Peptide Sex Pheromone Research Articles

Hormonal and pheromonal studies on amphibians with special reference to metamorphosis and reproductive behavior.

In this article, we have reviewed studies which have been conducted to investigate the hormonal influence on metamorphosis in the bullfrog (Rana catesbeiana) and Japanese toad (Bufo japonicus) larvae, in addition to studies conducted on the hormonal and pheromonal control of reproductive behavior in red-bellied newts (Cynops pyrrhogaster). Metamorphosis was studied with an emphasis on the roles of prolactin (PRL) and thyrotropin (TSH). The release of PRL was shown to be regulated by thyrotropin-releasing hormone (TRH) and that of TSH was evidenced to be regulated by corticotropin-releasing factor. The significance of the fact that the neuropeptide that control the secretion of TSH is different from those encountered in mammals was discussed in consideration of the observation that the release of TRH, which stimulates the release of PRL, is enhanced when the animals are subjected to a cold temperature. Findings that were made by using melanin-rich cells of Bufo embryos and larvae, such as the determination of the origin of the adenohypophyseal primordium, identification of the pancreatic chitinase, and involvement of the rostral preoptic recess organ as the hypothalamic inhibitory center of α-melanocyte-stimulating hormone (α-MSH) secretion have been mentioned in this article. In addition, the involvement of hormones in eliciting the courtship behavior in the male red-bellied newts and discovery of the peptide sex pheromones and hormonal control of their secretion have also been included in the present article. This article is protected by copyright. All rights reserved.

Mechanisms of peptide sex pheromone regulation of conjugation in Enterococcus faecalis.

In many gram positive bacteria, horizontal transfer and virulence are regulated by peptide‐mediated cell‐cell signaling. The heptapeptide cCF10 (C) activates conjugative transfer of the Enterococcus faecalis plasmid pCF10, whereas the iCF10 (I) peptide inhibits transfer. Both peptides bind to the same domain of the master transcription regulator PrgX, a repressor of transcription of the prgQ operon encoding conjugation genes. We show that repression of prgQ by PrgX tetramers requires formation of a pCF10 DNA loop where each of two PrgX DNA‐binding sites is occupied by a dimer. I binding to PrgX enhances prgQ repression, while C binding has the opposite effect. Previous models suggested that differential effects of these two peptides on the PrgX oligomerization state accounted for their distinct functions. Our new results demonstrate that both peptides have similar, high‐binding affinity for PrgX, and that both peptides actually promote formation of PrgX tetramers with higher DNA‐binding affinity than Apo‐PrgX. We propose that differences in repression ability of PrgX/peptide complexes result from subtle differences in the structures of DNA‐bound PrgX/peptide complexes. Changes in the induction state of a donor cell likely results from replacement of one type of DNA‐bound peptide/PrgX tetramer with the other.

Interspecies transfer of vancomycin, erythromycin and tetracycline resistance among Enterococcus species recovered from agrarian sources

BackgroundEnterococci are now well recognised for their ability to transfer antibiotic resistance and for their association with nosocomial infections, but less is known regarding their relevance in the wider environment. Enterococcus faecalis and Enterococcus faecium were isolated from a range of agrarian associated sources (low-flow water, septic tank, poultry litter, high flow water, slurry/soil) and were assessed for latent ability to transfer antimicrobial resistance.ResultsThe isolates were tested for phenotypic clumping in the presence of cell-free supernatant from other isolates. Some isolates were identified which demonstrated clumping, indicating that they possessed peptide sex pheromone conjugal machinery. All isolates were also tested for antibiotic resistance phenotypes using both disc diffusion and minimum inhibitory concentration (MIC) assays. These tests revealed that the enterococci demonstrated both phenotypic clumping and antibiotic resistance phenotypes. Based on these selection criteria, the isolates were identified as having the potential for horizontal gene transfer and were used to investigate the transfer of multiple antibiotic resistance phenotypes. Conjugal transfer of antibiotic resistance phenotypes was determined using a solid agar mating method followed by a standard antibiotic selection test resulting in different transfer patterns. An interspecies conjugal transfer of vancomycin resistance from E. faecalis to E. faecium was identified while the remaining reactions were within the same species. Transfer efficiencies ranging from 2 × 10−1 to 2.3 × 10−5 were determined based on the reactions of three donor isolates (MF06036, MF0410 and MF06035) and two recipient isolates (MW01105Rif and ST01109Rif), with the transfer of vancomycin, erythromycin and tetracycline resistance genes.ConclusionsThe conjugation reactions and selection conditions used in this study resulted in a variety of co-transferred resistance phenotypes suggesting the presence of different mobile elements in the set of natural isolates. This study highlights the potential for extensive horizontal gene transfer in a previously neglected reservoir for enterococci.

