Structural Differences in Complexes between the Master Regulator PrgX, Peptide Pheromones, and Operator Binding Sites Determine the Induction State for Conjugative Transfer of pCF10.

Pheromone-inducible conjugation in the Enterococcus faecalis pCF10 system is regulated by the PrgX transcription factor through binding interactions at two operator binding sites (XBS1 and XBS2) upstream of the transcription start site of the prgQ operon encoding the conjugation machinery. Repression of transcription requires the interaction of a PrgX tetramer with both XBSs via formation of a DNA loop. The ability of PrgX to regulate prgQ transcription is modulated by its interaction with two antagonistic regulatory peptides, ICF10 (I) and cCF10 (C); the former peptide inhibits prgQ transcription, while the latter peptide enhances prgQ transcription. In this report, we used electrophoretic mobility shift assays (EMSAs) and DNase footprinting to examine binding interactions between the XBS operator sites and various forms of PrgX (Apo-X, PrgX/I, and PrgX/C). Whereas a previous model based on high-resolution structures of PrgX proposed that the functional differences between PrgX/C and PrgX/I resulted from differences in PrgX oligomerization state, the current results show that specific differences in XBS2 occupancy by bound tetramers account for the differential regulatory properties of the two peptide/PrgX complexes and for the effects of XBS mutations on regulation. The results also confirmed a DNA looping model of PrgX function. IMPORTANCE Peptide pheromones regulate antibiotic resistance transfer in Enterococcus faecalis. Here, we present new data showing that pheromone-dependent regulation of transfer genes is mediated via effects on the structures of complexes between peptides, the intracellular peptide receptor, and operator sites on the target DNA.

Effects of endogenous levels of master regulator PrgX and peptide pheromones on inducibility of conjugation in the enterococcal pCF10 system.

Enterococcal pheromone responsive conjugative plasmids like pCF10 promote horizontal spread of antibiotic resistance genes following induction of plasmid-containing cells by potential recipients. Transcription of conjugation genes from promoter PQ is inhibited by the master regulator PrgX, further repressed when PrgX is in complex with the inhibitory I peptide, and allowed when PrgX is in complex with the C inducing peptide. Single-cell analysis has shown that heterogeneity in the pheromone response is prevalent. Here, we systematically varied levels of regulatory molecules to better understand why some individual cells have increased propensity for induction. In this study, PrgX was confirmed to repress PQ in the absence of exogenous peptides in vivo, but cells with increased levels of PrgX were shown to be more prone to induction. Further, ablation of endogenous I reduced PrgX levels, resulting in reduced basal repression and loss of inducibility. Reduction of both endogenous peptides by washing increased the inducibility of cells. Together, these results show that endogenous PrgX, C, and I levels can impact the induction potential of a cell and establish the importance of basal I for regulation. These results also suggest that PrgX/C complexes may directly activate prgQ transcription, contrary to a long-standing working model.

Expression of Adhesive Pili and the Collagen-Binding Adhesin Ace Is Activated by ArgR Family Transcription Factors in Enterococcus faecalis.

