Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY

Abstract

Conjugative transfer of the Enterococcus faecalis plasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently.

Post-liberation cleavage of signal peptides is catalyzed by the site-2 protease (S2P) in bacteria

“…Clearance of SPs from the membrane might be important for protein secretion in this organism, as the B. subtilis rasP disruptant exhibits a weak defect in protein secretion (19). In accordance with our conclusion, it was reported that several peptide sex pheromones in Enterococcus faecalis are produced from the lipoprotein SP through S2P (Eep)-dependent, endoproteolytic processing (20,21). We suggest that S2P proteases in bacteria have a role equivalent to that of SPPs in higher eukaryotes.…”

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

“…1). In uninduced cells, basal transcription from the P Q promoter generates a 380-nt transcript, Q S , which includes a single ORF (prgQ) encoding a 22-aa polypeptide processed into I and secreted into the growth medium (10). An increased intracellular level of C, resulting from addition of exogenous C or C-producing recipient cells, shifts the PrgX structure and oligomerization state from a repressing to a nonrepressing conformation, resulting in induction (11,12).…”

“…To suppress selfinduction by endogenously produced C in donors, a heptapeptide (AITLIFI) inhibitor molecule iCF10 (I) is encoded by the first gene in the polycistronic prgQ operon of pCF10 (9,10). Both C and I are synthesized initially as prepeptides that, after cleavage of the leader sequence, are secreted into the extracellular environment as active peptides (9) (Fig.…”

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

“…In an intriguing interplay between sensor and signal, it has been observed that the oligopeptide pheremone signals of enterococci are generated from proteolytic processing of lipoprotein signal peptides and taken up by lipoprotein-dependent olipopeptide ABC permeases [66,67]. These oligopeptides are released from the lipoprotein signal peptide by intramembrane proteases such as Eep, most likely following the cleavage of the signal peptide from the lipoprotein precursor by the action of Lsp [68].…”