Enterococcal Sex Pheromones: Signaling, Social Behavior, and Evolution

In Enterococcus faecalis, lateral transfer of conjugative plasmids that encode antibiotic resistance and virulence determinants can be induced by peptide sex pheromones. The tetracycline-resistance plasmid pCF10 represents a paradigm for illustrating important conserved features of a large family of pheromone-responsive enterococcal plasmids. The pheromone is released into the growth medium by plasmid-free recipient cells and sensed by plasmid-containing donors. The activity of the pheromone is antagonized by a plasmid-encoded inhibitor peptide that prevents conjugation in the absence of an inducing signal and is also required to return the system to the ground state following an induction cycle. The pheromone response involves multiple transcriptional and posttranscriptional mechanisms as well as bi-stable biological switch behavior. Multiple layers of regulation are essential for proper function, and evolution of this tight control system may have been favored by reduction of the fitness cost of plasmid maintenance to the host cell.

Antagonistic self-sensing and mate-sensing signaling controls antibiotic-resistance transfer

Conjugation is one of the most common ways bacteria acquire antibiotic resistance, contributing to the emergence of multidrug-resistant "superbugs." Bacteria of the genus Enterococcus faecalis are highly antibiotic-resistant nosocomial pathogens that use the mechanism of conjugation to spread antibiotic resistance between resistance-bearing donor cells and resistance-deficient recipient cells. Here, we report a unique quorum sensing-based communication system that uses two antagonistic signaling molecules to regulate conjugative transfer of tetracycline-resistance plasmid pCF10 in E. faecalis. A "mate-sensing" peptide sex pheromone produced by recipient cells is detected by donor cells to induce conjugative genetic transfer. Using mathematical modeling and experimentation, we show that a second antagonistic "self-sensing" signaling peptide, previously known to suppress self-induction of donor cells, also serves as a classic quorum-sensing signal for donors that functions to reduce antibiotic-resistance transfer at high donor density. This unique form of quorum sensing may provide a means of limiting the spread of the plasmid and present opportunities to control antibiotic-resistance transfer through manipulation of intercellular signaling, with implications in the clinical setting.

A Complex Set of Sex Pheromones Identified in the Cuttlefish Sepia officinalis

BackgroundThe cephalopod mollusk Sepia officinalis can be considered as a relevant model for studying reproduction strategies associated to seasonal migrations. Using transcriptomic and peptidomic approaches, we aim to identify peptide sex pheromones that are thought to induce the aggregation of mature cuttlefish in their egg-laying areas.ResultsTo facilitate the identification of sex pheromones, 576 5′-expressed sequence tags (ESTs) were sequenced from a single cDNA library generated from accessory sex glands of female cuttlefish. Our analysis yielded 223 unique sequences composed of 186 singletons and 37 contigs. Three major redundant ESTs called SPα, SPα′ and SPβ were identified as good candidates for putative sex pheromone transcripts and are part of the 87 unique sequences classified as unknown. The alignment of translated SPα and SPα′ revealed a high level of conservation, with 98.4% identity. Translation led to a 248-amino acid precursor containing six peptides with multiple putative disulfide bonds. The alignment of SPα-α′ with SPβ revealed a partial structural conservation, with 37.3% identity. Translation of SPβ led to a 252-amino acid precursor containing five peptides. The occurrence of a signal peptide on SPα, SPα′ and SPβ showed that the peptides were secreted. RT-PCR and mass spectrometry analyses revealed a co-localization of transcripts and expression products in the oviduct gland. Preliminary in vitro experiments performed on gills and penises revealed target organs involved in mating and ventilation.ConclusionsThe analysis of the accessory sex gland transcriptome of Sepia officinalis led to the identification of peptidic sex pheromones. Although preliminary functional tests suggested the involvement of the α3 and β2 peptides in ventilation and mating stimulation, further functional investigations will make it possible to identify the complete set of biological activities expected from waterborne pheromones.

