Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF
- Agathe Bourgogne1, 2,
- Danielle A Garsin3,
- Xiang Qin4,
- Kavindra V Singh1, 2,
- Jouko Sillanpaa1, 2,
- Shailaja Yerrapragada4,
- Yan Ding4,
- Shannon Dugan-Rocha4,
- Christian Buhay4,
- Hua Shen4,
- Guan Chen4,
- Gabrielle Williams4,
- Donna Muzny4,
- Arash Maadani3,
- Kristina A Fox3,
- Jason Gioia4,
- Lei Chen4,
- Yue Shang4,
- Cesar A Arias1, 2,
- Sreedhar R Nallapareddy1, 2,
- Meng Zhao1, 2,
- Vittal P Prakash1, 2,
- Shahreen Chowdhury1, 2,
- Huaiyang Jiang4,
- Richard A Gibbs4, 5,
- Barbara E Murray1, 2, 3Email author,
- Sarah K Highlander4, 6 and
- George M Weinstock4, 5, 6
© Bourgogne et al.; licensee BioMed Central Ltd. 2008
Received: 14 February 2008
Accepted: 8 July 2008
Published: 08 July 2008
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.
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.
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.
Enterococci have emerged over the past few decades as the second to third most common cause of nosocomial infections, including urinary tract and soft tissue infections, bacteremia, and endocarditis [1–3]. They are well equipped to thrive in environments with heavy antibiotic usage due to both their intrinsic resistance to antibiotics and their talent for swapping genetic information, which allows them to gain and share resistance determinants. Entecococcal infections are predominantly caused by E. faecalis and E. faecium. However, many, if not most, strains of these species are harmless commensals, with some enterococci being marketed in Europe to alleviate symptoms of irritable bowel syndrome and recurrent chronic sinusitis or bronchitis (Cylactin® and Fargo688® (E. faecium) and Symbioflor 1 (E. faecalis)). To differentiate the two faces of this organism, genome-wide comparisons are necessary. Although hundreds of microbial genomes have been sequenced, only two E. faecalis genomes have been reported (V583 as a clinical isolate  and Symbioflor 1 as a commensal isolate ), but only the V583 genome has been made publicly available. In this strain, more than one-quarter of the genome is mobile DNA, more than any other sequenced bacterial genome . The occurrence of multiple antibiotic resistance determinants in V583  makes it difficult to manipulate genetically. Moreover, the vancomycin resistance phenotype makes this strain more of a risk to handle in the laboratory. To avoid these issues, most laboratories use strain OG1 or its close derivatives. OG1 is a human isolate subsequently shown to cause dental caries in rats . OG1RF is a rifampicin and fusidic acid resistant derivative of OG1 [8, 9]. By pulsed-field gel electrophoresis, Murray et al.  estimated the size of the OG1RF genome as 2,825 kb and created a restriction map of the chromosome. Multilocus sequence typing (MLST) showed that OG1RF is clonally distinct from V583 (differs in six out of seven alleles of housekeeping genes)  and characterization of regions flanking transposon insertions in OG1RF suggested that approximately 10% of their sequences differed .
OG1 and its derivatives have been successfully used over the past 20 years in various animal models, starting with the demonstration that it can cause caries in germ-free rats , and later to characterize factors important for E. faecalis virulence in a mouse model of peritonitis , a rabbit model of endophthalmitis , a rat model of endocarditis  and in a mouse urinary tract infection model . OG1RF was also shown to be as virulent as V583 in the model host Caenorhabditis elegans . In addition to its virulence, the main reasons for the extensive use of OG1RF as a laboratory strain are that it does not carry plasmids, is readily transformable by electroporation, and is not resistant to commonly used antibiotics, other than rifampicin and fusidic acid. These resistances were serially selected in OG1 to provide strain markers . The lack of resistance to common antibiotics facilitates the selection of plasmids, transposons, and allelic replacement markers introduced into the strain.
