Plasmid pAD1 Research Articles

High Incidence of Multiple-Drug-Resistant Pheromone-Responsive Plasmids and Transmissions of VanA-Type Vancomycin-Resistant Enterococcus faecalis between Livestock and Humans in Taiwan.

A total of seventy VanA-type vancomycin-resistant enterococci (VRE) isolates obtained in Taiwan in the early 2000s were retrospectively characterized. Forty isolates were obtained from human patients and thirty from livestock. Of these VRE isolates, twenty-three (57.5%) of the human VRE and thirty (100%) of the livestock VRE were Enterococcus faecalis, and the remaining seventeen (42.5%) of the human VRE were E. faecium. Of the 53 E. faecalis isolates, twenty-two (96%) of the human VRE and thirty (100%) of the livestock VRE exhibited a high level of resistance to vancomycin and sensitivity to teicoplanin. They also had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. The vancomycin resistance of all of the 22 human isolates, and 20 of the 30 livestock isolates, transferred to E. faecalis FA2-2 at a frequency of 10-5 to 10-3 per donor cell in broth. Each of the transconjugants responded to E. faecalis pheromone (i.e., E. faecalis FA2-2 culture filtrate), indicating that the conjugative plasmids were pheromone-responsive plasmids. Three of the conjugative plasmids originated from human isolates, and five plasmids from livestock isolates were corresponded and classified as type A plasmid. Two plasmids originated from human isolates and six plasmids from livestock isolates were corresponded and classified as type B plasmid. E. faecalis FA2-2 containing either the type A or type B plasmid responded to the synthetic pheromone cAD1. The type A and type B plasmids transferred between E. faecalis FA2-2 and JH2SS at a frequency of about 10-2 per donor cell and conferred vancomycin, bacitracin, and erythromycin resistances. The complete DNA sequence of the representative type A plasmid pTW9 (85,068 bp) showed that the plasmid carried a Tn1546-like element encoding vanA-type resistance, erythromycin resistance (ermB), and bacitracin resistance (bcrABDR). The plasmid contained the regulatory region found in the pheromone-responsive plasmid and encoded the genes traA, traD and iad1, which are the key negative regulatory elements, and traE1, a key positive regulator of plasmid pAD1, indicating that plasmid pTW9 was pAD1-type pheromone-responsive plasmid. PFGE analysis of SmaI-digested chromosomal DNAs showed that several E. faecalis strains harboring an identical type A pheromone-responsive plasmid were indistinguishable, and that these were identified both in human and livestock isolates, indicating the transmissions of the VRE strains between livestock and humans. These data showed that the multiple-drug-resistant pheromone-responsive conjugative plasmids have been widely spread in both human and livestock VRE, and there was high potential for transfers of VRE from food animals to humans in Taiwan in the early 2000s.

Antagonistic Donor Density Effect Conserved in Multiple Enterococcal Conjugative Plasmids.

Enterococcus faecalis, a common causative agent of hospital-acquired infections, is resistant to many known antibiotics. Its ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious concern for public health. In some cases, induction of transfer of E. faecalis plasmids results from peptide pheromones produced by plasmid-free recipient cells, which are sensed by the plasmid-bearing donor cells. These plasmids generally encode an inhibitory peptide that competes with the pheromone and suppresses self-induction of donors. We recently demonstrated that the inhibitor peptide encoded on plasmid pCF10 is part of a unique quorum-sensing system in which it functions as a "self-sensing signal," reducing the response to the pheromone in a density-dependent fashion. Based on the similarities between regulatory features controlling conjugation in pAD1 and pAM373 and those controlling conjugation in pCF10, we hypothesized that these plasmids are likely to exhibit similar quorum-sensing behaviors. Experimental findings indicate that for both pAD1 and pAM373, high donor densities indeed resulted in decreased induction of the conjugation operon and reduced conjugation frequencies. This effect was restored by the addition of exogenous inhibitor, confirming that the inhibitor serves as an indicator for donor density. Donor density also affects cross-species conjugative plasmid transfer. Based on our experimental results, we propose models for induction and shutdown of the conjugation operon in pAD1 and pAM373. Enterococcus faecalis is a leading cause of hospital-acquired infections. Its ability to transfer antibiotic resistance and virulence determinants by sharing its genetic material with other bacteria through direct cell-cell contact via conjugation poses a serious threat. Two antagonistic signaling peptides control the transfer of plasmids pAD1 and pAM373: a peptide pheromone produced by plasmid-free recipients triggers the conjugative transfer in plasmid-containing donors, and an inhibitor peptide encoded on the plasmid and produced by donor cells serves to modulate the donor response in accordance with the relative abundance of donors and recipients. We demonstrate that high donor density reduces the conjugation frequency of both of these plasmids, which is a consequence of increased inhibitor concentration in high-donor-density cultures. While most antibiotic strategies end up selecting resistant strains and disrupting the community balance, manipulating bacterial signaling mechanisms can serve as an alternate strategy to prevent the spread of antibiotic resistance.

