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.     

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.     

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.     

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.     

Transfer origins in the conjugative Enterococcus faecalis plasmids pAD1 and pAM373: identification of the pAD1 nic site,a specific relaxase and a possible TraG-like protein

The Enterococcus faecalis conjugative plasmids pAD1 and pAM373 encode a mating response to the peptide sex pheromones cAD1 and cAM373 respectively. Sequence determination of both plasmids has recently been completed with strong similarity evident over many of the structural genes related to conjugation. pAD1 has two origins of transfer, with oriT1 being located within the repA determinant, whereas the more efficiently utilized oriT2 is located between orf53 and orf57, two genes found in the present study to be essential for conjugation. We have found a similarly located oriT to be present in pAM373. oriT2 corresponds to about 285 bp based on its ability to facilitate mobilization by pAD1 when ligated to the shuttle vector pAM401; however, it was not mobilized by pAM373. In contrast, a similarly ligated fragment containing the oriT of pAM373 did not facilitate mobilization by pAD1 but was efficiently mobilized by pAM373. The oriT sites of the two plasmids each contained a homologous large inverted repeat (spanning about 140 bp) adjacent to a series of non-homologous short (6 bp) direct repeats. A hybrid construction containing the inverted repeat of pAM373 and direct repeats of pAD1 was mobilized efficiently by pAD1 but not by pAM373, indicating a significantly greater degree of specificity is associated with the direct repeats. Mutational (deletion) analyses of the pAD1 oriT2 inverted repeat structure suggested its importance in facilitating transfer or perhaps ligation of the ends of the newly transferred DNA strand. Analyses showed that Orf57 (to be called TraX) is the relaxase, which was found to induce a specific nick in the large inverted repeat inside oriT; the protein also facilitated site-specific recombination between two oriT2 sites. Orf53 (to be called TraW) exhibits certain structural similarities to TraG-like proteins, although there is little overall homology.     

Cloning and functional analysis of Asa373, a novel adhesin unrelated to the other sex pheromone plasmid-encoded aggregation substances of Enterococcus faecalis

pAM373 of Enterococcus faecalis deviates from the various other representatives of sex pheromone plasmids in that it encodes a clumping-mediating adhesin, Asa373, unrelated to the highly conserved aggregation substances typical of this plasmid class. The use of a new general cloning strategy and sequencing of the corresponding gene has confirmed that Asa373 represents a novel type of adhesin embedded in a DNA sequence very similar to sex pheromone plasmid pPD1. To prove the specific function of the relatively small protein (75.6 kDa vs 137 kDa for pAD1-encoded Asa1) in cell aggregation, an expression vector, pERM-ex1, was constructed, allowing reliable and stable expression of proteins in E. faecalis. The expression of Asa373 in E. faecalis indeed resulted in constitutive clumping, whereas non-polar disruption of the gene in the original pAM373 abolished clumping capacity. Expression in a strain (INY3000) defective in binding substance - which for the other aggregation substances constitutes the attachment site on the mating partner - did not alter Asa373-dependent clumping; this implies a separate mechanism in cell-cell interaction for this adhesin. Some amino acid motifs of Asa373 link the protein to adhesins of oral streptococci and other cell surface proteins. Comparison of the leader sequence of asa373 with those of several other aggregation substances revealed a highly conserved translational unit possibly involved in the regulation of asa373 expression.     

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.     

Regulation of the pAD1 sex pheromone response in Enterococcus faecalis: effects of host strain and traA, traB, and C region mutants on expression of an E region pheromone-inducible lacZ fusion.

Pheromone-induced conjugal transfer of the hemolysin-bacteriocin plasmid pAD1 of Enterococcus faecalis is regulated by a cluster of determinants designated traA, traB, and regions C and E. The E region is believed to include a positive regulator that controls many structural genes related to conjugation. The pheromone-inducible Tn917-lac fusion NR5, located in the E region, is regulated by the products of traA, traB, and the C region. To more closely examine the effects of these genes on the induction of E region products, inserts in each of these genes were combined with the NR5 fusion in a novel approach involving triparental matings with a pAD1 miniplasmid and recombinational mutagenesis. Results indicate that (i) the traA gene product is a key repressor of the pheromone response; (ii) the traB gene product, in cooperation with a gene within or regulated by the E region, controls pheromone shutdown; (iii) a primary function of the C region gene product is in pheromone sensing, with secondary functions in pheromone shutdown and negative regulation; and (iv) the host in which the plasmid resides has a dramatic effect on the regulation of the NR5 fusion in traB and C region mutants. Numerous parallels were observed between the regulation of the NR5 fusion and the regulation of the aggregation and transfer response. These parallels aided in further defining the functions of particular regulatory determinants as well as further establishing the link between the regulation of the E region and the regulation of the aggregation and transfer response.     

