Comparative analysis of Enterococcus faecalis sex pheromone plasmids identifies a single homologous DNA region which codes for aggregation substance.

An analysis of the 11 known sex pheromone plasmids of Enterococcus faecalis was performed by DNA-DNA hybridization. Plasmids pAD1, pJH2, and pBEM10 turned out to be closely related, whereas pAM373 showed only weak homology with pAD1. A comparison of the hemolysin/bacteriocin determinants of pAD1, pJH2, and pOB1 revealed strong similarities at the DNA level. Our main finding was that one DNA region is conserved among all sex pheromone plasmids, with pAM373 again being an exception; for pAD1 this region was shown earlier to code for aggreagation substance. Detailed hybridization studies of the genes for this plasmid-coded adhesin, which is responsible for cell-cell contact during conjugative transfer via the so-called sex pheromone system of E. faecalis, support the idea of their common origin.     

The Enterococcus faecalis pheromone-responsive plasmid pAM373 does not encode an entry exclusion function

pAM373 is a conjugative plasmid in Enterococcus faecalis that confers a mating response to the peptide sex pheromone cAM373 which is produced also by Staphylococcus aureus and Streptococcus gordonii. Unlike other sex pheromone-inducible plasmids, pAM373 does not confer an entry exclusion phenotype.     

Streptococcus faecalis sex pheromone (cAM373) also produced by Staphylococcus aureus and identification of a conjugative transposon (Tn918).

Streptococcus faecalis RC73 was found to harbor a conjugative plasmid (pAM373) which confers a mating response to a sex pheromone (cAM373) excreted by plasmid-free members of the same species. The pheromone was also detected in culture filtrates of all of 23 Staphylococcus aureus strains but in only 2 of 22 coagulase negative staphylococcus strains. Streptococcus sanguis Challis and G9B also produced the activity, but 10 other Streptococcus sanguis strains did not. The activity was also produced by Streptococcus faecium 9790. A tetracycline resistance (Tc) determinant present in S. faecalis RC73 was not associated with pAM373 but served as a useful marker in efforts to identify pAM373 among other plasmids present in the strain. Analyses of the Tc determinant showed that it was located on a conjugative transposon very similar to Tn916. Designated Tn918, the transposon could insert into pAM373 as well as into two other hemolysin plasmids. Whereas pAM373 derivatives transferred very well between strains of Streptococcus faecalis, the plasmid would not establish in Staphylococcus aureus or Streptococcus sanguis. However, a derivative of pAM373 carrying Tn918 proved to be a useful delivery vehicle for generating transposon insertions into multiple sites on the staphylococcal chromosome.     

Regulation of transfer of the Enterococcus faecalis pheromone-responding plasmid pAD1: temperature-sensitive transfer mutants and identification of a new regulatory determinant, traD.

The enterococcal, conjugative, cytolysin plasmid pAD1 confers a mating response to the peptide sex pheromone cAD1 secreted by plasmid-free strains of Enterococcus faecalis. Cells carrying pAM714, a pAD1::Tn917 derivative with wild-type conjugation properties, were mutagenized with ethyl methanesulfonate to obtain variants that were induced (in the absence of pheromone) to transfer plasmid DNA upon shifting from 32 to 42 degrees C. Of 31 such mutants generated, the results of analyses of 7 are presented in detail. All seven strains were thermosensitive in the E. faecalis host FA2-2; colony morphology, clumping, and DNA transfer correlated well with each other at the two temperatures. In the nonisogenic host E. faecalis OG1X, however, only one derivative (pAM2725) exhibited correlation of all three traits at both temperatures. Three (pAM2700, pAM2703, and pAM2717) clumped and had colonies characteristic of pheromone-induced cells at 32 degrees C but transferred plasmid DNA at a higher frequency only at the elevated temperature. The other three (pAM2708, pAM2709, and pAM2712) were derepressed at both temperatures for all three characteristics. Four of the mutations, including that of pAM2725, mapped within the traA determinant, whereas two mapped identically in a previously unnoted open reading frame (designated traD) putatively encoding a short (23-amino-acid) peptide downstream of the inhibitor peptide determinant iad and in the opposite orientation. One mutant could not be located in the regions sequenced. Studies showed that the traA and traD mutations could be complemented in trans with a DNA fragment carrying the corresponding regions.     

Isolation and Structure of staph-cAM373 Produced by Staphylococcus aureus That Induces Conjugal Transfer of Enterococcus faecalis Plasmid pAM373

Enterococus faecalis plasmid pAM373 encodes a mating response to the sex pheromone, cAM373, which is secreted from pAM373-free E. faecalis. cAM373-like activity was detected in a culture filtrate of Staphylococcus aureus. The major active substance, termed staph-cAM313 was isolated, and its structure was identified as a H-Ala-Ile-Phe-Ile-Leu-Ala-Ala-OH heptapeptide.     