It was shown previously that the disruption of the ahrC gene encoding a predicted ArgR family transcription factor results in a severe defect in biofilm formation in vitro, as well as a significant attenuation of virulence of Enterococcus faecalis strain OG1RF in multiple experimental infection models. Using transcriptome sequencing (RNA-seq), we observed ahrC-dependent changes in the expression of more than 20 genes. AhrC-repressed genes included predicted determinants of arginine catabolism and several other metabolic genes and predicted transporters, while AhrC-activated genes included determinants involved in the production of surface protein adhesins. Most notably, the structural and regulatory genes of the ebp locus encoding adhesive pili were positively regulated, as well as the ace gene, encoding a collagen-binding adhesin. Using lacZ transcription reporter fusions, we determined that ahrC and a second argR transcription factor gene, argR2, both function to activate the expression of ebpR, which directly activates the transcription of the pilus structural genes. Our data suggest that in the wild-type E. faecalis, the low levels of EbpR limit the expression of pili and that biofilm biomass is also limited by the amount of pili expressed by the bacteria. The expression of ace is similarly enhanced by AhrC and ArgR2, but ace expression is not dependent on EbpR. Our results demonstrate the existence of novel regulatory cascades controlled by a pair of ArgR family transcription factors that might function as a heteromeric protein complex.IMPORTANCE Cell surface adhesins play critical roles in the formation of biofilms, host colonization, and the pathogenesis of opportunistic infections by Enterococcus faecalis Here, we present new results showing that the expression of two major enterococcal surface adhesins, ebp pili, and the collagen-binding protein Ace is positively regulated at the transcription level by two argR family transcription factors, AhrC and ArgR2. In the case of pili, the direct target of regulation is the ebpR gene, previously shown to activate the transcription of the pilus structural genes, while the activation of ace transcription appears to be directly impacted by the two ArgR proteins. These transcription factors may represent new targets for blocking enterococcal infections.

PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis.

Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. Here, we report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10-encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10-encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA-binding fold, and prgB-prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA-binding regulators that act to mitigate toxicity accompanying overproduction of PrgB-like adhesins in E. faecalis and other clinically-important Gram-positive species.

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range.

Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the PQ pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin (TA) loci present in E. faecalis, parpAD1 of the pAD1 plasmid and parEF0409 located on the E. faecalis chromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalisIMPORTANCEE. faecalis is an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the PQ pheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function in E. faecalis and to further elucidate its potential roles in cell physiology.

Antagonistic self-sensing and mate-sensing signaling controls antibiotic-resistance transfer.

Conjugation is one of the most common ways bacteria acquire antibiotic resistance, contributing to the emergence of multidrug-resistant "superbugs." Bacteria of the genus Enterococcus faecalis are highly antibiotic-resistant nosocomial pathogens that use the mechanism of conjugation to spread antibiotic resistance between resistance-bearing donor cells and resistance-deficient recipient cells. Here, we report a unique quorum sensing-based communication system that uses two antagonistic signaling molecules to regulate conjugative transfer of tetracycline-resistance plasmid pCF10 in E. faecalis. A "mate-sensing" peptide sex pheromone produced by recipient cells is detected by donor cells to induce conjugative genetic transfer. Using mathematical modeling and experimentation, we show that a second antagonistic "self-sensing" signaling peptide, previously known to suppress self-induction of donor cells, also serves as a classic quorum-sensing signal for donors that functions to reduce antibiotic-resistance transfer at high donor density. This unique form of quorum sensing may provide a means of limiting the spread of the plasmid and present opportunities to control antibiotic-resistance transfer through manipulation of intercellular signaling, with implications in the clinical setting.

Arginine impacts aggregation, biofilm formation, and antibiotic susceptibility in Enterococcus faecalis.

Enterococcus faecalis is a commensal bacterium in the gastrointestinal tract (GIT) of humans and other organisms. E. faecalis also causes infections in root canals, wounds, the urinary tract, and on heart valves. E. faecalis metabolizes arginine through the arginine deiminase (ADI) pathway, which converts arginine to ornithine and releases ATP, ammonia, and CO2. E. faecalis arginine metabolism also affects virulence of other pathogens during co-culture. E. faecalis may encounter elevated levels of arginine in the GIT or the oral cavity, where arginine is used as a dental therapeutic. Little is known about how E. faecalis responds to growth in arginine in the absence of other bacteria. To address this, we used RNAseq and additional assays to measure growth, gene expression, and biofilm formation in E. faecalis OG1RF grown in arginine. We demonstrate that arginine decreases E. faecalis biofilm production and causes widespread differential expression of genes related to metabolism, quorum sensing, and polysaccharide synthesis. Growth in arginine also increases aggregation of E. faecalis and promotes decreased susceptibility to the antibiotics ampicillin and ceftriaxone. This work provides a platform for understanding of how the presence of arginine in biological niches affects E. faecalis physiology and virulence of surrounding microbes.

