Transfer of the pheromone-inducible plasmid pCF10 among Enterococcus faecalis microorganisms colonizing the intestine of mini-pigs.

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10(6) and 10(7) CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10(6) CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10(3) and 10(4) CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.     

Effect of pheromone induction on transfer of the Enterococcus faecalis plasmid pCF10 in intestinal mucus ex vivo

The effect of synthetic sex pheromone on pheromone-inducible conjugation between the isogenic Enterococcus faecalis strains OG1RF and OG1SS was investigated in (i) Todd-Hewitt broth medium and (ii) intestinal mucus isolated from germ-free rats. In broth, the presence of synthetic pheromone cCF10 had no detectable effect on the transfer kinetics observed for the tetracycline resistance encoding plasmid pCF10. In mucus, presence of the same pheromone significantly increased the transfer efficiency observed during the first 2 h of conjugation, while the effect was less pronounced later in the experiment. We suggest that due to differences in diffusion rates and medium-binding of the pheromones, the effect of the synthetic cCF10 was immediately dominated by the effect of pheromones produced by the recipient E. faecalis strain in broth, while this happened later in mucus.     

The prgQ gene of the Enterococcus faecalis tetracycline resistance plasmid pCF10 encodes a peptide inhibitor, iCF10.

Conjugative transfer of the Enterococcus faecalis tetracycline resistance plasmid pCF10 is stimulated by a peptide pheromone, cCF10. Once a recipient strain acquires pCF10 and thus becomes a pheromone-responsive donor, cCF10 activity is no longer detected in culture filtrates. Here we show that pCF10 encodes a peptide inhibitor, iCF10, secreted by donor cells; this inhibitor antagonizes the cCF10 activity in culture filtrates. In order to detect and quantitate iCF10, we developed a reverse-phase high-performance liquid chromatography assay in which the inhibitor peptide elutes separately from the pheromone; this type of assay enabled us to determine that lack of pheromone activity in donor culture filtrates was due to secretion of a mixture of iCF10 and cCF10, rather than abolition of cCF10 secretion. The gene encoding iCF10, prgQ, is located on the EcoRI-C fragment of pCF10. The open reading frame comprising the prgQ gene encodes a 23-amino-acid precursor that resembles a signal peptide. This precursor is cleaved to the mature heptapeptide iCF10 during the secretion process.     

Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.     

Role of the pheromone-inducible surface protein Asc10 in mating aggregate formation and conjugal transfer of the Enterococcus faecalis plasmid pCF10.

The high transfer frequency of pheromone-inducible conjugative plasmids of Enterococcus faecalis in liquid culture is due in part to the formation of mating aggregates. These aggregates result from the interaction of two surface components, aggregation substance (AS), which is plasmid encoded, and the chromosomally encoded binding substance (BS). In the accompanying paper (S.-M. Kao, S. B. Olmsted, A. S. Viksnins, J.C. Gallo, G. M. Dunny, J. Bacteriol, 173:7650-7664, 1991), the sequence of the prgB gene encoding the AS molecule (Asc10) produced by pheromone-induced cells carrying plasmid pCF10 is presented. Here we report the results of genetic and immunological experiments which define the role of Asc10 in aggregation and plasmid transfer. These data indicate expression of AS on the surface of an E. faecalis cell and its binding to BS expressed on a second cell are required for the formation of a mating pair and the efficient transfer of pCF10 in liquid matings. However, the orientation of the receptors was not critical for transfer; ie., AS expressed on recipient cells could facilitate plasmid transfer via binding to BS on the donor. Our results suggest that additional (as yet unidentified) products are involved in forming the channel that ultimately serves to transfer the DNA, with AS-BS binding serving primarily to generate the initial attachment between cells. The putative prgC gene product, identified by DNA sequencing (data presented in the accompanying paper), could be involved in transfer events occurring subsequent to aggregation.     

Specific control of endogenous cCF10 pheromone by a conserved domain of the pCF10-encoded regulatory protein PrgY in Enterococcus faecalis

Conjugative transfer of Enterococcus faecalis plasmid pCF10 is induced by the heptapeptide pheromone cCF10. cCF10 produced by plasmid-free recipient cells is detected by pCF10-containing donor cells, which respond by induction of plasmid-encoded transfer functions. The pCF10-encoded membrane protein PrgY is essential to prevent donor cells from responding to endogenously produced pheromone while maintaining the ability to respond to pheromone from an exogenous source; this function has not been identified in any nonenterococcal prokaryotic signaling system. PrgY specifically inhibited endogenous cCF10 and cPD1 (a pheromone that induces transfer of closely related plasmid pPD1) but not cAD1 (which is specific for less-related plasmid pAD1). Ectopic expression of PrgY in plasmid-free recipient cells reduced pheromone activity in culture supernatants and reduced the ability of these cells to acquire pCF10 by conjugation but did not have any effect on the interaction of these cells with exogenously supplied cCF10. The cloned prgY gene could complement a pCF10 prgY null mutation, and complementation was used to identify point mutations impairing PrgY function. Such mutations also abolished the inhibitory effect of PrgY expression in recipients on pheromone production and on acquisition of pCF10. Most randomly generated point mutations identified in the genetic screen mapped to a predicted extracellular domain in the N terminus of PrgY that is conserved in a newly identified family of related proteins from disparate species including Borrelia burgdorferi, Archaeoglobus fulgidus, Arabidopsis thaliana, and Homo sapiens. The combined genetic and physiological data suggest that PrgY may sequester or inactivate cCF10 as it is released from the membrane.     

