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 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.     

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.     

Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen

The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis has been shown to confer a mating response to the sex pheromone cAD1 excreted by recipient strains. The response is characterized by the synthesis of a proteinaceous adhesin which coats the surface of the pAD1 -containing donor cell and facilitates the formation of mating aggregates. Donors exposed to cAD1 -containing filtrates of recipients undergo self-aggregation (clumping), an event believed to be associated with an interaction between the adhesin and a binding substance always present on the surface of both recipients and donors. To analyze the molecular processes involved in the mating response, mutants were generated by the erythromycin resistance transposon Tn917 . Transpositions to pAD1 in S. faecalis DS16 gave rise to a number of derivatives that exhibited "constitutive clumping" and the ability to transfer at high frequencies in short (10-min) matings. These mutants fell into two subclasses, which exhibited colony morphologies that were "dry" or "normal". The Tn917 insertions were mapped by restriction enzyme analysis to two separate clusters, designated traA and traB. The dry colony subclass corresponded to traA and represented a span of 1.5 kilobases, whereas the normal subclass corresponded to traB and spanned 1.3 kilobases. The two clusters were separated by 1.7 kilobases in which insertions of Tn917 did not affect the ability to respond normally to cAD1 . Neither type of constitutive clumper produced cAD1 . Another series of insertions exhibited reduced donor potential. In two cases, the reduction in transfer was three to four orders of magnitude; these mapped in traA . In two other cases, the reduction was one to two orders of magnitude. These mapped outside of traA and traB, and one was associated with an increase in plasmid copy number.     

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.     

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.     

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.     

Nucleotide sequence of the sex pheromone inhibitor (iAD1) determinant of Enterococcus faecalis conjugative plasmid pAD1

The determinant for the peptide sex pheromone inhibitor iAD1 (iad) on the hemolysin/bacteriocin plasmid pAD1 of Enterococcus faecalis was sequenced. The sequence reveals a 22-amino-acid precursor with the carboxyl-terminal 8 residues corresponding to iAD1. It appears that iAD1 is a component of its own signal sequence.     

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.     

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.     

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.     

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.     

Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria

The conjugative transposon Tn916 (15 kilobases), originally identified in Streptococcus faecalis DS16, has been cloned as an intact element on the pBR322-derived vector pGL101 in Escherichia coli. The EcoRI F' (EcoRI F::Tn916) fragment of pAM211 (pAD1::Tn916) was cloned into the single EcoRI site of pGL101 to form the chimera, pAM120, by selecting for the expression of Tn916-encoded tetracycline resistance (Tcr). Interestingly, in the absence of continued selection for Tcr, Tn916 excised from pAM120 at high frequency. This excision event resulted in a plasmid species consisting of the pGL101 vector and a 2.7-kilobase restriction fragment comigrating with the EcoRI F fragment of pAD1 during agarose gel electrophoresis. Filter blot hybridization experiments showed the 2.7-kilobase fragment generated as a result of Tn916 excision to be homologous with the EcoRI F fragment of pAD1. Analogous results were obtained with another chimera, pAM170, generated by ligating the EcoRI D' (EcoRI D::Tn916) fragment of pAM210 (pAD1::Tn916) to EcoRI-digested pGL101. Comparison of the AluI and RsaI cleavage patterns of the EcoRI F fragment isolated after Tn916 excision with those from an EcoRI F fragment derived from pAD1 failed to detect any difference in the two fragments: data in support of a precise Tn916 excision event in E. coli. Subcloning experiments showed that an intact transposon was required for Tn916 excision and located the Tcr determinant near the single HindIII site on Tn916. Although excision occurred with high frequency in E. coli, Tn916 insertion into the E. coli chromosome was a much rarer event. Tcr transformants were not obtained when pAM120 DNA was used to transform a polA1 strain, E. coli C2368.     

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.     

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.