Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF

Abstract

Background: Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies.

Results: The 2,739,625 base pair chromosome of OG1RF was found to contain approximately 232 kilobases unique to this strain compared to V583, the only publicly available sequenced strain. Almost no mobile genetic elements were found in OG1RF. The 64 areas of divergence were classified into three categories. First, OG1RF carries 39 unique regions, including 2 CRISPR loci and a new WxL locus. Second, we found nine replacements where a sequence specific to V583 was substituted by a sequence specific to OG1RF. For example, the iol operon of OG1RF replaces a possible prophage and the vanB transposon in V583. Finally, we found 16 regions that were present in V583 but missing from OG1RF, including the proposed pathogenicity island, several probable prophages, and the cpsCDEFGHIJK capsular polysaccharide operon. OG1RF was more rapidly but less frequently lethal than V583 in the mouse peritonitis model and considerably outcompeted V583 in a murine model of urinary tract infections.

Conclusion: E. faecalis OG1RF carries a number of unique loci compared to V583, but the almost complete lack of mobile genetic elements demonstrates that this is not a defining feature of the species. Additionally, OG1RF's effects in experimental models suggest that mediators of virulence may be diverse between different E. faecalis strains and that virulence is not dependent on the presence of mobile genetic elements.

Figure 1

Map of the OG1RF chromosome. The following features are displayed (from the inside out): restriction maps using SfiI, AscI, and NotI (black) from Murray et al. [10] overlaid with the digestion profile predicted from the sequence (red); G+C content in percentage in green; the total OG1RF-unique genes are shown in purple with those in (+) orientation labeled in blue, and those in (-) orientation labeled in red.

Figure 2

Dot plot of OG1RF versus V583 generated by BLASTN. The dot plot was generated by aligning the OR1RF genome against the V583 genome using BLASTN (e-value 1e-10). The alignment pairs were plotted according to their genome coordinates. The visible areas of divergences are labeled using 'Δ ' to indicate a sequence absent in OG1RF and '∇ ' to indicate a sequence unique to OG1RF (locus tag OG1_xxxx) when compared with V583 (locus tag EFxxxx). Phages 1, 3, 4, 5, 6, 7 of V583 (φ1 to 7; see [31]) and the PAI locations, all of which are missing from OG1RF, are also indicated.

Figure 3

The two CRISPR loci of OG1RF. (a) The CRISPR1 locus. The CRISPR1 element is represented with a hatched box while the CRISPR1 associated genes are represented in orange; the white arrows indicate ORFs present in both OG1RF and V583. The black diamonds represent the 37 bp repeat sequences, while the open boxes with a number indicate the 29 bp unique sequences. (b) The CRISPR2 locus containing only a CRISPR element. (c) CRISPR consensus and unique sequences. The underlined bases indicate mismatches at these locations. The sequences numbered 1 to 14 represent the unique sequences located in the CRISPR1 and CRISPR2 elements.

Figure 4

Two-component systems unique to OG1RF. (a) Two-component system with homology to the Van_G system. (b) Two-component system with homology to the comCD genes of S. pneumoniae. The two-component system (OG1RF_0198 and OG1RF_0199) is indicated in light blue; the two ORFs encoding potential transporter proteins (OG1RF_0200 and OG1RF_0201) are represented in pink. In green are indicated two small ORFs encoding polypeptides of less than 51 amino acids. The white arrows indicate ORFs also present in V583.

Figure 5

The iol operon. The iol genes are labeled based on the homology/conserved motif of their encoded proteins with known enzymes necessary for myo-inositol degradation. For all strains, the described or probable regulator is represented in blue. E. faecalis OG1RF: the iol operon is represented in yellow, OG1RF_0166 (green arrow) located downstream of the iol operon encodes a probable PTS IIC component, while the white arrows indicate ORFs also present in V583. For B. subtilis 168, C. perfringens strain 13, and L. casei BL23, the iol genes are represented in green, orange and purple, respectively. C. perfringens iol mRNA transcript includes five other genes encoding proteins whose functions do not appear to be related to myo-inositol degradation; these genes are represented in gray.

Figure 6

The OG1RF competence operon and its similarity with the competence operon of S. mutans. The ORFs essential for natural competence in S. mutans are shown in green as well as their homologues in OG1RF and V583. The ORF corresponding to the homologue of ComYD was not described in V583 [4], due to the presence of a probable prophage (EF1986-EF2043). The premature stop codon in EF1984 in V583 is indicated with an asterisk. ackA/EF1983 is represented in orange. The proteins encoded by the ORFs represented in white do not share any features of the known competence proteins or homology between S. mutans and E. faecalis; in S. mutans, ackA and ytxK are co-transcribed with the comY genes [47].

Figure 7

Comparison of OG1RF and V583 in a mouse urinary tract infection model. (a) Mixed infection by wild-type E. faecalis strains OG1RF and V583 in the kidneys and urinary bladders of mice (n = 21; competition assay). Data are expressed as the log₁₀(CFU)/gm for OG1RF or V583; the log₁₀(CFU)/gm for both kidneys were combined and averaged from two independent experiments. Black solid diamonds and triangles represent E. faecalis strains OG1RF and V583, respectively, for kidney homogenates, and empty diamonds and triangles represent OG1RF and V583, respectively, for urinary bladder homogenates. Horizontal bars represent geometric means. Log₁₀(CFU) were compared for statistical significance by the paired t-test. The minimum detection limit in these experiments was 10¹ and 10² CFU/gm of kidney and urinary bladder homogenates, respectively. (b) Mono-infection using E. faecalis strains OG1RF or V583 in the kidneys of mice (10³ CFU per mice, n = 9). Data are expressed as log₁₀(CFU)/gm for OG1RF recovered from kidney homogenates 48 h after infection; the log₁₀(CFU)/gm for a kidney pair were combined and averaged. Black and white triangles represent OG1RF and V583, respectively. Horizontal bars represent geometric means. The CFU of V583 recovered from kidneys was significantly reduced compared to the CFU of OG1RF, as determined by the unpaired t-test.