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.     

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.     

Properties of erythromycin-inducible transposon Tn917 in Streptococcus faecalis.

Streptococcus faecalis strain DS16 harbors two plasmids, a conjugative plasmid, pAD1, which encodes hemolysin and bacteriocin activities, and a nonconjugative plasmid, pAD2, encoding resistance to streptomycin, kanamycin, and erythromycin, the latter of which is inducible. The erythromycin resistance determinant is located on a 3.3-megadalton transposable element designated Tn917, which could be transposed to pAD1 as well as to two other plasmids, pAm gamma 1 and pAM alpha 1. When strain DS16 was exposed to low (inducing) concentrations of erythromycin for a few hours, the frequency of Tn917 transposition from pAD2 to pAD1 increased by an order of magnitude. This induction paralleled induction of erythromycin resistance and was prevented by exposing the cells to inhibitors of deoxyribonucleic acid, ribonucleic acid or protein synthesis. The exposure of strain DS16 to inducing concentrations of erythromycin also enhanced the frequency of erythromycin-resistant transconjugants appearing during mating. Initially, cointegrate molecules, whose molecular weights were approximately the sum of pAD1 and pAD2, accounted for these transconjugants; however, as the induction time increased, pAD1::Tn917 became increasingly prominent.     

Identification of the cAD1 sex pheromone precursor in Enterococcus faecalis

The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci. The determinant for the pheromone in E. faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety. The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. The nonisogenic E. faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8). Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1. The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response. A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential. A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor. Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety.     

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.     

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.     

Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid

Streptococcus faecalis strain DS16 harbors the conjugative hemolysin-bacteriocin plasmid pAD1 (35 megadaltons) and the nonconjugative R-plasmid pAD2 determining resistance to streptomycin, kanamycin, and erythromycin; a tetracycline resistance (Tetr) determinant is located on the chromosome. When strain DS16 was mated (on membrane filters) with the plasmid-free strain JH2-2, Tetr transconjugants could be obtained at a frequency of about 10(-6) per recipient. Analyses of transconjugants showed that some contained the Tetr determinant linked to pAD1. Subsequent studies showed that the Tetr determinant was located on a 10-megaldalton transposon, designated Tn916, which could insert into two hemolysin plasmids: pAM gamma 1 and pOB1. In addition, derivatives of DS16 devoid of pAD1 were capable of transferring Tetr to recipient strains. Transconjugants (plasmid-free) from such matings could subsequently act as donors in the transfer of Tetr. Both transposition and transfer were found to be rec independent.     

Transfer functions of the Streptococcus faecalis plasmid pAD1: organization of plasmid DNA encoding response to sex pheromone.

The conjugative plasmid pAD1 (59.6 kilobases) of Streptococcus faecalis shows a 10,000-fold increase in transfer frequency following induction by the sex pheromone cAD1. Mutagenesis of the plasmid with transposon Tn917 was undertaken to determine the region(s) of pAD1 required for the mating response. The relevant genetic material was found to be distributed over a 31.2-kilobase contiguous region of the plasmid. Although insertions in two previously identified regions (traA and traB) exhibited increased transfer frequencies, insertions in five new regions (D, E, F, G, and H) decreased the ability of pAD1 to transfer. Insertions in region H allowed the cells to form visible mating aggregates, but the plasmid transfer frequency was decreased to levels below detection during a 1-h broth mating. Mutants with mutations in region G were able to form aggregates; however, insertions in regions D, E, and F prevented aggregate formation. Insertions in region C decreased the sensitivity of the cell to exogenous cAD1 and exhibited increased activity of the pheromone inhibitor iAD1. Surface protein profiles produced by a number of these mutants were examined, and in some cases were found to be different from those of the wild type. A map showing the various regions is presented, and related aspects of the regulation of the pAD1 mating response are discussed.