The Type I toxin–antitoxin par locus from Enterococcus faecalis plasmid pAD1: RNA regulation by both cis- and trans-acting elements

The pAD1 par determinant was the first Type I toxin–antitoxin system identified in Gram-positive bacteria and has recently been shown to be the prototype of a family of loci that is widespread in these organisms. All family members have (i) convergently transcribed toxin message and regulatory RNAs, (ii) three non-contiguous complementary regions for potential interaction, and (iii) intramolecular structures within the toxin message that modulate translation and transcript stability. Therefore, the detailed information available on the par locus provides a paradigm for studying the function and mechanism of regulation of the related loci.

Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases

Plasmid pAD1 is a 60-kb conjugative element commonly found in clinical isolates of Enterococcus faecalis. The relaxase TraX and the primary origin of transfer oriT2 are located close to each other and have been shown to be essential for conjugation. The oriT2 site contains a large inverted repeat (where the nic site is located) adjacent to a series of short direct repeats. TraX does not show any of the typical relaxase sequence motifs but is the prototype of a unique family of relaxases (MOBC). The present study focuses on the genetic, biochemical, and structural analysis of TraX, whose 3D structure could be predicted by protein threading. The structure consists of two domains: (i) an N-terminal domain sharing the topology of the DNA binding domain of the MarR family of transcriptional regulators and (ii) a C-terminal catalytic domain related to the PD-(D/E)XK family of restriction endonucleases. Alignment of MOBC relaxase amino acid sequences pointed to several conserved polar amino acid residues (E28, D152, E170, E172, K176, R180, Y181, and Y203) that were mutated to alanine. Functional analysis of these mutants (in vivo DNA transfer and cleavage assays) revealed the importance of these residues for relaxase activity and suggests Y181 as a potential catalytic residue similarly to His-hydrophobe-His relaxases. We also show that TraX binds specifically to dsDNA containing the oriT2 direct repeat sequences, confirming their role in transfer specificity. The results provide insights into the catalytic mechanism of MOBC relaxases, which differs radically from that of His-hydrophobe-His relaxases.

Solution Structure and Membrane Binding of the Toxin Fst of theparAddiction Module

The par toxin−antitoxin system is required for the stable inheritance of the plasmid pAD1 in its native host Enterococcus faecalis. It codes for the toxin Fst and a small antisense RNA which inhibits translation of toxin mRNA, and it is the only known antisense regulated toxin−antitoxin system in Gram-positive bacteria. This study presents the structure of the par toxin Fst, the first atomic resolution structure of a component of an antisense regulated toxin−antitoxin system. The mode of membrane binding was determined by relaxation enhancements in a paramagnetic environment and molecular dynamics simulation. Fst forms a membrane-binding α-helix in the N-terminal part and contains an intrinsically disordered region near the C-terminus. It binds in a transmembrane orientation with the C-terminus likely pointing toward the cytosol. Membrane-bound, α-helical peptides are frequently found in higher organisms as components of the innate immune system. Despite similarities to these antimicrobial peptides, Fst shows neither hemolytic nor antimicrobial activity when applied externally to a series of bacteria, fungal cells, and erythrocytes. Moreover, its charge distribution, orientation in the membrane, and structure distinguish it from antimicrobial peptides.