Structural analysis of the Anti-Q–Qs interaction: RNA-mediated regulation of E. faecalis plasmid pCF10 conjugation

Conjugation of the E. faecalis plasmid pCF10 is triggered in response to peptide sex pheromone cCF10 produced by potential recipients. Regulation of this response is complex and multi-layered and includes a small regulatory RNA, Anti-Q that participates in a termination/antitermination decision controlling transcription of the conjugation structural genes. In this study, the secondary structure of the Anti-Q transcript and its sites of interaction with its target, Qs, were determined. The primary site of interaction occurred at a centrally-located loop whose sequence showed high variability in analogous molecules on other pheromone-responsive plasmids. This loop, designated the specificity loop, was demonstrated to be important but not sufficient for distinguishing between Qs molecules from pCF10 and another pheromone-responsive plasmid pAD1. A loop 5′ from the specificity loop which carries a U-turn motif played no demonstrable role in Anti-Q–Qs interaction or regulation of the termination/antitermination decision. These results provide direct evidence for a critical role of Anti-Q–Qs interactions in posttranscriptional regulation of pCF10 transfer functions.

Twenty years of ISAREN: An amphibian biologist in Wonderland

The 6th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology (ISAREN), the former International Symposium on Amphibian Endocrinology (ISAE), was recently held in Berlin. ISAREN developed from two symposia on amphibian biology held in European countries in 1988–1990. In this article, the history of ISAREN was briefly stated. In addition, some of the topics of our researches carried out in collaboration with several groups, using various amphibian species during the past 20 years and/or presented in the past symposia were reviewed. The topics included the discovery of pancreatic chitinase, involvement of growth hormone in vitellogenin synthesis, changes of ANF-like immunoreactivity in the frogs sent into the space, discovery of a peptide sex-pheromone, origin of the epithelial pituitary, and hypothalamic regulation of thyroid-stimulating hormone.

Characterization of an Active Partition System for theEnterococcus faecalisPheromone-Responding Plasmid pAD1

Enterococcus faecalis plasmid pAD1 is a 60-kb conjugative, low-copy-number plasmid that encodes a mating response to the peptide sex pheromone cAD1 and a cytolytic exotoxin that contributes to virulence. Although aspects of conjugation have been studied extensively, relatively little is known about the control of pAD1 maintenance. Previous work on pAD1 identified a 5-kb region of DNA sufficient to support replication, copy control, and stable inheritance (K. E. Weaver, D. B. Clewell, and F. An, J. Bacteriol. 175:1900-1909, 1993), and recently, the pAD1 replication initiator (RepA) and the origin of vegetative replication (oriV) were characterized (M. V. Francia, S. Fujimoto, P. Tille, K. E. Weaver, and D. B. Clewell, J. Bacteriol. 186:5003-5016, 2004). The present study focuses on the adjacent determinants repB and repC, as well as a group of 25 8-bp direct repeats (iterons with the consensus sequence TAGTARRR) located between the divergently transcribed repA and repB. Through mutagenesis and trans-complementation experiments, RepB (a 33-kDa protein, a member of the ParA superfamily of ATPases) and RepC (a protein of 14.4 kDa) were shown to be required for maximal stabilization. Both were active in trans. The iteron region was shown to act as the pAD1 centromere-like site. Purified RepC was shown by DNA mobility shift and DNase I footprinting analyses to interact in a sequence-specific manner with the iteron repeats upstream of the repBC locus. The binding of RepC to the iteron region was shown to be modified by RepB in the presence of ATP via a possible interaction with the RepC-iteron complex. RepB did not bind to the iteron region in the absence of RepC.

Structure of peptide sex pheromone receptor PrgX and PrgX/pheromone complexes and regulation of conjugation inEnterococcus faecalis

Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone binding destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.