Numerous factors important for virulence have been characterized in OG1RF. A recently described example are the Ebp pili, whose subunits are encoded by the ebp operon  and whose genes are regulated by EbpR . A non-piliated mutant produces less biofilm than the parent strain and is attenuated in a rat model of endocarditis  and in a murine urinary tract infection model . Also present is Ace, a member of the MSCRAMM (microbial surface component recognizing adhesive matrix molecules) family. The ace gene, like the ebp locus, is ubiquitous in E. faecalis and it occurs in at least four different forms that vary in the number of repeats of the B domain . Ace mediates conditional (that is, after growth at 46°C or in the presence of serum or collagen) adherence of E. faecalis to collagen type IV and to laminin  and, in unpublished data, influences the ability of OG1RF to cause experimental endocarditis (KV Singh and BE Murray, unpublished observation). Finally, the Fsr system, a major positive and negative transcriptional regulator in OG1RF , affects expression of several virulence factor genes, including gelE, which encodes gelatinase , and contributes to infection in various animal models [15, 24].
The distinct MLST profile and the wide range of phenotypic and genotypic analyses of OG1RF, including many molecular genetic studies and experiments in various animal models, suggested that genomic analysis of this strain would prove insightful and would be useful to future studies. Thus, we analyzed the sequence of E. faecalis OG1RF. This revealed approximately 232 kb encoding 227 open reading frames (ORFs) that are unique to this important strain compared to V583. The unique regions were then characterized further.
Results and discussion
General genome features
General features of OG1RF compared to V583
Size (base pairs)
G+C content (%)
Genes common to both strains
Genes unique to OG1RF
Similar to known proteins
No database match
Total number of ORFs
Frequency of the CRISPR locus among E. faecalis
A 14.8 kb region inserted in the 23.9 kb region containing fsrA and fsrB
Nakayama et al.  described a conserved 23.9 kb chromosomal deletion when comparing fsrA-lacking/fsrC+/gelE+ strains (by PCR) from various origins with V583; the deleted sequences start in the middle of EF1841, include the fsrAB genes and end in the middle of the fsrC gene (EF1820). Loss of the fsr regulatory components results in a gelatinase-negative phenotype under routine test conditions despite the fact that these strains still carry the gelE gene [23, 29]. The absence/presence of the 23.9 kb region, from EF1820/fsrC to EF1841, did not appear to correlate with the clinical origin of the isolates . In a more recent analysis of relationships between various E. faecalis strains, the 23.9 kb region was not detected in 86% of the strains of the clonal complex (CC)2, 58% of the CC9 strains, nor in any of the CC8 strains . The Symbioflor 1 strain, used as a probiotic, is one representative of the 7.4% of E. faecalis isolates that are missing the gelE gene in addition to the 23.9 kb region [5, 30]. Our analysis of this area in OG1RF revealed the presence of an additional 14.8 kb fragment inserted between the corresponding EF1826 and EF1827 of OG1RF (confirmed by PCR; results not shown). In OG1RF, this 14.8 kb region contains two loci, a WxL locus (described below) and a seven-gene locus that may encode a possible ABC transporter with similarity to one annotated in Pediococcus pentosaceus.
Components of the cell surface
It has been shown in E. faecalis that at least one cell surface protein (Ace) is subject to domain variation  and it has been postulated that domain variation may help bacteria escape the immune system. We found more polymorphisms in two families of E. faecalis proteins present on the cell surface: the MSCRAMMs and the WxL domain surface proteins. The MSCRAMMs are composed of two large regions, namely, the non-repeat A region (which is usually the ligand binding region for extracellular matrix molecules such as collagen or fibrinogen) and the B region (which typically contains repeated sub-domains). The B region of Ace contains five repeats in OG1RF, while it contains only four in V583 . We found two other MSCRAMM proteins that show polymorphisms in the number of their B repeats. OG1RF_0186 (corresponding to EF2505 of V583) has four repeats compared to seven in V583, and OG1RF_0165 (corresponding to EF2224 of V583) has eight repeats compared to five in V583. It has been proposed that the repeats are used as a stalk that projects the A region across the peptidoglycan and away from the cell surface . A hypothesis that the number of repeats may be proportional to the depth of the peptidoglycan has been proposed . However, OG1RF_0186 carries fewer repeats than EF2505 while Ace and OG1RF_0165 carry more repeats than their counterparts in V583, suggesting that our observation does not fit this hypothesis or that the peptidoglycan depth is not uniform. Apart from these three MSCRAMMs with B-repeat polymorphisms, we identified two unique MSCRAMM proteins in OG1RF: a homologue of EF0089 (OG1RF_0063, which shares 48% similarity) and a homologue of EF1896 (OG1RF_0039, which shares 75% similarity); both are located in the approximately 49 kb region unique to OG1RF described below (Figure 1; Additional data file 1).