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.

Identification and characterization of a family of toxin–antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Gram-positive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMS1 was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Gram-positive species.

An Intramolecular Upstream Helix Ensures the Stability of a Toxin-Encoding RNA inEnterococcus faecalis

The par stability determinant is required for the stable inheritance of the plasmid pAD1 in its native host, Enterococcus faecalis. It is the only antisense RNA-regulated addiction module identified to date in gram-positive bacteria. It encodes two small, convergently transcribed RNAs, RNA I and RNA II. RNA I encodes the Fst toxin and RNA II acts as the antitoxin by interacting with RNA I posttranscriptionally. As the toxin-encoding component of the system, it is important that RNA I is more stable than RNA II. This study reveals that a helix sequestering the 5' end of RNA I plays a crucial role in maintaining the stability of the RNA I. An adjacent structure previously determined to regulate Fst translation was not required to enhance stability. Results indicated that endoribonuclease J2 contributes significantly to the degradation of a mutant disrupting the upstream helix (UH) of RNA I in Bacillus subtilis. Finally, it was shown that interaction with RNA II stabilized the UH mutant of RNA I.

Isolation of VanB-Type Enterococcus faecalis Strains from Nosocomial Infections: First Report of the Isolation and Identification of the Pheromone-Responsive Plasmids pMG2200, Encoding VanB-Type Vancomycin Resistance and a Bac41-Type Bacteriocin, and pMG2201, Encoding Erythromycin Resistance and Cytolysin (Hly/Bac)

Eighteen identical VanB-type Enterococcus faecalis isolates that were obtained from different hospitalized patients were examined for their drug resistance and plasmid DNAs. Of the 18 strains, 12 strains exhibited resistance to erythromycin (Em), gentamicin (Gm), kanamycin (Km), tetracycline (Tc), and vancomycin (Van) and produced cytolysin (Hly/Bac) and a bacteriocin (Bac) active against E. faecalis strains. Another six of the strains exhibited resistance to Gm, Km, Tc, and Van and produced a bacteriocin. Em and Van resistance was transferred individually to E. faecalis FA2-2 strains at a frequency of about 10(-4) per donor cell by broth mating. The Em-resistant transconjugants and the Van-resistant transconjugants harbored a 65.7-kbp plasmid and a 106-kbp plasmid, respectively. The 106-kbp and 65.7-kbp plasmids isolated from the representative E. faecalis NKH15 strains were designated pMG2200 and pMG2201, respectively. pMG2200 conferred vancomycin resistance and bacteriocin activity on the host strain and responded to the synthetic pheromone cCF10 for pCF10, while pMG2201 conferred erythromycin resistance and cytolysin activity on its host strain and responded to the synthetic pheromone cAD1 for pAD1. The complete DNA sequence of pMG2200 (106,527 bp) showed that the plasmid carried a Tn1549-like element encoding vanB2-type resistance and the Bac41-like bacteriocin genes of pheromone-responsive plasmid pYI14. The plasmid contained the regulatory region found in pheromone-responsive plasmids and encoded the genes prgX and prgQ, which are the key negative regulatory elements for plasmid pCF10. pMG2200 also encoded TraE1, a key positive regulator of plasmid pAD1, indicating that pMG2200 is a naturally occurring chimeric plasmid that has a resulting prgX-prgQ-traE1 genetic organization in the regulatory region of the pheromone response. The functional oriT region and the putative relaxase gene of pMG2200 were identified and found to differ from those of pCF10 and pAD1. The putative relaxase of pMG2200 was classified as a member of the MOB(MG) family, which is found in pheromone-independent plasmid pHTbeta of the pMG1-like plasmids. This is the first report of the isolation and characterization of a pheromone-responsive highly conjugative plasmid encoding vanB resistance.