Bacterial Pheromone for Sex and Abstinence

Bacteria can transfer DNA through a process called conjugation, and the transfer of these extrachromasomal plasmid DNAs contributes to virulence and antibiotic resistance. Chandler et al. report that in Enterococcus faecalis , a mammalian pathogen, the same pheromone that stimulates donor bacteria to initiate conjugation with plasmid-free recipients is also produced by the donor bacteria and regulates the donor sensitivity to recipient-produced pheromone. The bacterial chromosome encodes the pheromone (cCF10), so both the donor and the recipient can produce this molecule. To prevent conjugation with other donor cells, the donor cells have two mechanisms for suppressing the response to the endogenously produced pheromone. The plasmid in the donor cells contains the prgQ operon, which encodes genes responsible for conjugation and genes responsible for inhibition of conjugation. One of the conjugation inhibitors is a secreted inhibitor protein, iCF10, which binds and sequesters secreted cCF10, and another is the membrane protein PrgY, which degrades or binds cCF10 as it is released. Using mutant bacterial strains that lacked functional cCF10, Chandler et al. show that cCF10 produced by the donor cells stimulates the production of iCF10. Donor cells grown in human plasma or in vivo produce the plasmid-encoded aggregation factor Asc10, which contributes to cellular invasion and virulence of the bacteria. The mutant bacteria that did not produce functional cCF10 did not aggregate when exposed to plasma. Albumin was identified by iCF10 affinity chromatography as the protein that bound to iCF10, thereby shifting the balance between iCF10 and cCF10, allowing self-induction of the conjugation genes, including the one encoding the aggregation factor Asc10. Donor bacteria use self-produced cCF10 to set the sensitivity to recipient-produced cCF10 through an autocrine signaling pathway from cCF10 to iCF10. When only self-produced cCF10 is present, the majority is inactivated, and that which is not stimulates iCF10 production. Under conditions where iCF10 abundance is decreased or cCF10 is secreted in abundance by recipient cells, the iCF10 to cCF10 ratio is decreased and conjugation is initiated. J. R. Chandler, J. Hirt, G. M. Dunny, A paracrine peptide sex pheromone also acts as an autocrine signal to induce plasmid transfer and virulence factor expression in vivo . Proc. Natl. Acad. Sci. U.S.A. 102 , 15617-15622 (2005). [Abstract] [Full Text]

A paracrine peptide sex pheromone also acts as an autocrine signal to induce plasmid transfer and virulence factor expressionin vivo

The peptide pheromone cCF10 of Enterococcus faecalis is an intercellular signal for induction of conjugative transfer of plasmid pCF10 from donor cells to recipient cells. When a donor cell is exposed to recipient-produced cCF10, expression of the pCF10-encoded aggregation substance of pCF10 (Asc10) and other conjugation gene products is activated. Asc10 also increases enterococcal virulence in several models, and when donor cells are grown in animals or in plasma, Asc10 expression is induced by means of the cCF10-sensing machinery. Plasmid pCF10 carries two genes that function to prevent self-induction by endogenous cCF10 in donor cells. The membrane protein PrgY reduces endogenous pheromone activity in donor cells, and the inhibitor peptide iCF10 neutralizes the residual endogenous cCF10 that escapes PrgY. In the current study, we found that E. faecalis strains with allelic replacements abolishing active cCF10 production showed reduced ability to acquire pCF10 by conjugation; prgY-null mutations had no phenotype in the cCF10-negative strains. We observed that expression of the mRNA for iCF10 was reduced in this background and that these mutations also blocked plasma induction of Asc10 expression. These findings support a model in which plasma induction in wild-type donors results from iCF10 inactivation by a plasma component, causing disruption of a precisely maintained balance of iCF10 to cCF10 activity and allowing subsequent induction by endogenous cCF10. Although cCF10 has traditionally been viewed as an intercellular signal, these results show that pCF10 has also adapted cCF10 as an autocrine signal that activates expression of virulence and conjugation functions.

Functional analysis of TraA, the sex pheromone receptor encoded by pPD1, in a promoter region essential for the mating response in Enterococcus faecalis.

Conjugative transfer of a bacteriocin plasmid, pPD1, of Enterococcus faecalis is induced in response to a peptide sex pheromone, cPD1, secreted from plasmid-free recipient cells. cPD1 is taken up by a pPD1 donor cell and binds to an intracellular receptor, TraA. Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells. In this study, we discuss how TraA transduces the signal of cPD1 to the mating response. Gel mobility shift assays indicated that TraA is bound to a traA-ipd intergenic region, which is essential for cPD1 response. DNase I footprinting analysis suggested the presence of one strong (tab1) and two weak (tab2 and tab3) TraA-binding sites in the intergenic region. Primer extension analysis implied that the transcriptional initiation sites of traA and ipd were located in the intergenic region. Northern analysis showed that cPD1 upregulated and downregulated transcription of ipd and traA, respectively. The circular permutation assay showed that TraA bent a DNA fragment corresponding to the tab1 region, and its angle was changed in the presence of cPD1 or iPD1. From these data, we propose a model that TraA changes the conformation of the tab1 region in response to cPD1 and upregulates the transcription of ipd, which may lead to expression of genes required for the mating response.

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 pAMα1, 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.

