Molecular Mechanism of Peptide-Specific Pheromone Signaling in Enterococcus faecalis: Functions of Pheromone Receptor TraA and Pheromone-Binding Protein TraC Encoded by Plasmid pPD1

Conjugative transfer of the Enterococcus faecalis plasmid pPD1 is activated by cPD1, one of several peptide sex pheromones secreted by plasmid-free recipient cells, and is blocked by a donor-produced peptide inhibitor, iPD1. Using a tritiated pheromone, [³H]cPD1, we investigated how pPD1-harboring donor cells receive these peptide signals. Donor cells rapidly incorporated [³H]cPD1. The cell extract but not the membrane fraction of the donor strain exhibited significant [³H]cPD1-binding activity. On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a high-molecular-weight compound. The cell extract of a strain carrying the traA-bearing multicopy plasmid (pDLHH21) also exhibited high [³H]cPD1-binding activity. A recombinant TraA exhibited a dissociation constant of 0.49 ± 0.08 nM against [³H]cPD1. iPD1 competitively inhibited [³H]cPD1 binding to TraA, whereas pheromones and inhibitors relating to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an antagonist for TraA. A strain carrying the traC-bearing multicopy plasmid (pDLES23) exhibited significant [³H]cPD1-binding activity. A strain carrying traC-disrupted pPD1 (pAM351CM) exhibited lower [³H]cPD1-binding activity as well as lower sensitivity to cPD1 than a wild-type donor strain. Some of the other pheromones and inhibitors inhibited [³H]cPD1 binding to the traC transformant like cPD1 and iPD1 did. These results show that TraC, as an extracellular less-specific pheromone-binding protein, supports donor cells to receive cPD1.     

Cloning, E. coli overexpression, purification and binding properties of TraA and TraC, two proteins involved in the pheromone-dependent conjugation process in enterococci

The bacteriocin encoding plasmid pPD1 from Enterococcus faecalis is involved in a mating response to the sex pheromone cPD1 produced by recipient bacterial cells devoid of pPD1. Previous studies showed that cPD1 is internalized into donor cells in a process in which TraC plays the role of cell surface pheromone receptor. Inside the recipient cells, the pheromone binds to the plasmid-encoded cytoplasmic protein TraA, able to recognize specific DNA sequences and to modulate the conjugation process. To avoid self-induction of the conjugation process, donor cells produce the inhibitor iPD1, which competes with cPD1. This study was designed to produce recombinant TraA and TraC in a functionally active state and to evaluate their main functional properties. We have isolated the sequences encoding TraA and TraC from the plasmid pPD1 and cloned them in suitable expression vectors. The two recombinant proteins were successfully obtained in a soluble form using Escherichia coli as expression host and a T7 inducible expression system. TraC and TraA were purified to homogeneity by three or two chromatographic steps, respectively, leading to a final yield up to 4 mg/l of cell culture for TraC and up to 10 mg/l of cell culture for TraA. The ability of TraA and TraC to bind the specific pheromone and inhibitor peptides has been assessed by means of ESI-mass spectrometry. Moreover, the ability of recombinant TraA to bind DNA has been demonstrated by means of electrophoretic mobility shift assay. Overall these results are consistent with the heterologously expressed TraC and TraA being functionally active.     

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.     

Genetic Analysis of Plasmid-specific Pheromone Signaling Encoded by pPD1 in Enterococcus faecalis

Certain plasmids in Enterococcus faecalis encode a mating response to recipient-produced peptide sex pheromones. Targeted disruption of tra genes on pPD1 suggested that TraA plays a central role in the plasmid-specific pheromone signaling pathway. TraA functioned as a negative regulator for the pheromone-inducible conjugal transfer. Complementation analysis of pPD1 tra gene mutants by pAD1 suggested that the pheromone binding function of TraC was non-specific between these plasmids, but the function of TraA and the pheromone shutdown function of TraB are plasmid-specific.     

Isolation and structure of the sex pheromone inhibitor, iPD1, excreted by Streptococcus faecalis donor strains harboring plasmid pPD1.

The sex pheromone inhibitor iPD1, which is excreted by Streptococcus faecalis donor strains harboring bacteriocin plasmid pPD1 and which inhibits sex pheromone cPD1, was isolated, and its structure was determined. Its molecular weight is 828, and its amino acid sequence is H-Ala-Leu-Ile-Leu-Thr-Leu-Val-Ser-OH.     

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     

Targeted disruption of the PD78 gene (traF) reduces pheromone-inducible conjugal transfer of the bacteriocin plasmid pPD1 in Enterococcus faecalis

Abstract Bacterial sex pheromone, cPD1, induces sexual aggregation of Enterococcus faecalis harboring the bacteriocin plasmid, pPD1, and enables pPD1 to transfer at high frequency in a liquid culture. PD78 is a cPD1-inducible cell surface protein encoded by pPD1. The PD78 gene, traF , was disrupted by homologous recombination between pPD1 and an artificial vector having a deletion in the middle portion of traF . The disruption of traF did not affect the cPD1-inducible aggregation but reduced the transfer frequency of pPD1 to 2% of the wild-type level.     

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     

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.     

pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coli and E. faecalis at levels of 10³ and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.     