Translational Regulation by an Intramolecular Stem-Loop Is Required for Intermolecular RNA Regulation of theparAddiction Module

The par stability determinant of Enterococcus faecalis plasmid pAD1 is the only antisense RNA-regulated addiction module identified to date in gram-positive bacteria. par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, that function as the toxin (Fst)-encoding and antitoxin components, respectively. Previous work showed that structures at the 5' end of RNA I are important in regulating its translation. The work presented here reveals that a stem-loop sequestering the Fst ribosome binding site is required for translational repression but a helix sequestering the 5' end of RNA I is not. Furthermore, disruption of the stem-loop prevented RNA II-mediated repression of Fst translation in vivo. Finally, although Fst-encoding wild-type RNA I is not toxic in Escherichia coli, mutations affecting stem-loop stability resulted in toxicity in this host, presumably due to increased translation.

Characterization of an Active Partition System for theEnterococcus faecalisPheromone-Responding Plasmid pAD1

Enterococcus faecalis plasmid pAD1 is a 60-kb conjugative, low-copy-number plasmid that encodes a mating response to the peptide sex pheromone cAD1 and a cytolytic exotoxin that contributes to virulence. Although aspects of conjugation have been studied extensively, relatively little is known about the control of pAD1 maintenance. Previous work on pAD1 identified a 5-kb region of DNA sufficient to support replication, copy control, and stable inheritance (K. E. Weaver, D. B. Clewell, and F. An, J. Bacteriol. 175:1900-1909, 1993), and recently, the pAD1 replication initiator (RepA) and the origin of vegetative replication (oriV) were characterized (M. V. Francia, S. Fujimoto, P. Tille, K. E. Weaver, and D. B. Clewell, J. Bacteriol. 186:5003-5016, 2004). The present study focuses on the adjacent determinants repB and repC, as well as a group of 25 8-bp direct repeats (iterons with the consensus sequence TAGTARRR) located between the divergently transcribed repA and repB. Through mutagenesis and trans-complementation experiments, RepB (a 33-kDa protein, a member of the ParA superfamily of ATPases) and RepC (a protein of 14.4 kDa) were shown to be required for maximal stabilization. Both were active in trans. The iteron region was shown to act as the pAD1 centromere-like site. Purified RepC was shown by DNA mobility shift and DNase I footprinting analyses to interact in a sequence-specific manner with the iteron repeats upstream of the repBC locus. The binding of RepC to the iteron region was shown to be modified by RepB in the presence of ATP via a possible interaction with the RepC-iteron complex. RepB did not bind to the iteron region in the absence of RepC.

Pheromone-Responsive Conjugative Vancomycin Resistance Plasmids in Enterococcus faecalis Isolates from Humans and Chicken Feces

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.

Why does Staphylococcus aureus secrete an Enterococcus faecalis-specific pheromone?

The long known fact that Staphylococcus aureus can secrete a peptide (staph-cAM373) which acts as a sex pheromone for strains of E. faecalis carrying the sex pheromone plasmid pAM373 was studied in detail. 85 of 100 independent isolates of S. aureus produced an activity inducing the pAM373 encoded adhesin resulting in a clumping reaction of E. faecalis (i.e. secreted staph-cAM373), but not a single one of 100 independent isolates of coagulase-negative Staphylococci possessed this activity. The production of peptides acting as sex pheromone for E. faecalis was observed only for sex pheromone plasmid pAM373, but not for sex pheromone plasmids pAD1 and pPD1. It was shown that the amount of staph-pAM373 produced during growth paralleled cell mass, therefore this peptide should not be used as a cell density indicator by S. aureus (i.e. has no quorum sensing function). The potential function of staph-cAM373 to trigger gene transfer from Escherichia faecalis to S. aureus is discussed.