Peptide pheromone-induced transfer of plasmid pCF10 in Enterococcus faecalis: probing the genetic and molecular basis for specificity of the pheromone response

The tetracycline resistance plasmid pCF10 represents a class of unique mobile genetic elements of the bacterial genus Enterococcus, whose conjugative transfer functions are inducible by peptide sex pheromones excreted by potential recipient cells. These plasmids play a significant role in the dissemination of virulence and antibiotic resistance genes among the enterococci, which have become major nosocomial pathogens. Pheromone response by plasmid-carrying donor cells involves specific import of the peptide signal molecule, and subsequent interaction of the signal with one or more intracellular regulatory gene products. The pheromones are chromosomally encoded hydrophobic octa- or hepta-peptides, and different families of homologous plasmids encode the ability to respond to each pheromone. Among the four pheromone-responsive plasmids that have been characterized in some detail, there is considerable conservation in the genes encoding pheromone sensing and regulatory functions, and the peptides themselves show considerable similarity. In spite of this, there is extremely high specificity of response to each peptide, with virtually no “cross-induction” of transfer of non-cognate pheromone plasmids by the pheromones. This communication reviews the evidence for this specificity and discusses current molecular and genetic approaches to defining the basis for specificity.

Vancomycin Resistance Plasmid in Enterococcus faecalis That Encodes Sensitivity to a Sex Pheromone Also Produced by Staphylococcus aureus

Plasmid-free (recipient) strains of Enterococcus faecalis secrete a number of peptide sex pheromones that act as mating (conjugation) signals to donor bacteria harboring certain conjugative plasmids. (See reference [4][1] for a recent review.) Upon acquisition of such a plasmid, the related

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.

Enterococcus faecalis conjugative plasmid pAM373: complete nucleotide sequence and genetic analyses of sex pheromone response.

pAM373 is a 36.7 kb conjugative plasmid in Enterococcus faecalis that encodes a response to a peptide sex pheromone, cAM373, secreted by plasmid-free (recipient) strains of enterococci. It was identified over 15 years ago as one of five plasmids in E. faecalis strain RC73 and was of interest because a related pheromone activity could be detected in culture supernatants of Staphylococcus aureus and Streptococcus gordonii. Because of increased clinical concern relating to the possibility of mobilizing vancomycin resistance determinants from enterococci, where they are becoming common, into pathogens such as S. aureus, efforts were initiated to characterize pAM373 further. The results of a complete nucleotide sequence determination of pAM373, as well as a genetic analysis of key genes related to regulation of the pheromone response, are reported here. With regard to determinants related to conjugation, the plasmid has a structural organization similar to other known pheromone-responsive plasmids such as pAD1, pCF10 and pPD1; however, there are several unique features. Although there are significant homologues relating to a pheromone-binding surface protein (TraC) and a negatively regulating protein (TraA), there is an absence of a determinant equivalent to traB of pAD1 (reduces endogenous pheromone) and a determinant for surface-exclusion protein. The precursor structure of the inhibitor peptide iAM373 was identified, and its determinant (iam373) was found to be about 500 nt upstream of an apparent transcription terminator t1. Tn917-lac insertion analyses provided interesting insights into aspects of control of the pheromone response and showed that, although the traA product is sensitive to pheromone, it appears to act differently from the traA homologue of pAD1.

A pAD1-encoded small RNA molecule, mD, negatively regulates Enterococcus faecalis pheromone response by enhancing transcription termination.

pAD1 is a 60-kb hemolysin-bacteriocin plasmid in Enterococcus faecalis that encodes a conjugative mating response to a peptide sex pheromone, cAD1, secreted by plasmid-free bacteria. The pheromone response is regulated by two proteins: TraE1, which positively regulates all or most conjugative structural genes, and TraA, which negatively regulates traE1. TraA binds to pAD1 DNA at the iad (encoding the inhibitor peptide iAD1) promoter but is released upon binding to imported pheromone. This leads to enhanced transcription through two closely spaced downstream terminators (t1 and t2) into traE1. TraE1 is believed to then upregulate itself from a site located within t2; thus, a small amount of transcription through t1-t2 could lead to overall induction. It is important therefore that the t1-t2 terminators be tightly controlled to keep the response shut down in the absence of pheromone. A small (200-nucleotide) RNA molecule designated mD is encoded just upstream of t1 by a determinant (traD) oriented in the direction opposite to that of transcripts utilizing t1. mD is expressed at high levels in the uninduced state, but it decreases significantly upon induction. Here we present results of genetic studies relating to the activity of t1-t2 and show that mD strongly enhances transcriptional termination at t1. The mD activity is shown to influence transcription well downstream and can affect the determinant for aggregation substance asa1. The phenomenon is specific in that there is no effect of mD on the unrelated pheromone-responding plasmids pPD1 and pCF10.

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.