Molecular Recognition by a Polymorphic Cell Surface Receptor Governs Cooperative Behaviors in Bacteria

Cell-cell recognition is a fundamental process that allows cells to coordinate multicellular behaviors. Some microbes, such as myxobacteria, build multicellular fruiting bodies from free-living cells. However, how bacterial cells recognize each other by contact is poorly understood. Here we show that myxobacteria engage in recognition through interactions between TraA cell surface receptors, which leads to the fusion and exchange of outer membrane (OM) components. OM exchange is shown to be selective among 17 environmental isolates, as exchange partners parsed into five major recognition groups. TraA is the determinant of molecular specificity because: (i) exchange partners correlated with sequence conservation within its polymorphic PA14-like domain and (ii) traA allele replacements predictably changed partner specificity. Swapping traA alleles also reprogrammed social interactions among strains, including the regulation of motility and conferred immunity from inter-strain killing. We suggest that TraA helps guide the transition of single cells into a coherent bacterial community, by a proposed mechanism that is analogous to mitochondrial fusion and fission cycling that mixes contents to establish a homogenous population. In evolutionary terms, traA functions as a rare greenbeard gene that recognizes others that bear the same allele to confer beneficial treatment.     

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.     

Control of Enterococcus faecalis sex pheromone cAD1 elaboration: Effects of culture aeration and pAD1 plasmid-encoded determinants

Aeration of plasmid-free Enterococcus faecalis strains resulted in an 8- to 16-fold decrease in sex pheromone cAD1 activity in culture filtrates. Levels of two unrelated pheromones, cPD1 and cAM373, were unaffected by culture aeration. Aeration also resulted in a decrease in the expression of conjugative transfer functions observed in cells containing pAD1 traB mutations, verifying a link between traB function and pheromone “shutdown.” Tests with a series of pAD1 mini-plasmids indicated that the product of the traB gene was involved in, but not sufficient for, pheromone shutdown; the cooperation of one or more other gene products encoded within the pheromone response control region was required.     

Discovery of a disused desaturase gene from the pheromone gland of the moth Ascotis selenaria, which secretes an epoxyalkenyl sex pheromone

Female Ascotis selenaria (Geometridae) moths use 3,4-epoxy-(Z,Z)-6,9-nonadecadiene, which is synthesized from linolenic acid, as the main component of their sex pheromone. While the use of dietary linolenic or linoleic fatty acid derivatives as sex pheromone components has been observed in moth species belonging to a few families including Geometridae, the majority of moths use derivatives of a common saturated fatty acid, palmitic acid, as their sex pheromone components. We attempted to gain insight into the differentiation of pheromone biosynthetic pathways in geometrids by analyzing the desaturase genes expressed in the pheromone gland of A. selenaria. We demonstrated that a Δ11-desaturase-like gene (Asdesat1) was specifically expressed in the pheromone gland of A. selenaria in spite of the absence of a desaturation step in the pheromone biosynthetic pathway in this species. Further analysis revealed that the presumed transmembrane domains were degenerated in Asdesat1. Phylogenetic analysis demonstrated that Asdesat1 anciently diverged from the lineage of Δ11-desaturases, which are currently widely used in the biosynthesis of sex pheromones by moths. These results suggest that an ancestral Δ11-desaturase became dysfunctional in A. selenaria after a shift in pheromone biosynthetic pathways.     

Sex pheromone of the cabbage looper: Reactions with antennal proteins in vitro

In the presence of (Z)-7-dodecen-1-ol acetate, the sex attractant of the cabbage looper, Trichoplusia ni, soluble protein from male antennae showed a time-dependent difference spectral absorbance at 280 nm. The change was associated with the enzymatic conversion of the pheromone to (Z)-7-dodecen-1-ol, a potent inhibitor of behavioural responses to the pheromone. In contrast, the response obtained with the inhibitor was indicative of non-enzymatic binding to specific protein(s) in the fraction. GLC analyses of the relative rates of enzymatic hydrolysis of the pheromone by the antennae, haemolymph, and legs revealed 33·9, 10·1, and 6·5 per cent conversion per hour, respectively. These results may provide an insight into the fate of a pheromone in the olfactory process of this insect; however, the significance of the reaction with the inhibitor is not yet known.     

Concurrent modulation of neuronal and behavioural olfactory responses to sex and host plant cues in a male moth

Mating has profound effects on animal physiology and behaviour, not only in females but also in males, which we show here for olfactory responses. In cotton leafworm moths, Spodoptera littoralis, odour-mediated attraction to sex pheromone and plant volatiles are modulated after mating, producing a behavioural response that matches the physiological condition of the male insect. Unmated males are attracted by upwind flight to sex pheromone released by calling females, as well as to volatiles of lilac flowers and green leaves of the host plant cotton, signalling adult food and mating sites, respectively. Mating temporarily abolishes male attraction to females and host plant odour, but does not diminish attraction to flowers. This behavioural modulation is correlated with a response modulation in the olfactory system, as shown by electro-physiological recordings from antennae and by functional imaging of the antennal lobe, using natural odours and synthetic compounds. An effect of mating on the olfactory responses to pheromone and cotton plant volatiles but not to lilac flowers indicates the presence of functionally independent neural circuits within the olfactory system. Our results indicate that these circuits interconnect and weigh perception of social and habitat odour signals to generate appropriate behavioural responses according to mating state.