Mutations are changes in genomic sequences on DNA, which occur randomly. These mutations occur mainly due to errors during replication. Sometimes these mutations may give a selective advantage to an organism such as resistance to some antibiotics. If this event occurs the mutant then becomes the major in the population.
Genetic exchange in bacteria occurs by three methods, transformation, conjugation and transduction.
Genetic transformation occurs when a cell takes up (takes inside) and expresses a new piece of genetic material-DNA. This new genetic information often provides the organism with a new trait that is identiï¬able after transformation. Genetic transformation literally means change caused by genes and involves the insertion of one or more gene(s) into an organism in order to change the organism's traits.
This concept of genetic exchange was demonstrated by Fred Griffith in 1928, when he discovered that avirulent strains of Streptococcus pneumonia could be restored to virulence strain. His work was later backed by Avery Mcleod and McCarty who demonstrated that the restoration of avirulent strains to virulent was caused by a "transforming principle" known as DNA. Transformation of a bacterial cell can only occur if the bacterium is "competent", i.e. it is it is able to take up exogenous DNA and be transformed by it. DNA uptake into bacterial cells for transformation requires specialized systems for competence which may not be expressed at all times.
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Natural transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Natural transformation can be divided into three basic steps: (i) DNA binding, (ii) DNA uptake (comprising transport through the outer membrane, periplasm, and inner membrane), and (iii) DNA recombination into the chromosome. Neisseria species have been shown to preferentially take up and transform their own DNA by virtue of a non- palindromic Neisseria-speciï¬c DNA uptake sequence (DUS) (Elkins et al., 1991). There are two forms of the DUS, DUS10 (5 â€² -GCCGTCTGAA) and DUS12 (5 â€² -ATG CCGTCTGAA), which are necessary for the most efï¬cient transformation into Neisseria, with the DUS12 sequence showing the greatest efï¬ciency (Smith et al., 1999; Ambur et al., 2007). Neisseria genomes are enriched for the DUS10 and DUS12 sequences, and many reports have demonstrated increased DNA uptake and transformation with DNA fragments containing one or both DUS sequences (Goodman & Scocca, 1988; Ambur et al., 2007; Dufï¬n & Seifert, 2010). It appears that the DUS10 and DUS12 sequences function similarly but that the DUS12. Although the molecular basis of DUS recognition is unknown, the DUS is thought to bind a surface- localized, sequence-speciï¬c, DNA-binding protein, and this interaction is required for efï¬cient DNA uptake into the periplasm.
Researchers have identified numerous factors, which are involved in transformation in Neiserria gonorrheae. The factors involved in transformation are proteins which are involved in transport of DNA across the cell surface into the cytoplasm and proteins involved in DNA recombination. N.gonorrheae use a Type VI pilus for transformation and are constitutively competent for DNA transformation (Sparling, 1966). Double-stranded DNA binds nonspeciï¬cally to the cell surface via an unknown mechanism, although Opa proteins and the minor pilus protein ComP can affect DNA binding to the cell surface and transformation efï¬ciency (1, 2, 25). DNA is secreted into the medium directly by the Type VI pilus (TfP). Since the N gonorrheae cell is highly competent, the secreted DNA is rapidly taken up by the cell system, and incorporated into its genetic system. There are two proteins (TpC and ComL) which are localised on the cell periplasm, and these, according toâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦, may aid in the transportation of DNA through the peptidoglycan layer. These two proteins are part of the TfP system which includes the secretin PilQ, the major pilus subunit PilE, and the ATPase PilT (â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.). As the double stranded DNA is secreted into the cell, one of the strands is degraded by a DNA degrading enzyme, which is bound to a pore on the periplasm. The other strand is secreted into the cytoplasm through the inner membrane protein ComA. Once in the cytoplasm, DNA recombination of the single stranded DNA (ssDNA) into the chromosome occurs. This recombination in mediated by RecA and requires the helicase PriA. RecBCD recombination appears to play a partial role in transformation, since null mutants show intermediate levels of transformation (36).
DNA uptake sequence mediated enhancement of transformation in N.Gonorrhoeae
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The lack of stable clonal lineages indicates that exchange of chromosomal DNA is common between N. gonorrhoeae strains (Smith et al., 1993). This frequent transformation aids in spread of antibiotic resistance determinants. DNA transformation is a multi-step process that includes DNA binding, DNA uptake into the periplasm and cytoplasm, and DNA recombination into the chromosome (reviewed in Hamilton & Dillard, 2006.