AhrC and Eep are biofilm infection-associated virulence factors in Enterococcus faecalis.

Enterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species.

In vivo and in vitro analyses of regulation of the pheromone-responsive prgQ promoter by the PrgX pheromone receptor protein.

Expression of conjugative transfer and virulence functions of the Enterococcus faecalis antibiotic resistance plasmid pCF10 is regulated by the interaction of the pheromone receptor protein PrgX with two DNA binding operator sites (XBS1 and XBS2) upstream from the transcription start site of the prgQ operon (encoding the pCF10 transfer machinery) and by posttranscriptional mechanisms. Occupancy of both binding sites by PrgX dimers results in repression of the prgQ promoter. Structural and genetic studies suggest that the peptide pheromone cCF10 functions by binding to PrgX and altering its oligomerization state, resulting in reduced occupancy of XBSs and increased prgQ transcription. The DNA binding activity of PrgX has additional indirect regulatory effects on prgQ transcript levels related to the position of the convergently transcribed prgX operon. This has complicated interpretation of previous analyses of the control of prgQ expression by PrgX. We report here the results of in vivo and in vitro experiments examining the direct effects of PrgX on transcription from the prgQ promoter, as well as quantitative correlation between the concentrations of XBSs, PrgX protein, and prgQ promoter activity in vivo. The results of electrophoretic mobility shift assays and quantitative analysis of prgQ transcription in vitro and in vivo support the predicted roles of the PrgX DNA binding sites in prgQ transcription regulation. The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10 in vivo.

Use of recombinase-based in vivo expression technology to characterize Enterococcus faecalis gene expression during infection identifies in vivo-expressed antisense RNAs and implicates the protease Eep in pathogenesis.

Enterococcus faecalis is a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades. E. faecalis must be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-based in vivo expression technology (RIVET) to identify promoters on the E. faecalis OG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putative in vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected in in vitro- and in vivo-grown cells, providing the first evidence of in vivo-expressed antisense RNAs in E. faecalis. Deletions in the in vivo-activated genes that encode glutamate 5-kinase (proB [EF0038]), the transcriptional regulator EbrA (ebrA [EF1809]), and the membrane metalloprotease Eep (eep [EF2380]) did not hinder biofilm formation in in vitro assays. In a rabbit model of endocarditis, the ΔebrA strain was fully virulent, the ΔproB strain was slightly attenuated, and the Δeep strain was severely attenuated. The Δeep virulence defect could be complemented by the expression of the wild-type gene in trans. Microscopic analysis of early Δeep biofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

Enterococcus faecalis colonizes and forms persistent biofilm microcolonies on undamaged endothelial surfaces in a rabbit endovascular infection model.

Infectious endocarditis (IE) is an uncommon disease with significant morbidity and mortality. The pathogenesis of IE has historically been described as a cascade of host-specific events beginning with endothelial damage and thrombus formation and followed by bacterial colonization of the nascent thrombus. Enterococcus faecalis is a Gram-positive commensal bacterial member of the gastrointestinal tract microbiota in most terrestrial animals and a leading cause of opportunistic biofilm-associated infections, including endocarditis. Here, we provide evidence that E. faecalis can colonize the endocardial surface without pre-existing damage and in the absence of thrombus formation in a rabbit endovascular infection model. Using previously described light and scanning electron microscopy techniques, we show that inoculation of a well-characterized E. faecalis lab strain in the marginal ear vein of New Zealand White rabbits resulted in rapid colonization of the endocardium throughout the heart within 4 days of administration. Unexpectedly, ultrastructural imaging revealed that the microcolonies were firmly attached directly to the endocardium in areas without morphological evidence of gross tissue damage. Further, the attached bacterial aggregates were not associated with significant cellular components of coagulation or host extracellular matrix damage repair (i.e. platelets). These results suggest that the canonical model of mechanical surface damage as a prerequisite for bacterial attachment to host sub-endothelial components is not required. Furthermore, these findings are consistent with a model of initial establishment of stable, endocarditis-associated E. faecalis biofilm microcolonies that may provide a reservoir for the eventual valvular infection characteristic of clinical endocarditis. The similarities between the E. faecalis colonization and biofilm morphologies seen in this rabbit endovascular infection model and our previously published murine gastrointestinal colonization model indicate that biofilm production and common host cell attachment factors are conserved in disparate mammalian hosts under both commensal and pathogenic contexts.

