Identification of aggregation substances of Enterococcus faecalis cells after induction by sex pheromones

The sex pheromone system of Enterococcus faecalis is responsible for the clumping response of a plasmid carrying donor strain with a corresponding plasmid free recipient strain due to the production of sex pheromones by the recipient strain. The clumping response is mediated by a surface material (called aggregation substance) which is synthesized upon addition of sex pheromones to the cultures. Here we show that after induction a dense layer of hairlike structures is formed on the cell wall of the bacteria. These hairlike structures are responsible for the cell-cell contact which leads to the aggregation of cells. Formation of these structures was specific, only occurring after the addition of homologous sex pheromone.Abbreviations cAD1 sex pheromone specific for plasmid pAD1 - cPD1 sex pheromone specific for plasmid pPD1 - CW cell wall - pAD1 conjugative plasmid specifically transferred in the presence of cAD1 - pPD1 conjugative plasmid specifically transferred in the presence of cPD1 - PBS 10 mM Na-phosphate pH 7.5, 0.85% NaCl     

Cloning and characterization of a region of Enterococcus faecalis plasmid pPD1 encoding pheromone inhibitor (ipd), pheromone sensitivity (traC), and pheromone shutdown (traB) genes.

Bacteriocin plasmid pPD1 in Enterococcus faecalis encodes a mating response to recipient-produced sex pheromone cPD1. Once a recipient acquires pPD1, transconjugants apparently shut off cPD1 activity in broth culture and no longer behave as recipients for pPD1. This event is performed by synthesis of the pheromone inhibitor iPD1 and also by repression of cPD1 production, the so-called "pheromone shutdown." A 5.4-kb EcoRV-HincII segment of pPD1, which expressed iPD1 in Escherichia coli, was sequenced and found to be organized as traC-traB-traA-ipd; each open reading frame is analogous to that found in other pheromone plasmids, pAD1 and pCF10, and thus is designated in accordance with the nomenclature in pAD1. The ipd gene encodes a peptide consisting of 21 amino acids, in which the C-terminal eight residues correspond to iPD1. The putative TraC product has a strong similarity to oligopeptide-binding proteins found in other bacterial species, as do pheromone-binding proteins of pCF10 and pAD1. A strain carrying traC-disrupted pPD1 required a concentration of cPD1 fourfold higher than that needed by the wild-type strain for induction of sexual aggregation. These results suggest that the TraC product contributes to pheromone sensitivity as a pheromone-binding protein. A strain transformed with traB-disrupted pPD1 produced a high level of cPD1 similar to that produced by plasmid-free recipients and underwent self-induction. Thus, the TraB product contributes to cPD1 shutdown.     

Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone.

pAD1, a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis, encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. A gene, traC, encoding a 60.7-kDa protein with a typical amino terminal signal peptide, was identified within a region that appears to encode a product that binds to exogenous pheromone. A cloned segment of DNA containing traC resulted in specific binding of cells to synthetic cAD1. The putative traC product has strong similarity to a product of the E. faecalis plasmid pCF10 as well as oligopeptide binding proteins of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis.     

Comparative analysis of 18 sex pheromone plasmids fromEnterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3′-terminus of the surface exclusion gene,sep1, of sex pheromone plasmid pPD1 inEnterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.     

Localization of aggregation substances of Enterococcus faecalis after induction by sex pheromones

The distribution of sex pheromone induced aggregation substance was studied on the cell surface of various Enterococcus faecalis strains. In the accompanying paper we have shown that the aggregation substance appears as a layer of hairlike structures. Using direct and indirect immunogold technique, transmission electron microscopy and high resolution scanning electron microscopy we investigated the appearance and distribution of the aggregation substance. The hairs increase in number with increasing exposure to sex pheromones (maximum density: 1300/m²). We show that these structures are unequally distributed over the cell surface, even if the cells were induced by sex pheromones for a long period of time. Statistical analysis of the unequal distribution indicates that aggregation substance is incorporated into pre-existing old cell-walls and that this incorporation shows a saturation ca. 40 min after addition of sex pheromones.Abbreviations cAD1 sex pheromone specific for plasmid pAD1 - cPD1 sex pheromone specific for plasmid pPD1 - FESEM field emission scanning electron microscope - pAD1 conjugative plasmid specifically transferred in the presence of cAD1 - pPD1 conjugative plasmid specifically transferred in the presence of cPD1 - TEM transmission electron microscope     

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.     

