/ prophages). Yang tak kalah penting lagi dalam mempengaruhi fenotipe sel adalah unsur DNA ekstrakromosomal yang dikenal dengan nama plasmid. Meskipun dianggap sebagai fenomena yang terpisah dari bakteriofaga (P1) yang todak terintegrasi ke dalam kromosom tetapi dalam keadaan profaga ada bagian molekul DNA terpisah yang pada dasarnya adalah plasmid. Plasmid dan faga/phages memberikan dimensi yang berbeda dan menjadi bagian penting bagi fleksibilitas mikroorganisme menanggapi perubahan lingkungan, apakah perubahan itu lawan bagi mereka (misalnya adanya paparan antibiotik) atau sebaliknya, bahkan menguntungkan bagi sel mereka (tersedianya substrat baru). Karenanya, dimensi yang berbeda ini memiliki karakteristik perifer yang berbeda untuk replikasi dan produksi struktur sel dasar. Peran plasmid memberikan kontribusi karakteristik tambahan, dan sangat penting bagi kemudahan transfer antara strain atau spesies yang berbeda.

5.1 Some bacterial characteristics are determined by Plasmids

5.1.1 Antibiotic resistance

The most widely studied plasmid-borne characteristic is that of drug resistance. Many bacteria can become resistant to antibiotics by acquisition of a plasmid, although plasmid-borne resistance to some drugs such as nalidixic acid and rifampicin does not seem to occur. (In those cases, resistance usually occurs by mutation of the gene that codes for the target protein). The antibiotic resistance genes themselves are many and varied, ranging from plasmid-encoded betalactamases which destroy penicillins to membrane proteins which reduce the intracellular accumulation of tetracycline. The ability of plasmids to be transferred from one bacterium to another, even sometimes between very different species (Chapter 6), has contributed greatly to the widespread dissemination of antibiotic resistance genes. Bacteria can become resistant to a number of separate antibiotics, either by the acquisition of several independent plasmids or through acquiring a single plasmid with many resistance determinants on it. Some examples will be discussed later in this chapter. Transposons (Chapter 7) are thought to have played a major role in the development of drug resistance plasmids, by promoting the movement of the genes responsible between different plasmids or from the chromosome of a naturally resistant organism onto a plasmid. It should be appreciated that other mechanisms of antibiotic resistance also

occur and that such resistance is not always due to plasmids: indeed many of the bacteria that are currently causing problems of hospital cross-infection are either inherently resistant or owe their antibiotic resistance to chromosomal genes.

5.1.2 Colicins and bacteriocins

Another property conferred by some plasmids that has been widely studied is the ability to produce a protein which has an antimicrobial action, usually against only closely-related organisms. One group of such proteins, produced by strains of E. coli, are capable of killing other E. coli strains, and are hence referred to as colicins, and the strains that produce them are colicinogenic. (These terms are more familiar than the general ones, bacteriocin and bacteriocinogenic and will therefore be used in this chapter). The colicin gene is carried on a plasmid (known as a Col plasmid), together with a second gene that confers immunity to the action of the colicin, thus protecting the cell against the lethal effects of its own product.

One particular Col plasmid, ColE1, is of special importance because of the detailed information that is available concerning its replication and control (see later in this chapter) and also because most of the commonly used E. coli cloning vectors are based on ColE1 or a close relative.

5.1.3 Virulence determinants

The previous chapter discussed how bacteriophages can carry genes that code for toxins and that the presence of the phage is necessary for pathogenicity. In some bacterial species toxin genes are carried on plasmids rather than phages. For example, some strains of E. coli are capable of causing a disease that resembles cholera (although milder). These strains produce a toxin known as LT (labile toxin – to distinguish it from a different, heat-stable, toxin known as ST). The LT toxin is closely related to the cholera toxin, but whereas the gene in V. cholerae is

carried by a prophage, the LT gene in E. coli is found on a plasmid. Plasmids can also carry other types of genes that are necessary for (or enhance) virulence. One of the most dramatic examples of this is the 70-kb virulence plasmid of Yersinia species. This plasmid which is found in species of Yersinia (including Yersinia pestis, the causative organism of plague) has been aptly described as a mobile arsenal since it encodes an integrated system which allows these bacteria to inject effector proteins into cells of the immune response to disarm them, to disrupt their communications or even to kill them. Box 5.1 provides examples of virulence factors which are carried by bacteriophages and plasmids in various pathogenic bacteria. This is by no means an

exhaustive list.