Specific Control of Endogenous cCF10 Pheromone by a Conserved Domain of the pCF10-Encoded Regulatory Protein PrgY inEnterococcus 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.

Replication of Enterococcusfaecalis Pheromone-Responding Plasmid pAD1: Location ofthe Minimal Replicon and oriV Site and RepAInvolvement in Initiation of Replication

Replication of <i>Enterococcusfaecalis</i> Pheromone-Responding Plasmid pAD1: Location ofthe Minimal Replicon and <i>oriV</i> Site and RepAInvolvement in Initiation of Replication

Drug resistance of Enterococcus faecium clinical isolates and the conjugative transfer of gentamicin and erythromycin resistance traits

Drug resistance and the transferability of resistance were examined in 218 Enterococcus faecium clinical isolates obtained from in-patients of a Japanese university hospital between 1990 and 1999. One hundred and sixty one isolates (73.9%) were drug-resistant and 127 (58.2%) isolates were resistant to two or more drugs. Vancomycin resistant E. faecium (VRE) was not isolated. The transferability of drug-resistance to an E. faecium strain was examined by broth or filter mating. Six (12.5%) of the 48 gentamicin resistance traits, and fifty (50%) of the 101 erythromycin resistance traits were transferred by filter mating. The gentamicin resistance traits of five isolates and the erythromycin resistance traits of four isolates were transferred to the recipient strains by both broth mating and filter mating at a frequency of about 10 −6 and 10 −5 per donor cell, respectively. The five gentamicin resistant strains were shown to harbor pMG1-like plasmids on the basis of their Southern hybridization with pMG1 (65.1 kbp, Gm r), which transfers efficiently between enterococci by broth mating. Each of the four erythromycin resistant transconjugants obtained by broth mating harbored a large conjugative plasmid (more than 100 kbp). The plasmids showed no homology with well-characterized enterococcal conjugative plasmids such as pAD1, pPD1, pAMβ1, pIP501 and pMG1 by Southern hybridization. Of the erythromycin resistance traits that transferred only by filter mating, it was found that the erythromycin resistance trait was conferred by a 47-kbp transposable element that transferred from the chromosome of the donor strain to different sites within the pheromone responsive plasmid pAD1 (60 kbp) of the recipient strain, suggesting that the erythromycin resistance trait was encoded on a conjugative transposon, which was named Tn 950.

Antisense RNA regulation by stable complex formation in the Enterococcus faecalis plasmid pAD1 par addiction system.

The par stability determinant, encoded by the Enterococcus faecalis plasmid pAD1, is the only antisense RNA regulated postsegregational killing system identified in gram-positive bacteria. Because of the unique organization of the par locus, the par antisense RNA, RNA II, binds to its target, RNA I, at relatively small, interspersed regions of complementarity. The results of this study suggest that, rather than targeting the antisense bound message for rapid degradation, as occurs in most other antisense RNA regulated systems, RNA I and RNA II form a relatively stable, presumably translationally inactive complex. The stability of the RNA I-RNA II complex would allow RNA I to persist in an untranslated state unless or until the encoding plasmid was lost. After plasmid loss, RNA II would be removed from the complex, allowing translational activation of RNA I. The mechanism of RNA I activation in vivo is unknown, but in vitro dissociation experiments suggest that active removal of RNA II, for example by a cellular RNase, may be required.

Sex Pheromone Response, Clumping, and Slime Production in Enterococcal Strains Isolated from Occluded Biliary Stents