Another family of E. faecalis surface proteins includes the newly described WxL domain surface proteins. Siezen et al.  reported a novel gene cluster encoding exclusively cell-surface proteins that is conserved in a subgroup of Gram-positive bacteria. Each gene cluster has at least one member of three gene families: a gene encoding a small LPxTG protein (approximately 120 amino acids); a gene encoding a member of the DUF916 transmembrane protein family; and a gene encoding a WxL domain surface protein. In addition, members of these gene families were found as singletons or associated with genes encoding other proteins (Additional data file 2). Recently, it was shown that the WxL domain attaches to the peptidoglycan on the cell surface  and one member of this WxL domain family, the homologue of EF2686 in OG1RF (a probable internalin protein), was shown to be important for virulence in a mouse peritonitis model and is required for dissemination to the spleen and liver . OG1RF shares five complete WxL loci with V583 (EF0750-7, EF2682-6, EF2970-68, EF3181-8, and EF3248-53). OG1RF does not contain homologues of EF2248-54 (carrying instead the iol operon), though it has a novel WxL locus within the 14.8 kb unique region upstream of the fsr locus (Additional data file 2). In addition to the variation in the number of WxL loci, we also observed polymorphisms in six of the WxL domain surface proteins. For example, OG1RF_0213 shares 88% similarity with EF3188, while OG1RF_0224, OG1RF_0225, and OG1RF_0227 share 64-68% similarity with their V583 counterparts, EF3248, EF3250, and EF3252, respectively. Also, in place of EF3153, EF3154, and EF3155 (which share 70% similarity among themselves), were found non-distantly related homologues, OG1RF_0209 and OG1RF_0210, which share 60-80% similarity with EF3153, EF3154, and EF3155. It is interesting to note that while several of these WxL loci, including the EF0750 and EF3184 loci, were present by hybridization in all the strains (clinical or food isolates) tested by Lepage et al. , other loci, including the EF3153 and EF3248 loci, were not detected in the majority of these strains. In addition, it appears that the EF3248 locus diverges in the Symbioflor 1 strain. When compared to V583, the sequence identity in this area between the two strains appears to be as low as 75% (depicted in Figure 2 from reference ).
However, because the Symbioflor 1 genome sequence is not currently available, it was not possible to compare their respective sequences in more detail. Since these proteins are located at the surface of the cell, the low level of homology shared between them may be the result of antigenic variation. More analyses are required for a better understanding of the number, frequency and function of these WxL domain proteins and their possible relationship with the diversity of E. faecalis.
Finally, as previously found using PCR, the cpsCDEFGHIJK operon capsule polysaccharide genes  were confirmed here as missing, although OG1RF carries the cpsA and cpsB genes, which were proposed to be essential for E. faecalis since all strains tested by Hufnagel et al.  carry these two genes. In OG1RF, the region that would encode the cps operon is only 59 bp in length and has no homology with V583. Thus, while V583 and OG1RF share much similarity between their surface components, there are unique differences that could potentially be important in affecting the behavior of the strains and might be useful for strain typing.
Two-component regulatory systems
LytR family response regulator
BglG family transcriptional antiterminator
Probable regulator of the downstream PTS system
Probable transcriptional regulator of the downstream ABC superfamily transporter
GntR family transcriptional regulator
Probable regulator of the downstream PTS system
DNA binding protein
Probable regulator of the iol operon
Sensor histidine kinase VanSG
Best homology with VanG and
Response regulator VanRG
VanG2 two-component systems.
OG1RF_0192 and OG1RF_0193 appear cotranscribed with a gene encoding a M15 family muramoylpentapeptide carboxypeptidase
Best homology with AgrA from Bacillus cereus G9241. However, no presence of AgrB or AgrD homologues in the vicinity. Also similar to ComE of S. pneumoniae (52% similarity)
Sensor histidine kinase
Best homology with AgrC from Bacillus cereus G9241. Also similar to ComD of Streptococcus pneumoniae (48% similarity)
Probable endoribonuclease MazF
Toxin-antitoxin described in E.