5.1.4 Plasmids in plant-associated bacteria

A different type of pathogenicity is seen with the plant pathogen Agrobacterium tumefaciens, which causes a tumour-like growth known as a crown gall in some plants. Again, it is only strains that carry a particular type of plasmid (known as a Ti plasmid, for Tumour Inducing) that are pathogenic; in this case however, pathogenicity is associated with the transfer of a specific part of the plasmid DNA itself into the plant cells. This phenomenon has additional importance because of its application to the genetic manipulation of plant cells (see Chapter 8). Members of the genus Rhizobium also ‘infect’ plants, although in this case the relationship is symbiotic rather than pathogenic. These bacteria form nodules on the roots of leguminous plants. Under these conditions the bacteria are able to fix nitrogen and supply the plant with a usable source of reduced nitrogen, a process of considerable ecological and agricultural importance. The genes necessary for both nodulation and nitrogen fixation are carried by plasmids.

5.1.5 Metabolic activities

Plasmids are capable of expanding the host cell’s range of metabolic activities in a variety of other ways. For example, a plasmid that carries genes for the fermentation of lactose, if introduced into a lactose non-fermenting strain, will convert it to one that is able to utilize lactose. Such plasmids can cause problems in diagnostic laboratories where organisms are often identified on the basis of a limited set of biochemical characteristics. Commonly the potentially pathogenic Salmonella genus is differentiated from the (usually) non-pathogenic E. coli species primarily because of the inability of Salmonella to ferment lactose. In some cases, the detection of serious epidemics of Salmonella infections has been delayed because the causative agent had acquired a lactose-fermenting plasmid. A large number of other genes have also been found on plasmids, including those for fermentation of other sugars such as sucrose, hydrolysis of urea, or production of H2S. Many of these were initially identified because of the confusion they caused in biochemical identification tests.

Biodegradation and bioremediation

Another type of plasmid-mediated metabolic activity is the ability to degrade potentially toxic chemicals. One such plasmid, pWWO, obtained from Pseudomonas putida, encodes a series of enzymes that convert the cyclic hydrocarbons toluene and xylene to benzoate (upper pathway in Figure 5.1) and a second operon responsible for the degradation of benzoate, via ring cleavage of a catechol intermediate, into metabolic intermediates that can be used for energy production and biosynthesis (lower pathway – see Figure 5.1). This organism can therefore grow using toluene as a sole carbon source. The enzymes of the upper pathway are specialized; other plasmids code for upper pathway enzymes with different specificities, enabling the organism to convert other chemicals into benzoate and catechol derivatives which can be degraded by the lower pathway

enzymes. Plasmid-mediated degradation includes naphthalene and camphor, as well as chlorinated aromatic compounds such as 3-chlorobenzoate and the herbicide 2,4-D (dichlorophenoxyacetic acid).

The ability to degrade environmentally damaging chemicals is potentially useful in clearing up polluted sites (bioremediation). There is therefore considerable interest in extending the range of chemicals that can be degraded by microorganisms, both by modification of existing pathways and also by screening bacteria isolated from contaminated sites for novel activities. The usefulness of such strains is also potentiated by plasmids which confer resistance to toxic metal

ions, notably copper and mercury.

