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

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.     

Complete genome sequencing and comparative analysis of the linezolid-resistant Enterococcus faecalis strain DENG1

Genome level analysis of bacterial strains provides information on genetic composition and resistance mechanisms to clinically relevant antibiotics. To date, whole genome characterization of linezolid-resistant Enterococcus faecalis isolated in the clinic is lacking. In this study, we report the entire genome sequence, genomic characteristics and virulence factors of a pathogenic E. faecalis strain, DENG1. Our results showed considerable differences in genomic characteristics and virulence factors compared with other E. faecalis strains (V583 and OG1RF). The genome of this LZD-resistant E. faecalis strain can be used as a reference to study the mechanism of LZD resistance and the phylogenetic relationship of E. faecalis strains worldwide.     

Suppression of a thermosensitive dnaA mutation of Escherichia coli by bacteriophage P1 and P7.

Like other plasmids, the P1 and P7 prophages suppress E. coli dnaA(Ts) mutations by integrating into the host chromosome. This conclusion is supported by three lines of evidence: (1) Alkaline sucrose gradients reveal the absence of plasmid DNA in suppressed lysogens; (2) the prophage is linked to host chromosomal markers in conjugation; and (3) auxotrophs whose defect is linked to the prophage are found among suppressed colonies. No phage or bacterial mutation is required for suppression. Integrative suppression by P1 and P7, unlike suppression by F, does not require the host recA⁺ function. Among suppressed P7 lysogens are some that do not produce phage; these contain defective prophages. The genetic extent of the deletions contained by these defective prophages delineates the prophage regions which are not necessary for suppression of dnaA(Ts). The possible mechanisms of integration and deletion formation are discussed.     

A method for construction of E. coli strains with multiple DNA insertions in the chromosome

A system for construction of E. coli strains with multiple DNA insertions in the chromosome, based on elements of modules for site specific recombination of Tn1545 and phage λ, has been developed. Circular non-replicating DNA fragments containing the transposon attachment site (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly into the chromosome of a host E. coli strain when provided with transposon integrase, Int-Tn (the host strain was obtained by insertion of the fragment containing transposon int-Tn gene coding for Int-Tn into the chromosome). Integration of these fragments into the chromosome of int-Tn⁺ cells gives rise to a collection of antibiotic-resistant clones with single insertions at different locations in the chromosome. These insertions are transferred subsequently by P1 transduction into one strain and selected for antibiotic resistance provided by the cassette with the selectable marker. After transduction of each copy, a helper plasmid bearing phage λ xis and int genes is introduced into the cells to excise the drug resistance gene flanked with the λattL and λattR sites from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of P1 transduction/drug resistance gene excision. Each cycle adds another chromosomal copy of the foreign gene. To show the utility of the system, we constructed an E. coli strain bearing several chromosomal copies of lacZ at different locations.     

Survey of Genomic Diversity among Enterococcus faecalis Strains by Microarray-Based Comparative Genomic Hybridization

We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the “test” strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.     

Prophage Tracer: precisely tracing prophages in prokaryotic genomes using overlapping split-read alignment

The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. However, the identification of prophages that are highly divergent from known phages remains challenging. In this study, by taking advantage of the lysis-lysogeny switch of temperate phages, we designed Prophage Tracer, a tool for recognizing active prophages in prokaryotic genomes using short-read sequencing data, independent of phage gene similarity searching. Prophage Tracer uses the criterion of overlapping split-read alignment to recognize discriminative reads that contain bacterial (attB) and phage (attP) att sites representing prophage excision signals. Performance testing showed that Prophage Tracer could predict known prophages with precise boundaries, as well as novel prophages. Two novel prophages, dsDNA and ssDNA, encoding highly divergent major capsid proteins, were identified in coral-associated bacteria. Prophage Tracer is a reliable data mining tool for the identification of novel temperate phages and mobile genetic elements. The code for the Prophage Tracer is publicly available at https://github.com/WangLab-SCSIO/Prophage_Tracer.     

