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17.9-kb plasmid

As well as the large number of insertion elements, the E.faecalis V583 genome contains seven regions that are derived from probable phage integration. Homol-ogy searches of all seven phage regions show their best matches to phages from other lactic acid bacteria. Interestingly, the fourth phage region contains the hemH gene (EF1989, a putative ferrochelatase), a cold shock protein, CspC (EF1991), and homologues of both PblB (EF2001) and PblA (EF2003). A second protein with sequence identity to PblB is also present in the sixth phage region

(EF2811). In Streptococcus mitis, PblA and PblB resemble phage capsid and tail fiber proteins and are present on an inducible phage, SM1. In S. mitis these proteins have been linked to platelet adherence, as disruption of either of these genes resulted in reduced platelet binding [17]; however, their role in E.faecalis virulence is unknown. Phage integration events in Streptococcus pyogenes are responsible for the major gaps in the alignments of different M serotypes [18], and the presence of seven integrated prophages in the E.faecalis V583 genome suggests an important role for phage integration in genome plasticity.

The E.faecalis V583 chromosome also contains three regions that are possibly integrated plasmids. Present in two of these regions are genes encoding aggregation substance (EF0485, EF0149). One of the integrated plasmid regions is located within the pathogenicity island (EF0485-EF0506) and is very similar to pTEFl, but the aggregation substance is more closely related to that from pTEF2. The second region (EF0131-EF0166), has genes of plasmid, phage, and conjugative trans-poson origin, and is flanked by a phage integrase (EF0166). The third plasmid region (EF2512-EF2545) is very similar to this, has no aggregation substance gene, but does contain cell-wall surface anchor protein (EF2525) and a putative sortase (EF2524). The presence of three integrated plasmids points to a potentially important role for plasmids in the genome evolution of E.faecalis.

In Staphylococcus epidermidis, it has been noted that that IS256 occurs preferentially in nosocomial isolates [19], and this IS element has also been linked to phe-notypic variation in biofilm formation [20, 21]. It could be interesting to assess the frequency of IS256 in E.faecalis commensal strains relative to clinical isolates, to see whether this trend is also seen in enterococci. It has been speculated that the presence of multiple copies of IS256 could play a role in genomic flexibility and development of virulence, as inactivation of genes by IS elements allows the development of novel phenotypes [19]. Insertion element movement and integration allows rapid evolution of a bacterial genome. Another example of this is the genome comparisons of Yersinia pestis and Yersinia pseudotuberculosis, which illustrated that differences in virulence are linked to insertion sequence integration and homologous recombination [22]. Inactivation of certain genes and pathways in Y. pestis appear to have played a substantial role in the development of virulence in this organism [22].

Conjugative plasmids in E.faecalis have long been studied as one of the ways in which E. faecalis disseminates antimicrobial resistance genes and other genetic information rapidly. E.faecalis V583 contains three plasmids, pTEF1, pTEF2 (similar to the pheromone response plasmids pAD1 [23] and pAM373 [24]), and pTEF3, which belongs to the pAMyb1 family of broad host range plasmids. PTEF2 shares regions of similarity with pCF10 [25], with identical prgA, prgB, and prgC genes (EFB0010-EFB0012), but lacks prgQ and has a novel pheromone inhibitor (EFB0005.1). Only one insertion element, IS256 (EFB0052), is present in this plasmid. The smallest plasmid, pTEF3, contains a prgZ-like pheromone receptor, similar to pTEF2. This prgZ homologue (EFC0001) neighbors several insertion sequences (EFC0002, EFC0004, EFC0007).

pTEFl shares extensive sequence homology with the well-characterized enterococcal pheromone response plasmid pAD1 [23]. pTEFl has both an identical pher-omone inhibitor (iADl) and aggregation substance (Asal); however, there is a 31-kbp inversion falling between the sex pheromone inhibitor and traE1. This plasmid contains a total of eight insertion sequences, five of which are IS1216. The insertion sequences are clustered in two regions at either end of the 31-kbp inversion, one of which (EFA0006-EFA0016) contains an erythromycin resistance gene (EFA0007), a putative multidrug resistance protein (EFA0010), and a putative drug resistance transporter (EFA0014). The second area (EFA0056-EFA0063) contains a 6-aminoglycoside N-acetyltransferase (EFA0060) conferring high-level gen-tamicin resistance.

In addition to the antimicrobial resistance genes, encoded on the V583 chromosome are four putative drug resistance transporters of the EmrB/QacA family (EF0420, EF0785, EF1370, EF1814), three major facilitator type drug resistance transporters (EF1078, EF1943, EF2068), three putative streptomycin resistance genes (EF1076, EF2300, EF2861), and genes predicted to encode daunorubicin resistance (EF1032), tunicamycin resistance (EF1055), and tellurite resistance (EF2698). Vancomycin resistance in E.faecalis V583 is due to the presence of the vanB operon [26] (EF2293-EF2299); however, this is not present on the conjuga-tive element Tn1549 [27]. Instead, the operon is located on a previously unknown mobile element sharing some similarities with Tn1549, but also containing multiple insertions, deletions, and rearrangements. This mobile element has an atypical nucleotide content, suggesting acquisition via horizontal gene transfer.

From the E.faecalis genome sequence, homologues for most of the competence proteins of S. pneumoniae can be found, but several are absent. E. faecalis V583 contains four proteins that have identity to the Bacillus subtilis comG operon (EF2044-EF2046, EF1986), but is missing comG4, comG5, and comG7. Interestingly, a prophage is present between comG3 and comG6, suggesting that these genes may in fact be present in other strains of E. faecalis that do not contain the integrated phage. The genome sequences of other lactic acid bacteria such as L. monocytogenes [28], Lactococcus lactis [29], and S. pyogenes [30], also reveal the presence of homologues for most of the genes involved in competence. Due to the discovery of a homologue of the B. subtilis competence regulatory protein MecA in L. monocytogenes, the ability of L. monocytogenes to take up DNA was tested, but competence was not detected in the various strains screened [31]. A protein with identity to MecA is also present in E.faecalis (EF2677). In L. monocytogenes, it was hypothesized that these competence-related proteins may be involved in other functions, and that perhaps these bacteria have developed similar complex regulatory mechanisms in response to certain signals in their environmental niches [31]. It is interesting to speculate on the possibility of natural transformation in E. faecalis, especially due to the large amount of mobile DNA present in its genome, and to suggest that perhaps under certain environmental conditions (e.g., the acidic pH of the gut, or the alkaline pH of the bile duct) this organism could be capable of up taking foreign DNA.

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