Isolation and Structure of the Sex Pheromone Inhibitor,iAM373, of Enterococcus faecalis

An Enterococcus faecalis plasmid, pAM373, has a high frequency of transfer in a liquid medium when induced by a recipient-produced sex pheromone, cAM373. The sex pheromone inhibitor against cAM373, termed iAM373, was isolated from a culture supernatant of E. faecalis harboring pAM377 (=pAM373::Tn917), and its structure was identified as a heptapeptide, H-Ser-Ile-Phe-Thr-LeuVal-Ala-OH.     

Identification and Characterization of a Determinant (eep) on the Enterococcus faecalis Chromosome That Is Involved in Production of the Peptide Sex Pheromone cAD1

Plasmid-free strains of Enterococcus faecalis secrete a peptide sex pheromone, cAD1, which specifically induces a mating response by donors carrying the hemolysin plasmid pAD1 or related elements. A determinant on the E. faecalis OG1X chromosome has been found to encode a 46.5-kDa protein that plays an important role in the production of the extracellular cAD1. Wild-type E. faecalis OG1X cells harboring a plasmid chimera carrying the determinant exhibited an eightfold enhanced production of cAD1, and plasmid-free cells carrying a mutated chromosomal determinant secreted undetectable or very low amounts of the pheromone. The production of other pheromones such as cPD1, cOB1, and cCF10 was also influenced, although there was no effect on the pheromone cAM373. The determinant, designated eep (for enhanced expression of pheromone), did not include the sequence of the pheromone. Its deduced product (Eep) contains apparent membrane-spanning sequences; conceivably it is involved in processing a pheromone precursor structure or in some way regulates expression or secretion.     

Enterococcal plasmid transfer: sex pheromones,transfer origins,relaxases, and the Staphylococcus aureus issue

Certain conjugative plasmids in Enterococcus faecalis encode a mating response to peptide sex pheromones encoded on the chromosome of potential recipient (plasmid-free) strains. The pheromone precursors correspond to the precursors of surface lipoproteins with the mature peptides coming from the last 7-8 residues of the related signal sequences. Processing that gives rise to the pAD1-related peptide involves a chromosome-encoded metalloprotease (Eep) that is believed to operate within the cytoplasmic membrane. Mutations in the determinants for cAD1 and cAM373, cad and camE, respectively, do not affect cell viability; and when the related plasmid is present, the pheromone response is normal. A cAM373-like activity is produce by Staphylococcus aureus, but the corresponding lipoprotein determinant (camS) is unrelated to the enterococcal determinant (camE). pAD1 has two origins of transfer, oriT1 and oriT2 and encodes a relaxase (TraX), which has been shown to specifically nick in oriT2. pAM373 has a site, oriT, that is similar to oriT2 of pAD1. Both sites (oriT2 of pAD1 and oriT of pAM373) have a series of short direct repeats (5-6 bp with 5-6 bp-spacings) adjacent to a long inverted repeat (140 bp). The direct repeats differ significantly and confer specificity to the two systems. pAD1 and pAM373 are both able to mobilize the nonconjugative plasmid pAMalpha1, which encodes two relaxases that are involved in transfer. Relevant information concerning the possible movement of vancomycin resistance from E. faecalis to S. aureus in a clinical environment is discussed.     

Identification of pheromone-induced surface proteins in Streptococcus faecalis and evidence of a role for lipoteichoic acid in formation of mating aggregates.

The conjugative transfer of the Streptococcus faecalis plasmid pAD1 is characterized by a 10,000-fold increase in frequency following sex pheromone (cAD1) induction. Before the increase in plasmid transfer, donor cells synthesize a proteinaceous adhesin that facilitates the formation of mating aggregates. Four novel surface proteins appearing after exposure of pAD1-containing cells to sex pheromone have been identified. Thirty minutes after induction, a 130-kilodalton (kDa) protein was detectable by Western blotting. A 74-kDa protein, the major species present, and a pair of bands at 153 and 157 kDa were evident 45 min after induction. Induced cells containing another conjugative S. faecalis plasmid, pPD1, gave rise to three high-molecular-weight proteins of the same size (130, 153, and 157 kDa) as those synthesized by pAD1-containing cells. These proteins cross-reacted with antisera raised against induced cells containing pAD1. However, the major protein species produced by pPD1-containing cells had a molecular weight of 78,000 and did not cross-react significantly with the corresponding band of the pAD1 system. Pheromone-induced transfer of the two plasmids, when both were present in the same cell, was independent; induction was limited to the pheromone-specified plasmid. The possibility that lipoteichoic acid might act as a receptor (binding substance) for the induced adhesin protein was also explored. Free lipoteichoic acid (isolated from S. faecalis) inhibited clumping of induced cells, apparently by acting as a competitive inhibitor of the cellular binding substance.     