Probable antitoxin MazE
coli and recently on an E. faecium plasmid
Yebra et al. reported that L. casei was the sole member of the Lactobacillales to carry a functional iol operon . To survey E. faecalis, also a member of this order, for the presence of the iol operon, 48 isolates with different MLST and/or from various origins (including OG1RF and V583) were tested for myo-inositol fermentation; 23 of 48 isolates were positive. In addition, PCR verified the presence of iolE and iolR in these strains and in one negative for myo-inositol fermentation, indicating that the iol operon is not unique to OG1RF. To verify that the iol genes are responsible for the fermentation of myo-inositol in OG1RF, transposon insertion mutants  in the iolB and iolG2 genes of OG1RF were tested. Both mutants failed to ferment myo-inositol (data not shown), demonstrating that these genes are essential for myo-inositol fermentation. To investigate whether the iol operon was 'inserted into' or 'removed from' a putative ancestral strain, the sequences surrounding the iol genes were examined. In OG1RF, the iol operon is located between the equivalent of EF2239 and EF2352 when compared with V583. In V583, this region encodes probable prophage proteins and carries the vanB transposon, which confers vancomycin resistance. Since we did not identify any remnants of the iol operon in V583, it would appear that at least two independent events at the same location differentiate OG1RF and V583, suggesting that it is a hot region for rearrangement. This region between EF2239 and EF2352 (111 Kb) is also missing in the Symbioflor 1 strain (referred to as gap 2) . The possible junction and presence of unique sequence in this region, if investigated, was not mentioned in the publication. Nonetheless, preliminary analysis of other strains' genotypes in this area seemed to confirm the hypothesis of a hot region for rearrangement (data not shown).
A homologue of Tn916in OG1RF
An analysis of the G+C content of OG1RF unique regions revealed several loci with a lower G+C content than the 37.8% average content of OG1RF. One of these is an approximately 49 kb fragment with a G+C content of 32.1% located between an rRNA operon and the homologue in OG1RF of EF1053, replacing 10 tRNA genes present in V583 (Figure 1). This fragment appears to be a patchwork composed of hypothetical genes, homologues of Tn916-associated genes and homologues of genes from other Gram-positive organisms, including Listeria, E. faecium, staphylococci, or lactococci (Additional data file 1). It is interesting to note that this region contains: a putative adhesin protein gene (OG1RF_0039) at one end of the fragment; homologues of 14 Tn916-associated genes (Tn916_2 to Tn916_12, Tn916_18 and Tn916_19, with an average of 70% similarity); and a gene encoding a putative integrase (OG1RF_0088) at the other end - these three features are also present in Tn5386 in E. faecium D344R . However, the approximately 49 kb fragment lacks an excisase gene and the probable lantibiotic ABC transporter genes present in Tn5386.
An uninterrupted competence operon in OG1RF
Natural competence has not been reported for E. faecalis. To assess the functionality of this operon in OG1RF, we evaluated the competence of cells in different phases of growth (early log growth to stationary phase) using pAM401  and pMSP3535VA . We were not able to show natural competence under the conditions tested. We have also noted that V583 is less transformable by electroporation than OG1RF. To investigate the possibility that directly or indirectly the com operon might be responsible for this phenotype, we also evaluated transformability by electroporation. When compared with OG1RF, transposon mutants  in the OG1RF equivalent of EF2045 (encoding the comGB homologue) and in the OG1RF equivalent of EF1986 (encoding the comGF homologue) showed similar levels of transformability by electroporation (data not shown), implying that the difference in electroporation efficiency observed between OG1RF and V583 is not related to this locus.