Impact of actin on adhesion and translocation of Enterococcus faecalis

This study focuses on the impact of actin on adhesion and translocation of Enterococcus (E.) faecalis OG1RF, E. faecalis Symbioflor^®, and E. faecalis V583. Insight into the role of actin aggregation in the mediation of bacterial adhesion and translocation was provided by a two-chamber translocation assay, which employed Ptk6 cells. Determination of translocation rates, cytochalasin D treatment, and laser scanning confocal microscopic observation revealed actin as a predominant brace for enterococci to pass through the epithelial cell layer. As the three enterococci had moderate adhesion ability to actin, actin-binding proteins were isolated and characterized by LC–MS/MS. The isolated proteins were identified as pyruvate formate lyase, enolase, glyceraldehyde-3-phosphate dehydrogenase, and GroEL. All these proteins belong to two major groups of moonlighting proteins, i.e., proteins, which display additional functions other than their described major biochemical catalysis. Both groups of moonlight proteins were determined to be associated with epithelial cell binding.     

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.     

Screening of In Vivo Activated Genes in Enterococcus faecalis during Insect and Mouse Infections and Growth in Urine

Enterococcus faecalis is part of the commensal microbiota of humans and its main habitat is the gastrointestinal tract. Although harmless in healthy individuals, E. faecalis has emerged as a major cause of nosocomial infections. In order to better understand the transformation of a harmless commensal into a life-threatening pathogen, we developed a Recombination-based In Vivo Expression Technology for E. faecalis. Two R-IVET systems with different levels of sensitivity have been constructed in a E. faecalis V583 derivative strain and tested in the insect model Galleria mellonella, during growth in urine, in a mouse bacteremia and in a mouse peritonitis model. Our combined results led to the identification of 81 in vivo activated genes. Among them, the ef_3196/7 operon was shown to be strongly induced in the insect host model. Deletion of this operonic structure demonstrated that this two-component system was essential to the E. faecalis pathogenic potential in Galleria. Gene ef_0377, induced in insect and mammalian models, has also been further analyzed and it has been demonstrated that this ankyrin-encoding gene was also involved in E. faecalis virulence. Thus these R-IVET screenings led to the identification of new E. faecalis factors implied in in vivo persistence and pathogenic potential of this opportunistic pathogen.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

Secondary Cell Wall Polymers of Enterococcus faecalis Are Critical for Resistance to Complement Activation via Mannose-binding Lectin

The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583ΔtagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of →6)[α-l-Rhap-(1→3)]β-d-GalpNAc-(1→5)-Rbo-1-P and →6) β-d-Glcp-(1→3) [α-d-Glcp-(1→4)]-β-d-GalpNAc-(1→5)-Rbo-1-P→, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system.     

Comparative Genomics and Transduction Potential of Enterococcus faecalis Temperate Bacteriophages