Direct evidence for control of the pheromone-inducible prgQ operon of Enterococcus faecalis plasmid pCF10 by a countertranscript-driven attenuation mechanism.

The mating response of Enterococcus faecalis cells carrying the conjugative plasmid pCF10 is controlled by multiple regulatory circuits. Initiation of transcription of the prgQ conjugation operon is controlled by the peptide receptor protein PrgX; binding of the pheromone peptide cCF10 to PrgX abolishes PrgX repression, while binding of the inhibitor peptide iCF10 enhances repression. The results of molecular analysis of prgQ transcripts and genetic studies suggested that the elongation of prgQ transcripts past a putative terminator (IRS1) may be controlled by the interaction of nascent prgQ mRNAs with a small antisense RNA (Anti-Q) encoded within prgQ. Direct evidence for interaction of these RNAs, as well as the resulting effects on readthrough of prgQ transcription, has been limited. Here we report the results of experiments that (i) determine the inherent termination properties of prgQ transcripts in the absence of Anti-Q; (ii) determine the direct effects of the interaction of Anti-Q with nascent prgQ transcripts in the absence of complicating effects of the PrgX protein; and (iii) begin to dissect the structural components involved in these interactions. The results confirm the existence of alternative terminating and antiterminating forms of nascent prgQ transcripts in vivo and demonstrate that the interaction of Anti-Q with these transcripts leads to termination via inhibition of antiterminator formation. In vitro transcription assays support the major results of the in vivo studies. The data support a model for Anti-Q function suggested from recent studies of these RNAs and their interactions in vitro (S. Shokeen, C. M. Johnson, T. J. Greenfield, D. A. Manias, G. M. Dunny, and K. E. Weaver, submitted for publication).

Structural analysis of the Anti-Q-Qs interaction: RNA-mediated regulation of E. faecalis plasmid pCF10 conjugation.

Conjugation of the E. faecalis plasmid pCF10 is triggered in response to peptide sex pheromone cCF10 produced by potential recipients. Regulation of this response is complex and multi-layered and includes a small regulatory RNA, Anti-Q that participates in a termination/antitermination decision controlling transcription of the conjugation structural genes. In this study, the secondary structure of the Anti-Q transcript and its sites of interaction with its target, Qs, were determined. The primary site of interaction occurred at a centrally-located loop whose sequence showed high variability in analogous molecules on other pheromone-responsive plasmids. This loop, designated the specificity loop, was demonstrated to be important but not sufficient for distinguishing between Qs molecules from pCF10 and another pheromone-responsive plasmid pAD1. A loop 5' from the specificity loop which carries a U-turn motif played no demonstrable role in Anti-Q-Qs interaction or regulation of the termination/antitermination decision. These results provide direct evidence for a critical role of Anti-Q-Qs interactions in posttranscriptional regulation of pCF10 transfer functions.

Multiple functional domains of Enterococcus faecalis aggregation substance Asc10 contribute to endocarditis virulence.

Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo.

Vancomycin-resistance gene cluster, vanC, in the gut microbiome of acute leukemia patients undergoing intensive chemotherapy.