In Streptococcus pneumoniae , the competence operon is tightly regulated by a quorum sensing two-component system (ComDE) and a competence-stimulating peptide (CSP; encoded by comC). We did not find any homologues of CSP in OG1RF. Two homologous ComDE sensor histidine kinase/response regulators were found in OG1RF, one of which is FsrC/FsrA. Based on our previous microarray data, the Fsr system does not regulate the comY operon, at least under our previously used conditions (mid-log phase growth to early stationary phase in brain heart infusion (BHI)) . The other ComDE homology is that with a two-component system unique to OG1RF (OG1RF_0199 and OG1RF_0198, respectively) that lies on a 4,706 bp unique fragment that maps between EF3114 and EF3115 in V583. This fragment also carries two genes (OG1RF_0200 and OG1RF_0201) encoding homologues of the YhaQ and YhaP sodium efflux ATP-binding cassette efflux/transporter proteins (Figure 4b). Although they are potential elements of a secretion apparatus, these two proteins do not share any homology at the protein level with the competence secretion apparatus ComAB of S. pneumoniae  nor CslAB from S. mutans . Searching for a possible CSP in the vicinity of these genes, we identified a small ORF encoding 50 amino acids between yhaP and OG1RF199 and another encoding 20 amino acids downstream of OG1RF198. More analysis will be required to determine if there are conditions in which the OG1RF com operon is expressed and to determine whether or not this two-component system is involved in competence.
Limited presence of mobile elements
By probing a microarray of the V583 genome and plasmids with OG1RF genomic DNA, we previously estimated that only 75% of V583 ORFs were also present in OG1RF . Later, Aakra et al.  compared nine strains, including OG1RF to V583, using comparative genomic hybridization. In these results, OG1RF appears to carry a few genes included in the PAI, and a few prophage genes. Using the complete genome sequence, we have now found that OG1RF lacks 639 genes and the three pTET plasmids described in V583. All but 45 of the missing genes are associated with putative mobile elements, such as the entire PAI, the recently described phages 1, 3, 4, 5, 6, and 7 , and the approximately 111 kb area between genes EF2240 and EF2351 (including the vanB transposon) present in V583. The absence of these elements appears also to be a characteristic of the commensal strain Symbioflor 1, although because the genome was not completely finished, the possibility remains that some of these regions were not sequenced. In conclusion, other than the approximately 49 kb fragment containing a Tn916 homologue, it appears that OG1RF has only one additional possible mobile element derivative, namely the phage 2 proposed to be part of the core genome .
Fusidic acid and rifampicin resistance
OG1RF was sequentially selected from OG1 for resistance to fusidic acid and rifampicin . The mutation leading to rifampicin resistance was identified in the rpoB gene by Ozawa et al.  and is caused by the A1467G mutation, which results in substitution of arginine for histidine at amino acid 489. The mutation also affected the clumping phenotype of traA mutants and this effect appears to be specific for the pAM373 system . All of the other 22 differences in rpoB between OG1RF and V583 are synonymous. Fusidic acid resistance is associated with mutation(s) in the fusA gene, which encodes elongation factor G. We compared fusA from OG1RF with that in V583 and identified two differences (C1368A and T1992C). The mutation T1992C is synonymous, while the mutation C1368A leads to the presence of glutamine (histidine in V583) at position 404 in OG1RF. Mutations in this region have been associated with fusidic acid resistance in Staphylococcus aureus [55, 56], and thus the C1368A mutation is likely the cause of the fusidic acid resistance phenotype in OG1RF.
Virulence and biofilm comparisons of OG1RF with V583
E. faecalis OG1RF carries a number of unique loci compared to V583. Those of particular interest include new surface proteins (MSCRAMMs and WxL domain proteins), an operon encoding myo-inositol utilization, an intact competence operon, and two CRISPR elements. The CRISPR elements may be of particular significance when one considers that most of what is missing from OG1RF compared to V583 consists of mobile genetic elements (MGEs), including 6 phages or remnants thereof. The presence of the CRISPR elements in OG1RF provides a tantalizing, but as yet unproven, explanation for the discordance in the number of mobile elements between these two strains.