Comparative analysis of 18 sex pheromone plasmids fromEnterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3-terminus of the surface exclusion gene,sep1, of sex pheromone plasmid pPD1 inEnterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.     

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.     

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.     

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.     

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

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

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.     

Flow cytometric analysys of Enterococcus faecalis pAD1(-) strains response to cAD1 pheromone

Conjugative plasmids transfer in Enterococcus faecalis is inducted by sex pheromones. The pheromone is excreted by recipient cells and induces expression of aggregation protein AS in donor cells. This protein is involved in formation of matting aggregates. Use of flow cytometry and anti-As monoclonal antibodies allowed collect of interesting data pheromone response. However, according to our knowledge, no study focused on unspecific influence on particular pheromone for plasmid-free recipient strains. Six pAD1 (-) and tree pAD1 (+) Enterococcus faecalis stains were cultivated for 18h in BHI, with and without cAD1 pheromone (Sigma, Germany), respectively. The bacteria were washed, stained with carboksyfluorescein (FCDA, and analyzed by flow cytometry in FACS BD scan cytometr. Relative fluorescence and size of aggregation was used to compare influence on particular strains. Surprisingly, the results shows divergence in fluorescence, size of aggregates and degree of correlation between fluorescence of aggregates and their sizes among pAD1(-) strains, allowing for distinguish of two groups. Three of studied strains have higher fluorescence than pAD (+) stains. Correlation between fluorescence and size of aggregates, significant higher than in pAD1(+) stains, decrease from r = 0.88 to r=0.74 in reaction to cAD1. The strains if other group fluorize with lower intensity than pAD1 (+). Furthermore, 30.4% pAD1 (-) of second group have no detectable fluorescence. In contrast to pAD1 (-) ) strains of the first group and pA1 (+) strains, low (r=0.55) correlation between fluorescence and size of aggregates of group II increase up to r=0.74 after incubation with cAD1 pheromone. Previous study of these pAD1 (-) strains, currently assigned to group II, shown their low frequency of collecting aph2" gene encoded on other conjugative plasmid, pMG. According to these results, such flow cytometric analysis may be used to predict ability of strain to collect unrelated conjugative plasmid.     

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.     

C-terminal identification of AD74, a proteolytic product of Enterococcus faecalis aggregation substance: application of liquid chromatography/mass spectrometry.

Sexual aggregation involved in conjugative transfer of Enterococcus faecalis plasmid pAD1 is enhanced by the sex pheromone cAD1, which is excreted from recipient cells. A membrane-anchored 137 kDa protein is a pAD1-encoded aggregation substance designated asal, which is responsible for cell-cell contact and leads to the aggregation of cells. An AD74 protein is a proteolytic product corresponding to the N-terminal half of asal. The C-terminal of AD74 was identified as lysine at position 510 (K-510) by liquid chromatography/mass spectrometry (LC/MS): it indicates that asal is cleaved specifically between K-510 and G-511.     

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.     

Streptococcus faecalis sex pheromone (cAD1) response: evidence that the peptide inhibitor excreted by pAD1-containing cells may be plasmid determined

Streptococcus faecalis strains harboring the conjugative plasmid pAD1 excrete a small peptide, iAD1, which inhibits the sex pheromone cAD1. Studies making use of the host strain Streptococcus faecium 9790, which normally does not excrete peptide pheromones, suggest that iAD1 may be determined directly by pAD1.