Two recent reports suggested that the less common, less virulent enterococcal species, Enterococcus gallinarum and E. casseliflavus, with low-level vancomycin resistance due to chromosomally encoded vanC1 and vanC2/3, may influence host immunity. We reported that peri-transplant gut colonization with E. gallinarum and E. casseliflavus is associated with lower mortality after allogeneic hematopoietic cell transplantation (HCT). Because most acute leukemia patients undergoing HCT have received intensive chemotherapy (usually requiring prolonged hospitalization) for their underlying disease before HCT, we hypothesized that some may have acquired vanC-positive enterococci during chemotherapy. Therefore, we evaluated the presence of the vanC gene cluster using vanC1 and vanC2/3 qPCR in thrice-weekly collected stool samples from 20 acute leukemia patients undergoing intensive chemotherapy. We found that an unexpectedly large proportion of patients have detectable vanC1 and vanC2/3 (15% and 35%, respectively) in at least one stool sample. Comparing qPCR results with 16S rRNA gene sequencing results suggested that E. gallinarum may reach high abundances, potentially persisting into HCT and influencing transplant outcomes.

Early E. casseliflavus gut colonization and outcomes of allogeneic hematopoietic cell transplantation.

Gut dysbiosis has been associated with worse allogeneic hematopoietic cell transplantation (allo-HCT) outcomes. We reported an association between intrinsically vancomycin-resistant enterococci (iVRE: E. gallinarum and E. casseliflavus) gut colonization and lower post-transplant mortality. In this study, using an expanded cohort, we evaluated whether our previously observed association is species-specific. We included allo-HCT recipients with ≥1 positive rectal swab or stool culture for iVRE between days -14 and +14 of transplant. To investigate whether iVRE modulate the gut microbiota, we performed agar diffusion assays. To investigate whether iVRE differ in their ability to activate the aryl hydrocarbon receptor, we analyzed iVRE genomes for enzymes in the shikimate and tryptophan pathways. Sixty six (23 E. casseliflavus and 43 E. gallinarum) of the 908 allograft recipients (2011-2017) met our inclusion criteria. Overall survival was significantly higher in patients with E. casseliflavus (91% vs. 62% at 3 years, P = 0.04). In multivariable analysis, E. casseliflavus gut colonization was significantly associated with reduced all-cause mortality (hazard ratio 0.20, 95% confidence interval 0.04-0.91, P = 0.04). While agar assays were largely unremarkable, genome mining predicted that E. casseliflavus encodes a larger number of enzymes in the tryptophan metabolism pathway. In conclusion, E. casseliflavus gut colonization is associated with reduced post-HCT morality. Further research is needed to understand the mechanisms for this association.

Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF.

Enterococcus faecalis is a common commensal bacterium in animal gastrointestinal (GI) tracts and a leading cause of opportunistic infections of humans in the modern health care setting. E. faecalis OG1RF is a plasmid-free strain that contains few mobile elements yet retains the robust survival characteristics, intrinsic antibiotic resistance, and virulence traits characteristic of most E. faecalis genotypes. To facilitate interrogation of the core enterococcal genetic determinants for competitive fitness in the GI tract, biofilm formation, intrinsic antimicrobial resistance, and survival in the environment, we generated an arrayed, sequence-defined set of chromosomal transposon insertions in OG1RF. We used an orthogonal pooling strategy in conjunction with Illumina sequencing to identify a set of mutants with unique, single Himar-based transposon insertions. The mutants contained insertions in 1,926 of 2,651 (72.6%) annotated open reading frames and in the majority of hypothetical protein-encoding genes and intergenic regions greater than 100 bp in length, which could encode small RNAs. As proof of principle of the usefulness of this arrayed transposon library, we created a minimal input pool containing 6,829 mutants chosen for maximal genomic coverage and used an approach that we term SMarT (sequence-defined mariner technology) transposon sequencing (TnSeq) to identify numerous genetic determinants of bile resistance in E. faecalis OG1RF. These included several genes previously associated with bile acid resistance as well as new loci. Our arrayed library allows functional screening of a large percentage of the genome with a relatively small number of mutants, reducing potential effects of bottlenecking, and enables immediate recovery of mutants following competitions. IMPORTANCE The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest.

Enterococcus faecalis readily colonizes the entire gastrointestinal tract and forms biofilms in a germ-free mouse model.