The acquisition of MGE is believed to be an important mechanism by which the species E. faecalis had been able to generate genetic diversity and, thereby, highly variable phenotypes . It has been proposed that the ability of E. faecalis to cause healthcare related infections is associated with these MGEs [4, 5]. This hypothesis was supported by several studies that have highlighted the importance of virulence determinants carried by these mobile elements, such as cytolysin  by the PAI. However, more recent results from Aakra et al.  and Lepage et al.  demonstrate that these factors may be present in harmless strains while absent in clinical isolates, indicating that E. faecalis virulence is not dependent on any single virulence factor. Indeed, few studies have compared the virulence pattern of strains from various origins. The increased ability of OG1RF to infect kidneys and to produce biofilm, despite the absence of MGEs and their associated virulence factors, was surprising. Different possibilities can be proposed relating to the factors important for these differences in enterococcal infections. One of these is that virulence in the assays used may be linked to the shared core genome of these two strains, with the differences arising from the unique genes. On the other hand, virulence could be associated primarily with the genes unique to each strain, but with each set being able to complement the absence of the other. It seems most likely that virulence, and/or some combination of virulence and fitness, is caused by the expression of a mixture of both the core and unique genes. It is also important to remember that E. faecalis is a well adapted commensal, carrying the genes necessary to survive and to colonize the gut, and that a subset, particularly MLST CC2 and CC9 , predominates among hospital acquired infections. It may be that these clonal complexes are not more virulent per se, as defined in the assays described here, but rather are better able to survive and/or colonize hospitalized patients, taking advantage of factors that predispose to nosocomial infections such as urinary or venous catheters, or mucositis, among others.
Sequencing of more E. faecalis strains may facilitate our understanding of the path from commensalism to pathogenicity, a crucial prerequisite for designing therapeutic interventions directed to control an organism that is already resistant to a large spectrum of antibiotics.
Materials and methods
E. faecalis OG1 is a strain of human origin (formerly designated 2SaR ) and was subsequently selected on rifampicin and fusidic acid [8, 9] to generate OG1RF (deposited at the American Type Culture Collection (ATCC) under ATCC accession number 47077). V583 is a vancomycin resistant E. faecalis strain , recovered from a blood culture of a patient hospitalized at the Barnes Hospital, St Louis, MO, USA in February 1987 (ATCC accession number 700802, NCBI complete genome accession number NC_004668). Bacteria were grown routinely at 37°C in BHI broth (Difco Laboratories, Detroit, MI, USA) or BHI agar unless otherwise indicated. Comparisons of OG1RF and V583 grown in broth (BHI, tryptic soy broth with glucose (TSBG), or BHI with 40% serum) did not reveal any obvious differences.
DNA sequencing and genome assembly
Genomic DNA was purified from cesium chloride (CsCl) gradients of whole cell lysates . DNA sequencing was performed by a combined approach using 454 Life Sciences pyrosequencing strategies  and the Solexa approach . Read-pair information was used to create higher order scaffolds. Sanger sequencing was used for OG1RF whole gun sequencing and finishing. The coverage was 28× by the 454, 104× by Solexa, and 4.5× by Sanger sequencing technique. The assembly was done using Atlas .
Gene identification and annotation
Gene prediction and manual annotation were performed as previously described . Glimmer  and GeneMark  were used independently to predict ORFs. Visualization of gene predictions was performed using the Genboree system  and the CONAN database . OG1RF-unique ORFs were analyzed with BLASTN and BLASTX. Protein sequences were analyzed by BLASTP versus the nr database at NCBI . When appropriate, other predictive tools were used as described previously . This whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the project accession ABPI00000000. The version described in this paper is the first version, ABPI01000000. This project includes also the annotation of the ORFs unique to OG1RF. The OG1RF-unique ORFs are listed in Additional data file 1.
Transposon mutations in OG1RF-unique sequences
Following the creation of an E. faecalis Tn917 library , 6,237 sequences representing the flanking regions of the transposon insertion sites were obtained and compared to the V583 genome by BLASTN. A total of 196 sequences were unique to OG1RF. Thirty-seven of the unique genes contained a transposon insertion. The locations of the transposon insertions are listed in Additional data file 1.
Carbohydrate fermentation tests
Forty-eight E. faecalis isolates, including OG1RF and V583, having different MLST profiles, pulsed field gel electrophoresis types or from various geographical origins, were streaked onto BHI agar and incubated overnight at 37°C. Five to ten colonies of each strain were resuspended in 100 μl of 0.9% saline in a microtiter plate and tested for fermentation using BBL™ Phenol Red Broth Base (Diagnostic Systems, Sparks, MD, USA) supplemented with agar and either 10 mM glucose (positive control), 10 mM dulcitol (negative control), or 10 mM myo-inositol (Sigma, St Louis, MO, USA). Plates were read after incubation at 37°C for 24 h; a yellow halo around the colony was considered positive for fermentation. iolB and iolG2 transposon mutants  were also tested.