5.2 Molecular properties of plasmids

Bacterial plasmids in general exist within the cell as circular DNA molecules with a very compact conformation, due to supercoiling of the DNA. In many cases, they are quite small molecules, just a few kilobases in length, but in some organisms, notably members of the genus Pseudomonas, plasmids up to several hundred kilobases are common. However, it is worth noting that the standard methods for isolating plasmids (see below) are geared to the separation of small covalently closed circular DNA, and the occurrence of large plasmids, or alternative forms such as linear plasmids, may be underestimated. It is convenient to regard plasmids from E. coli as consisting of two types. The first group, of which ColE1 is the prototype, are relatively small (usually less than 10 kb), and are present in multiple copies within the cell. Their replication is not linked to the processes of chromosomal replication and cell division (hence the high copy number), although there are some controls on plasmid replication (as discussed later in this chapter). Replication of these plasmids can continue under certain conditions (such as inhibition of protein synthesis) that prevent chromosome replication, giving rise to a considerable increase in the number of copies of the plasmid per cell. This phenomenon, known as plasmid amplification, is very useful for isolating the plasmid concerned. The second group of plasmids, exemplified by the F plasmid, are larger (typically greater than 30 kb; F itself is about 100 kb) and are present in only one or two copies per cell. This is because their replication is controlled in essentially the same manner as that of the chromosome; hence when a round of chromosome replication is initiated, replication of the plasmid will occur as well. It follows therefore that plasmids of this type cannot be amplified. In general, these large plasmids are able to promote their own transfer by conjugation (they are known as conjugative plasmids: see Chapter 6). 

The existence of these two groups can be rationalized on the basis of their different survival strategy. Members of the first group rely on their high copy number to ensure that, at cell division, if the plasmid molecules partition randomly between the two cells, then each daughter cell is virtually certain to contain at least one copy of the plasmid (Figure 5.2a). For example, with a plasmid that is present in 50 copies per cell, the chance of one daughter cell not receiving any copies of the plasmid is as low as 1 in 1015. However, high copy number imposes a size constraint. Replication of a plasmid imposes a metabolic burden that is related to the size and copy number of the plasmid. The greater the burden, the greater the selective pressure in favour of those cells that do not possess the plasmid. Hence it is logical that high copy number plasmids will also be small. ColE1, for example, is 6.4 kb in size. If there are 30 copies per cell, this represents about 4 per cent of the total DNA of the cell. The F plasmid on the other hand (c. 100 kb), if it were to be present at a similar copy number, would add nearly 70 per cent to the total DNA content which would inevitably make the cell grow much more slowly and any cells that had lost the plasmid would have a marked selective advantage. But the information required to establish conjugation in E. coli is quite extensive (see Chapter 6). With the F plasmid for example about 30 kb (out of 100 kb) consists of genes required for plasmid transfer. It follows therefore that a small plasmid will not be able to carry all the information needed for conjugative transfer. The second type of plasmid has evolved a different strategy (Figure 5.2b). Firstly, linking replication of the plasmid to that of the chromosome ensures that there are at least two copies of the plasmid available when the cell divides.

Secondly, random partitioning will not be sufficient to ensure that each of the daughter cells receives a copy; so the plasmid must be distributed between the progeny in a directed manner, in much the same way as the copies of the chromosome are distributed. The ability to transfer by conjugation provides a back-up mechanism since any plasmid-free cells that arise in the population by failure of the partitioning mechanism will then be able to act as recipients for transfer of the plasmid. It is necessary to be aware that this picture, although useful, is a highly simplified one, and there are many exceptions, even in E. coli. There are numerous examples of small plasmids that have a low copy number, although none of them are conjugative, and some examples of larger plasmids that exist in multiple copies. In addition, in other organisms the picture is less clear; for example in Streptomyces it seems that quite small plasmids are able to promote their own transfer by conjugation.

5.2.1 Plasmid replication and control

In order to understand the reasons for the different behaviour of plasmids as described above, we need to look at the mechanisms of plasmid replication and how it is controlled. This should be compared with the description of chromosome replication in Chapter 1. Many plasmids are replicated as doublestranded circular molecules. The overall picture with such plasmids is basically similar to that of the chromosome, in that replication starts at a fixed point known as oriV (the vegetative origin, to distinguish it from the point at which conjugative transfer is initiated, oriT), and proceeds from this point, either in one direction or in both directions simultaneously until the whole circle is copied. However there are some aspects of replication that differ from that of the chromosome, especially for the multicopy plasmids. Two examples that have been studied intensively are ColE1 and R100. Other plasmids with quite different modes of replication are dealt with later on.

sumber (source): 

Molecular Genetics
of Bacteria
4th Edition

Jeremy W. Dale
Simon F. Park
University of Surrey, UK