Enterococcus faecalis PrgJ, a VirB4-like ATPase, mediates pCF10 conjugative transfer through substrate binding

The Enterococcus faecalis prg and pcf genes of plasmid pCF10 encode a type IV secretion system (T4SS) required for conjugative transfer. PrgJ is a member of the VirB4 family of ATPases that are universally associated with T4SSs. Here, we report that purified PrgJ dimers displayed ATP binding and hydrolysis activities. A PrgJ nucleoside triphosphate (NTP) binding site mutation (K471E) slightly diminished ATP binding but abolished ATP hydrolysis in vitro and blocked pCF10 transfer in vivo. As shown with affinity pulldown assays, PrgJ and the K471E mutant protein interacted with the substrate receptor PcfC and with relaxase PcfG and accessory factor PcfF, which together form the relaxosome at the oriT sequence to initiate plasmid processing. The purified PrgJ and K471E proteins also bound single- and double-stranded DNA substrates without sequence specificity in vitro, and both PrgJ derivatives bound pCF10 in vivo by a mechanism dependent on an intact oriT sequence and cosynthesis of PcfC, PcfF, and PcfG, as shown by a formaldehyde-cross-linking assay. Our findings support a model in which the PcfC receptor coordinates with the PrgJ ATPase to drive early steps of pCF10 processing/transfer: (i) PcfC first binds the pCF10 relaxosome through contacts with PcfF, PcfG, and DNA; (ii) PcfC delivers the plasmid substrate to PrgJ; and (iii) PrgJ catalyzes substrate transfer to the membrane translocase. Substrate engagement with a VirB4-like subunit has not been previously described; consequently, our studies point to a novel function for these signature T4SS ATPases in mediating early steps of type IV secretion.     

Specificity determinants of conjugative DNA processing in the Enterococcus faecalis plasmid pCF10 and the Lactococcus lactis plasmid pRS01

The DNA-processing region of the Enterococcus faecalis pheromone-responsive plasmid pCF10 is highly similar to that of the otherwise unrelated plasmid pRS01 from Lactococcus lactis. A transfer-proficient pRS01 derivative was unable to mobilize plasmids containing the pCF10 origin of transfer, oriT. In contrast, pRS01 oriT-containing plasmids could be mobilized by pCF10 at a low frequency. Relaxases PcfG and LtrB were both capable of binding to single-stranded oriT DNAs; LtrB was highly specific for its cognate oriT, whereas PcfG could recognize both pCF10 and pRS01 oriT. However, pcfG was unable to complement an ltrB insertion mutation. Genetic analysis showed that pcfF of pCF10 and ltrF of pRS01 are also essential for plasmid transfer. Purified PcfF and LtrF possess double-stranded DNA binding activities for the inverted repeat within either oriT sequence. PcfG and LtrB were recruited into their cognate F-oriT DNA complex through direct interactions with their cognate accessory protein. PcfG also could interact with LtrF when pCF10 oriT was present. In vivo cross-complementation analysis showed that ltrF partially restored the pCF10DeltapcfF mutant transfer ability when provided in trans, whereas pcfF failed to complement an ltrF mutation. Specificity of conjugative DNA processing in these plasmids involves both DNA-protein and protein-protein interactions.     

Genetic diversity among Enterococcus faecalis

Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.     

牦牛肠道粪肠球菌的分离和鉴定

试验为获得1株与牦牛肠道共生的粪肠球菌,通过对健康牦牛粪样中的肠球菌进行分离培养、革兰染色、生化试验、16S rRNA基因测序比对等生物学鉴定,初步证实该分离菌株为粪肠球菌。此试验为进一步分析牦牛肠道菌群结构,探索牦牛耐饥、耐渴、耐粗饲、对高山草原极强适应性与其肠道菌群的关系,进一步研究与牦牛共生的粪肠球菌的生物学特性、生理特性、对宿主危害性等提供一些实验依据。     

Cloning and characterization of a region of the Enterococcus faecalis conjugative plasmid, pCF10, encoding a sex pheromone-binding function.