PCR was performed using genomic DNA purified using Bactozol™ (Molecular Research Center, Inc., Cincinnati, OH, USA), as recommended by the manufacturer. Specific PCR primer pairs (Additional data file 4) were used to assess the presence of the OG1RF-unique sequences and for confirmation of flanking DNA regions in common with V583.
To test strains for competence, overnight cultures, grown at 37°C in Todd-Hewitt broth, were diluted in Todd-Hewitt broth to an OD600 nm of 0.05 and then further diluted 10,000-fold in Todd-Hewitt broth to a final volume of 100 ml. After 30 minutes at 37°C, with shaking at 150 rpm, and every hour for 10 h, 0.5 ml samples were removed and 2.5 μg of plasmid DNA were added. The plasmids tested were pAM401  and pMSP3535VA . The samples were incubated for 2 h before plating on BHI or BHI plus antibiotic (chloramphenical 10 μg/ml for pAM401 or kanamycin 2 mg/ml for pMSP3535VA). Following overnight incubation at 37°C, the total numbers of CFU/ml recovered on selective agar for the plasmid were compared to the total number of CFU/ml (plated on BHI agar) for each time point.
Biofilm assay and statistical analysis
The biofilm assay was performed as described by Mohamed et al. . Each assay was performed using 16 wells on three occasions. The median was calculated using the 48 OD570 nm readings on data pooled from all experiments and statistical analysis was performed using a non-parametric t-test.
Mouse peritonitis model
E. faecalis strains OG1RF and V583  were tested using a previously published method . Mice were injected intraperitoneally with appropriate dilutions of premixed bacteria/sterile rat fecal extract and were observed for five days. Two-fold dilutions of test bacteria in the range 107-109 CFU were used as the inocula for LD50 determination using 6-9 mice per inoculum group. Inocula CFU geometric mean values were obtained and used for LD50 calculation by the method of Reed and Muench .
UTI model for competition assay and ID50 determination
E. faecalis strains OG1RF and V583 were tested in the UTI model as previously described . For the mixed inoculum experiments, an approximately 1:1 ratio of E. faecalis OG1RF:V583 at approximately 103 CFU each was used. Two independent experiments, using 10 and 11 mice, respectively, were performed and the results were combined. The log10(CFU) of OG1RF and V583 per gram of tissue of each animal (kidney or bladder) from mixed infection were analyzed for significance by the paired t-test. For mono-infection, approximately 103 CFU organisms grown in BHI + 40% horse serum were used for each strain independently and CFU obtained from kidney pairs (nine mice per strain) were analyzed for significance by the unpaired t-test. The minimum detectable limits of recovered bacteria were 101 and 102 CFU/gm of kidney pairs and urinary bladder homogenates, respectively.
Additional data files
The following additional data files are available with the online version of this paper. Additional data file 1 is a list of the ORFs unique to OG1RF compared to V583 with their OG1RF locus tag, location in the genome, and definition. Additional data file 2 is a list of genes encoding proteins with a WxL domain in OG1RF and/or V583. Additional data file 3 shows the results of the mouse peritonitis model using OG1RF and V583, with the statistical analysis. Additional data file 4 is a list of the significant primers used in this study.
American type culture collection
brain heart infusion
colony forming units
comprised of regularly interspaced short palindromic repeats
histidine kinase-response regulator
mobile genetic element
microbial surface component recognizing adhesive matrix molecules
multilocus sequence typing
open reading frame
We would like to express our sincere thanks to J Hernandez, S Wang, Z Li, D Ngo, and L Hemphill for their help during the sequencing process. We also would like to thank JM Urbach, Massachusetts General Hospital, Boston, MA for helping localize the transposon insertions in the OG1RF unique sequences. This research was supported by grant R21 AI064470 from the National Institutes of Health to GMW and by NIH grant R37 AI47923 from the Division of Microbiology and Infectious Diseases, NIAID, to BEM.
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