Pheromone-regulated expression of sex pheromone plasmid pAD1-encoded aggregation substance depends on at least six upstream genes and a cis-acting, orientation-dependent factor

Conjugative transfer of Enterococcus faecalis-specific sex pheromone plasmids relies on an adhesin, called aggregation substance, to confer a tight cell-to-cell contact between the mating partners. To analyze the dependence of pAD1-encoded aggregation substance, Asa1, on pheromone induction, a variety of upstream fragments were fused to an alpha-amylase reporter gene, amyL, by use of a novel promoter probe vector, pAMY-em1. For pheromone-regulated alpha-amylase activity, a total of at least six genes, traB, traC, traA, traE1, orfY, and orf1, are required: TraB efficiently represses asa1 (by a mechanism unrelated to its presumptive function in pheromone shutdown, since a complete shutdown is observed exclusively in the presence of traC); only traC can relieve traB-mediated repression in a pheromone-dependent manner. In addition to traB, traA is required but not sufficient for negative control. Mutational inactivation of traE1, orfY, or orf1, respectively, results in a total loss of alpha-amylase activity for constructs normally mediating constitutive expression. Inversion of a fragment covering traA, P(0), and traE1 without disrupting any gene or control element switches off amyL or asa1 expression, indicating the involvement of a cis-acting, orientation-dependent factor (as had been shown for plasmid pCF10). Unexpectedly, pAD1 represses all pAMY-em1 derivatives in trans, while its own pheromone-dependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a trans-acting positive regulator is discussed.     

Isolation and structure of the Streptococcus faecalis sex pheromone, cAM373

The Streptococcus faecalis sex pheromone, cAM373, which induces a mating response of donor cells harboring plasmid pAM373 and is also produced by Staphylococcus aureus, was isolated and its structure determined. Supernatant from an overnight culture of a recipient strain was subjected to successive purification procedures, and 4.4 micrograms cAM373 was obtained. The isolated pheromone showed activity at a concentration as low as 5 X 10(-11) M. Sequence analysis indicated that cAM373 was a heptapeptide, H-Ala-Ile-Phe-Ile-Leu-Ala-Ser-OH, and that its Mr was 733. A synthetic replicate of the peptide showed the same biological activity and chromatographic behavior as the native cAM373.     

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

Summary During conjugative transfer of sex pheromone plasmids ofEnterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3 .8 kb fragment of the sex pheromone plasmid pAD1 containing the structural genesea1 for surface exclusion protein and a small open reading frame (ORF) upstream ofsea1. Surface exclusion protein Seal was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene seal demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5 to that for aggregation substance.     

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.     

The genes coding for enterocin EJ97 production by Enterococcus faecalis EJ97 are located on a conjugative plasmid

Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.     

Constitutive expression of erythromycin resistance mediated by the ermAM determinant of plasmid pAM beta 1 results from deletion of 5' leader peptide sequences

We have sequenced the erythromycin resistance determinant (erm) of the Streptococcus faecalis plasmid pAM beta 1 to investigate its relationship to other known resistance determinants. We show that this determinant is strongly (99%) homologous at the DNA level to that of plasmid pAM77 (Streptococcus sanguis) and of transposon Tn917 (S. faecalis). Moreover, nucleotide sequence comparison with the determinants of pAM77 and Tn917 shows that most of the probable regulatory region is absent, providing an explanation for the constitutive expression of the pAM beta 1 erm determinant.     

Sequence analysis of Enterococcus faecalis aggregation substance encoded by the sex pheromone plasmid pAD1

The location of the structural gene for aggregation substance on the sex pheromone plasmid pAD1 of Enterococcus faecalis was determined using an oligonucleotide deduced from the N-terminal amino acid sequence of the purified protein. The nucleotide sequence was determined for the corresponding region and two open reading frames (ORFs) could be identified. ORF1 codes for a small (Mr 13,160) acidic protein of unknown function. The gene for aggregation substance (named asa1) was found to code for a protein of 1296 amino acids (Mr 142,248). The protein has a signal peptide of 43 amino acids (the resulting Mr for mature aggregation substance is 137,429) and contains in its C-terminal region a proline-rich sequence, previously characterized as being involved in cell wall association, which is followed by a membrane anchor. The membrane anchor showed significant similarity to that of other Gram-positive organisms, but no other similarities to surface proteins from Gram-positive bacteria were found. In particular, no repeats on the DNA or protein level could be detected for pAD1-specific aggregation substance. The protein contains the amino acid motifs Arg-Gly-Asp-Ser and Arg-Gly-Asp-Val (once each), which, it is proposed, play a crucial role in adherence to eukaryotic cells.