The mammalian gastrointestinal (GI) tract is a complex organ system with a twist-a significant portion of its composition is a community of microbial symbionts. The microbiota plays an increasingly appreciated role in many clinically-relevant conditions. It is important to understand the details of biofilm development in the GI tract since bacteria in this state not only use biofilms to improve colonization, biofilm bacteria often exhibit high levels of resistance to common, clinically relevant antibacterial drugs. Here we examine the initial colonization of the germ-free murine GI tract by Enterococcus faecalis-one of the first bacterial colonizers of the naïve mammalian gut. We demonstrate strong morphological similarities to our previous in vitro E. faecalis biofilm microcolony architecture using 3 complementary imaging techniques: conventional tissue Gram stain, immunofluorescent imaging (IFM) of constitutive fluorescent protein reporter expression, and low-voltage scanning electron microscopy (LV-SEM). E. faecalis biofilm microcolonies were readily identifiable throughout the entire lower GI tract, from the duodenum to the colon. Notably, biofilm development appeared to occur as discrete microcolonies directly attached to the epithelial surface rather than confluent sheets of cells throughout the GI tract even in the presence of high (>109) fecal bacterial loads. An in vivo competition experiment using a pool of 11 select E. faecalis mutant strains containing sequence-defined transposon insertions showed the potential of this model to identify genetic factors involved in E. faecalis colonization of the murine GI tract.

Analysis of the amino acid sequence specificity determinants of the enterococcal cCF10 sex pheromone in interactions with the pheromone-sensing machinery.

The level of expression of conjugation genes in Enterococcus faecalis strains carrying the pheromone-responsive transferable plasmid pCF10 is determined by the ratio in the culture medium of two types of signaling peptides, a pheromone (cCF10) and an inhibitor (iCF10). Recent data have demonstrated that both peptides target the cytoplasmic receptor protein PrgX. However, the relative importance of the interaction of these peptides with the pCF10 protein PrgZ (which enhances import of cCF10) versus PrgX is not fully understood, and there is relatively little information about specific amino acid sequence determinants affecting the functional interactions of cCF10 with these proteins in vivo. To address these issues, we used a pheromone-inducible reporter gene system where various combinations of PrgX and PrgZ could be expressed in an isogenic host background to examine the biological activities of cCF10, iCF10, and variants of cCF10 isolated in a genetic screen. The results suggest that most of the amino acid sequence determinants of cCF10 pheromone activity affect interactions between the peptide and PrgX, although some sequence variants that affected peptide/PrgZ interactions were also identified. The results provide functional data to complement ongoing structural studies of PrgX and increase our understanding of the functional interactions of cCF10 and iCF10 with the pheromone-sensing machinery of pCF10.

Genetic characterization of the conjugative DNA processing system of enterococcal plasmid pCF10.

Conjugation is a major contributor to lateral gene transfer in bacteria, and pheromone-inducible conjugation systems in Enterococcus faecalis play an important role in the dissemination of antibiotic resistance and virulence in enterococci and related bacteria. We have genetically dissected the determinants of DNA processing of the enterococcal conjugative plasmid pCF10. Insertional inactivation of a predicted relaxase gene pcfG, via insertion of a splicing-deficient group II intron, severely reduced pCF10 transfer. Restoration of intron splicing ability by genetic complementation restored conjugation. The pCF10 origin of transfer (oriT) was localized to a 40-nucleotide sequence within a non-coding region with sequence similarity to origins of transfer of several other plasmids in gram positive bacteria. Deletion of the oriT reduced pCF10 transfer by more than five orders of magnitude without affecting pCF10-dependent mobilization of co-resident oriT-containing plasmids. Although the host range for pCF10 replication is limited to enterococci, we found that the pCF10 conjugation system promotes mobilization of oriT-containing plasmids to multiple bacterial genera. Therefore, this transfer system may have applications for gene delivery to a variety of poorly-transformed bacteria.