To determine the relative importance of temperate bacteriophage in the horizontal gene transfer of fitness and virulence determinants of Enterococcus faecalis, a panel of 47 bacteremia isolates were treated with the inducing agents mitomycin C, norfloxacin, and UV radiation. Thirty-four phages were purified from culture supernatants and discriminated using pulsed-field gel electrophoresis (PFGE) and restriction mapping. From these analyses the genomes of eight representative phages were pyrosequenced, revealing four distinct groups of phages. Three groups of phages, ΦFL1 to 3, were found to be sequence related, with ΦFL1A to C and ΦFL2A and B sharing the greatest identity (87 to 88%), while ΦFL3A and B share 37 to 41% identity with ΦFL1 and 2. ΦFL4A shares 3 to 12% identity with the phages ΦFL1 to 3. The ΦFL3A and B phages possess a high DNA sequence identity with the morphogenesis and lysis modules of Lactococcus lactis subsp. cremoris prophages. Homologs of the Streptococcus mitis platelet binding phage tail proteins, PblA and PblB, are encoded on each sequenced E. faecalis phage. Few other phage genes encoding potential virulence functions were identified, and there was little evidence of carriage of lysogenic conversion genes distal to endolysin, as has been observed with genomes of many temperate phages from the opportunist pathogens Staphylococcus aureus and Streptococcus pyogenes. E. faecalis JH2-2 lysogens were generated using the eight phages, and these were examined for their relative fitness in Galleria mellonella. Several lysogens exhibited different effects upon survival of G. mellonella compared to their isogenic parent. The eight phages were tested for their ability to package host DNA, and three were shown to be very effective for generalized transduction of naive host cells of the laboratory strains OG1RF and JH2-2.Enterococcus faecalis is a member of the natural flora of humans and colonizes the gastrointestinal and vaginal tracts and the oral cavity. In recent years it has emerged as an important opportunistic nosocomial pathogen and is a causative agent of bacteremia, infective endocarditis, and surgical wound and urinary tract infections. The accumulation of acquired antibiotic resistance determinants, in addition to its intrinsic resistance and tenacity, has given rise to the evolution of clinical isolates of E. faecalis that are therapeutically problematic (19). Greater notoriety was afforded to this species following the observed transfer of the conjugative transposon Tn1546 to Staphylococcus aureus, imparting vancomycin resistance (11). Subsequent analysis has revealed that multiple independent E. faecalis-dependent vanA transfers had occurred in the United States prior to 2007 (50). This places enterococci in an important and dynamic position within the health care system, warranting their increased study.The specific determinants that are proposed to contribute to the virulence of E. faecalis are not universally present, and expression of the cognate genes is variable (21, 37). For example, in a recent study of 106 clonally diverse strains of E. faecalis the metallopeptidase gelatinase (GelE) was shown to be expressed in less than 60% of 106 genotypically positive isolates, whereas expression of cytolysin was less frequently observed (expression in ∼25% of isolates, with 30% being genotypically positive) (33). A proposed pathogenicity island identified with E. faecalis V583 (49) is composed of a variable gene set encoding the virulence determinants enterococcal surface protein, cytolysin, and aggregation substance. This highly variable 150-kb mobile element contains many components of unknown function that are hypothesized to facilitate survival and/or transmission in the health care setting (34, 40, 49).Two sequenced and annotated genomes of E. faecalis have been completed and published to date. These are the blood isolate and first-observed vancomycin-resistant strain V583 (40) and the oral isolate OG1RF, used as a common laboratory strain (8). A major difference between these genomes is the presence in V583 of seven regions containing phage-associated sequences. In contrast, OG1RF contains only one phage remnant, which was proposed by McBride et al. (33) to form part of the core genome, a theory supported by the presence in OG1RF of this phage remnant region together with two CRISPR loci. CRISPR sequences provide sequence-specific resistance to bacteriophages via the assembly of phage DNA sequences interspersed as spacers between repeats, in concert with associated cas genes, which collectively operate as an RNA-based gene silencing mechanism (5, 6, 28, 30, 36, 42). This elegant heritable mechanism is proposed to limit horizontal gene transfer of bacteriophage, transposable elements, and conjugative plasmids (9, 10, 32).Within the firmicute division of Gram-positive bacteria, temperate bacteriophages are key vectors for the horizontal transfer of virulence genes. In Staphylococcus aureus, bacteriophages encode and mobilize an impressive array of immune evasion genes (54, 55) and Panton-Valentine leukocidin (43). Several bacteriophage-encoded virulence determinants also contribute to pathogenesis in group A Streptococcus (2, 3, 4).The role of bacteriophages in the virulence of E. faecalis is not clear. Encoded within seven phage-related sequences of strain V583, there are multiple reported homologs of the Streptococcus mitis platelet-binding proteins PblA and PblB (7) and a ferrochelatase (40). In contrast, the absence of mobile genetic elements (MGEs) in strain OG1RF led Bourgogne et al. (8) to speculate that they did not engender virulence in E. faecalis.In this study we determined the morphology and complete genome sequences of eight induced bacteriophages purified from clinical isolates of E. faecalis. We sought to determine the potential carriage of genes that might contribute to the virulence or fitness of this organism and characterize the capacity of these phages to participate in transduction.     