Bile-resistant bacteria, particularly gram-positive Enterococcus faecalis and Enterococcus faecium, play an important role in biliary stent occlusion, because their sessile mode of growth protects them against host defenses and antimicrobial agents. Twelve E. faecalis and seven E. faecium strains isolated from occluded biliary stents have been investigated for slime production, presence of aggregation substance genes, and ability to adhere to Caco-2 cells. Ten isolates were strong producers of slime, and seven isolates produced clumps when exposed to pheromones of E. faecalis JH2-2 and/or OG1RF. The small E. faecium clumps differed from the large clumps of E. faecalis and were similar to those of E. faecium LS10(pBRG1) carrying a pheromone response plasmid. After induction with pheromones, the adhesion to Caco-2 cells of clumping-positive strains was found to increase from two- to fourfold. Amplicons of the expected size were detected in three clumping-positive and three clumping-negative E. faecalis isolates by using primers (agg) internal to a highly conserved region of the E. faecalis pheromone response plasmids pAD1, pPD1, and pCF10 and primers internal to prgB of the E. faecalis plasmid pCF10. The agg/prgB-positive E. faecalis strains were also positive in Southern hybridization experiments with a prgB-specific probe. No PCR products were obtained with the same primers from four clumping-positive isolates (one E. faecalis and three E. faecium strains), which were also Southern hybridization negative. Our results demonstrate that slime production and pheromone response are both present in isolated enterococci, suggesting that clinical strains with these features might have a selective advantage in colonizing biliary stents.

Replication of Enterococcus faecalis pheromone-responding plasmid pAD1: location of the minimal replicon and oriV site and RepA involvement in initiation of replication.

The hemolysin-determining plasmid pAD1 is a member of a widely disseminated family of highly conjugative elements commonly present in clinical isolates of Enterococcus faecalis. The determinants repA, repB, and repC, as well as adjacent iteron sequences, are believed to play important roles in pAD1 replication and maintenance. The repA gene encodes an initiator protein, whereas repB and repC encode proteins related to stability and copy number. The present study focuses specifically on repA and identifies a replication origin (oriV) within a central region of the repA determinant. A small segment of repA carrying oriV was able to support replication in cis of a plasmid vector otherwise unable to replicate, if an intact RepA was supplied in trans. We demonstrate that under conditions in which RepA is expressed from an artificial promoter, a segment of DNA carrying only repA is sufficient for stable replication in E. faecalis. We also show that RepA binds specifically to oriV DNA at several sites containing inverted repeat sequences (i.e., IR-1) and nonspecifically to single-stranded DNA, and related genetic analyses confirm that these sequences play an important role in replication. Finally, we reveal a relationship between the internal structure of RepA and its ability to recognize oriV. An in-frame deletion within repA resulting in loss of 105 nucleotides, including at least part of oriV, did not eliminate the ability of the altered RepA protein to initiate replication using an intact origin provided in trans. The relationship of RepA to other known initiator proteins is also discussed.

Presence of a vanA-carrying pheromone response plasmid (pBRG1) in a clinical isolate of Enterococcus faecium.

Sex pheromone plasmids, frequently found in Enterococcus faecalis, have rarely been detected in Enterococcus faecium. pBRG1 is an approximately 50-kb vanA-carrying conjugative plasmid of an E. faecium clinical isolate (LS10) that is transferable to E. faecalis laboratory strains. In cell infection experiments, E. faecium LS10 exhibited remarkably high invasion efficiency and produced cytopathogenic effects in Caco-2 cell monolayers. Growth in the presence of sex pheromones produced by E. faecalis JH2-2 was found to cause self-aggregation of both E. faecium LS10 and E. faecalis JH-RFV(pBRG1) (a transconjugant obtained by transfer of pBRG1 to E. faecalis JH2-2) and to increase the cell adhesion and invasion efficiencies of both E. faecium LS10 and E. faecalis JH-RFV(pBRG1). Sex pheromone cCF10 caused clumping of E. faecalis OG1RF(pBRG1) (a transconjugant obtained by transfer of pBRG1 to E. faecalis OG1RF) at a concentration approximately 100-fold higher than the one required for the control strain E. faecalis OG1RF(pCF10). PCR products of the expected sizes were obtained with primers internal to aggregation substance genes of E. faecalis pheromone response plasmids pAD1, pPD1, and pCF10 and primers internal to ash701 of E. faecium pheromone plasmid pHKK701. These findings suggest that pBRG1 of E. faecium LS10 is a sex pheromone response plasmid.