In order to investigate the mechanism by which peptide sex pheromones induce expression of the conjugation functions of certain Enterococcus faecalis plasmids, a biological assay was developed to measure the ability of cells carrying the conjugative plasmid pCF10 to bind the sex pheromone cCF10. The data indicated that pCF10 endows its host E. faecalis cell with the ability to specifically remove (apparently by irreversible binding) cCF10 activity from culture medium. The pCF10 DNA encoding this ability was localized to a 3.4-kb segment within a region involved in negative control of expression of conjugal transfer functions. This segment also encoded ability to bind the pheromone inhibitor peptide iCF10. DNA sequencing revealed three open reading frames, which have been denoted prgW (pheromone responsive gene W), prgZ, and prgY. The deduced product of prgW resembled regulatory proteins from other bacteria and eucaryotes, with a very high degree of identity within a putative DNA-binding domain. The prgY gene actually extended into an adjacent region of pCF10 and could encode a protein with significant similarity to a protein called TraB, believed to be involved in shutdown of pheromone cAD1 production by cells carrying the pheromone-inducible hemolysin plasmid pAD1, according to F.Y. An and D.B. Clewell (Abstr. Gen. Meet. Am. Soc. Microbiol. 1992, H70, 1992). The prgZ gene product showed significant relatedness to binding proteins encoded by oligopeptide permease (opp) operons in gram-positive and gram-negative bacteria and is highly similar to a pAD1-encoded protein, TraC, which is believed to mediate sex pheromone cAD1 binding (K. Tanimoto, F. Y. An, and D. B. Clewell, submitted for publication). A Tn5 insertion into prgZ abolished cCF10 binding ability.     

High-resolution visualization by field emission scanning electron microscopy of Enterococcus faecalis surface proteins encoded by the pheromone-inducible conjugative plasmid pCF10.

Enterococcus faecalis can acquire antibiotic resistance and virulence genes by transfer of pheromone-inducible conjugative plasmids such as pCF10, which encodes tetracycline resistance. Two pCF10-encoded cell surface proteins, Sec10 and Asc10, have been previously shown to play an important role in the transfer of this plasmid. We used high-resolution, field emission scanning electron microscopy to visualize these proteins on the surfaces of a series of isogenic strains of E. faecalis. Immunogold labeling, using both 6- and 12-nm colloidal gold, unambiguously demonstrated the expression and distribution of Sec10 and Asc10 on the surface of the E. faecalis cells. On unlabeled E. faecalis cells which expressed either Sec10 or Asc10, the former appeared to be more readily detected. Immunogold labeling of E. faecalis cells expressing both Asc10 and Sec10 clearly demonstrated the abundance and intermixing of both proteins on the cell surface except at septal regions. Sec10 was observed to be distributed over the cell surface. At regions of cell-cell contact, fine strands representing Asc10 were observed directly attaching adjacent cells to one another.     

Peptide pheromone-induced transfer of plasmid pCF10 in Enterococcus faecalis: probing the genetic and molecular basis for specificity of the pheromone response

The tetracycline resistance plasmid pCF10 represents a class of unique mobile genetic elements of the bacterial genus Enterococcus, whose conjugative transfer functions are inducible by peptide sex pheromones excreted by potential recipient cells. These plasmids play a significant role in the dissemination of virulence and antibiotic resistance genes among the enterococci, which have become major nosocomial pathogens. Pheromone response by plasmid-carrying donor cells involves specific import of the peptide signal molecule, and subsequent interaction of the signal with one or more intracellular regulatory gene products. The pheromones are chromosomally encoded hydrophobic octa- or hepta-peptides, and different families of homologous plasmids encode the ability to respond to each pheromone. Among the four pheromone-responsive plasmids that have been characterized in some detail, there is considerable conservation in the genes encoding pheromone sensing and regulatory functions, and the peptides themselves show considerable similarity. In spite of this, there is extremely high specificity of response to each peptide, with virtually no "cross-induction" of transfer of non-cognate pheromone plasmids by the pheromones. This communication reviews the evidence for this specificity and discusses current molecular and genetic approaches to defining the basis for specificity.     

The peptide pheromone-inducible conjugation system of Enterococcus faecalis plasmid pCF10: cell-cell signalling, gene transfer, complexity and evolution

Expression of a large set of gene products required for conjugative transfer of the antibiotic resistance plasmid pCF10 is controlled by cell-cell communication between plasmid-free recipient cells and plasmid-carrying donor cells using a peptide mating pheromone cCF10. Most of the recent experimental analysis of this system has focused on the molecular events involved in initiation of the pheromone response in the donor cells, and on the mechanisms by which the donor cells control self-induction by endogenously produced pheromone. Recently, studies of the molecular machinery of conjugation encoded by the pheromone-inducible genes have been initiated. In addition, the system may serve as a useful bacterial model for addressing the evolution of biological complexity.     

pS86, A New Theta-Replicating Plasmid from Enterococcus faecalis

The complete nucleotide sequence of the small (5149 bp) and cryptic plasmid pS86 from Enterococcus faecalis ssp. faecalis S-86 has been determined. Sequence analysis revealed six putative open reading frames (ORFs) encoding polypeptides of 28.3, 11.5, 8.4, 65.1, 7.3, and 11.96 kDa each. Based on sequence similarity, two cassettes have been identified in pS86: ORF1 codes for the replication initiation protein (Rep); ORF4 codes for a putative mobilization protein that shows similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. No function could be assigned to the other putative ORFs found. According to our results, pS86 plasmid could use a theta-mode of replication, similar to the recently described theta-type replicons from pUCL287 (Tetragenococcus halophila) and pLA1 or pLA105 (Lactobacillus acidophilus) plasmids. Received: 24 November 1999 / Accepted: 26 April 2000     

Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin

Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.