Lambdoid phages as elements of bacterial genomes (integrase/phage21/ Escherichia coli K-12/icd gene)

The lambdoid phages are a group of related temperate bacteriophages that lysogenize by site-specific recombination with the bacterial chromosome. Various members of the group have different specific chromosomal insertion sites, despite the fact that the enzymes catalyzing the insertion (integrases) appear to be all descended from a common ancestor. Insertion sites are not located randomly on the E. coli chromosome but are restricted to one segment of the map; also, most prophages are oriented in the same direction along the chromosome. Lambdoid phage 21 inserts within the isocitrate dehydrogenase gene and introduces an alternative 165 bp 3 end for that gene. A defective element (el4) inserts at the same position. We suggest that this mode of insertion arose from insertion of an ancestral phage to the right of icd which then picked up part of the icd gene by abnormal excision speculate that, at an earlier time, phages may have arrived at their present locations by a process of chromosomal walking.     

Capsid size determination by Staphylococcus aureus pathogenicity island SaPI1 involves specific incorporation of SaPI1 proteins into procapsids

The Staphylococcus aureus pathogenicity island SaPI1 carries the gene for the toxic shock syndrome toxin (TSST-1) and can be mobilized by infection with S. aureus helper phage 80α. SaPI1 depends on the helper phage for excision, replication and genome packaging. The SaPI1-transducing particles comprise proteins encoded by the helper phage, but have a smaller capsid commensurate with the smaller size of the SaPI1 genome. Previous studies identified only 80α-encoded proteins in mature SaPI1 virions, implying that the presumptive SaPI1 capsid size determination function(s) must act transiently during capsid assembly or maturation. In this study, 80α and SaPI1 procapsids were produced by induction of phage mutants lacking functional 80α or SaPI1 small terminase subunits. By cryo-electron microscopy, these procapsids were found to have a round shape and an internal scaffolding core. Mass spectrometry was used to identify all 80α-encoded structural proteins in 80α and SaPI1 procapsids, including several that had not previously been found in the mature capsids. In addition, SaPI1 procapsids contained at least one SaPI1-encoded protein that has been implicated genetically in capsid size determination. Mass spectrometry on full-length phage proteins showed that the major capsid protein and the scaffolding protein are N-terminally processed in both 80α and SaPI1 procapsids.     

Complete genome sequence and comparative genomic analysis of Enterococcus faecalis EF-2001, a probiotic bacterium

Enterococcus faecalis is a common human gut commensal bacterium. While some E. faecalis strains are probiotic, others are known to cause opportunistic infections, and clear distinction between these strains is difficult using traditional taxonomic approaches. In this study, we completed the genome sequencing of EF-2001, a probiotic strain, using our in-house hybrid assembly approach. Comparative analysis showed that EF-2001 was devoid of cytolysins, major factors associated with pathogenesis, and was phylogenetically distant from pathogenic E. faecalis V583. Genomic analysis of strains with a publicly available complete genome sequence predicted that drug-resistance genes- dfrE, efrA, efrB, emeA, and lsaA were present in all strains, and EF-2001 lacked additional drug-resistance genes. Core- and pan-genome analyses revealed a higher degree of genomic fluidity. We found 49 genes specific to EF-2001, further characterization of which may provide insights into its diverse biological activities. Our comparative genomic analysis approach could help predict the pathogenic or probiotic potential of E. faecalis leading to an early distinction based on genome sequences.     

Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36–42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13–15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10–33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.     

Cloning with Mud-P22 hybrid prophages: mapping of IS200 elements on the chromosome of Salmonella typhimurium LT2

Specific DNA fragments from the chromosome of Salmonella typhimurium LT2 were packaged in P22 capsids by induction of “locked-in” Mud-P22 hybrid prophages. High yields of the packaged DNA were obtained upon capsid disruption. DNA hybridization using a fragment of insertion sequence IS200 as probe permitted physical mapping of IS200 elements on the chromosome of S. typhimurium LT2 within?±1 centisome (CS). IS200 copies were found at the following locations: CS 24 (copy VI), CS 53 (copy V), CS 63 (copy I), CS 80 (copy II) and CS 93 (copy III). Copy IV, previously mapped near fliA (CS 42), was not included in our study.