Genetic Transformation of NG shows a strand preference
Conjugation is a major mechanism of horizontal genetic transfer in gram-positive cocci and the genes transferred often include antibiotic resistance and virulence determinants (9, 14, 15, 40). A number of enterococcal conjugative plasmids can transfer very efï¬ciently (frequencies as high as 5 3 10 2 1 ) in liquid cultures. This efficiency comes about through peptide signals known as pheromones. The pheromones are released by recipient cells and induce donor cells to find them and exchange genetic material.
Conjugative systems have been studied so extensively in Enterococcus faecalis. Some strains of E. faecalis secrete diffusible peptides that have a pheromone-like action that can stimulate the expression of the transfer (tra) genes of a specific plasmid in a neighbouring cell (dale). The Donor cell has been shown to contain cell surface receptors that the pheromones bind onto. Different types of plasmid code for different receptors and are therefore stimulated by different pheromones (dale) . However the recipient produces a range of pheromones and is therefore capable of mating with cells carrying different plasmids. After the pheromone has bound to the cell-surface receptor it is transported into the cytoplasm, by a specific transport protein, where it interacts with a protein called TraA. This protein is a repressor of the tra genes on the plasmid and the binding of the peptide to it relieves that repression, thus stimulating expression of the tra genes. One result is the formation of aggregation products which cause the formation of a mating aggregate containing donor and recipient cells bound together. A further consequence of expression of the tra genes is stimulation of the events needed for transfer of the plasmid which occurs by a mechanism similar to that described previously. One advantage of this system is that the cells containing the plasmid do not express the genes needed for plasmid transfer unless there is a suitable recipient in the vicinity. Not only does this reduce the metabolic load on the cell but it also means that they are not expressing surface antigens (such as conjugative pili) that could be recognized by the host immune system. (Dale)
In the generally accepted model for pheromone-inducible plasmid transfer, recipient cells excrete the peptide pheromone molecule into the medium, where it can diffuse to a potential donor cell. Plasmid-encoded gene products allow the donor cell to bind the pheromone with high afï¬nity, and the speciï¬c recognition of this signalling molecule serves as a means of communicating the presence of a recipient cell in close proximity. The bound pheromone somehow initiates a response resulting in activation of expression of transfer functions, including the synthesis of aggregation substance (AS), which can promote attachment to recipients via a complementary receptor called enterococcal binding substance (EBS). The close cell-to-cell contact resulting from AS-EBS binding allows for subsequent formation of some sort of mating channel between the two cells that enables transfer of the plasmid from the donor to the recipient. Since the new donor cell created by this event generally shows a pheromone-inducible transfer pattern identical to that of the original donor (18, 19) (even though it has the genes for both production and response to pheromone), the plasmid must encode functions to prevent the donor cell from responding to its own pheromone.
The pathogenicity of enterococci has received increasing attention recently because of the very high prevalence of the organism in nosocomial infections (41, 56) and because the inherent and acquired antibiotic resistance of these organisms has resulted in situations where there is no effective antimicrobial chemotherapeutic agent available to treat some cases of life-threatening enterococcal infections (42). Interestingly, there is substantial evidence indicating that several components of pheromone-inducible conjugation may affect the virulence of the organism.
E faecalis also provides an example of an exception to the general rule that conjugation is plasmid mediated. E faecalis contains a transpososn known as Tn916. This transposon is unique in the sense that it has the ability to transfer from one cell to another by conjugative means. The major feature about the Tn916 is that it is replicated and inherited as part of the chromosome. The Tn 916 transposon, similarly to a plasmid, has an origin of transfer, Ori T and has the genes necessary for conjugal transfer (Dale). The transposon is excised from the chromosome using specialised transposon encoded enzymes (Int & Xis). Once excised, the result is the production of a circular molecule. Transfer of Tn 916 involves ssDNA synthesis initiated at the Ori T and transfer at the displaced strand to the reciepient (Dale)
Pheromone inducible conjugation in e.Faecalis
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The most obvious significance of conjugation is that it enables the transmission of plasmids from one strain to another. Since conjugation is not necessarily confined to members of the same species, this provides a route for genetic information to flow across wide taxonomic boundaries. One practical consequence is that plasmids that are present in the normal gut flora can be transmitted to infecting pathogens, which then become resistant to a range of different antibiotics. It can be assumed therefore that conjugative transposons as well, have played a significant role in the dissemination of genetic material including antibiotic resistance genes.