Identification of the cAD1 sex pheromone precursor in Enterococcus faecalis

Abstract

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.

FIG. 1.

Key regions of the nucleotide sequence of cad along with related amino acids in E. faecalis FA2-2. The region representing vAD1 of E. faecalis V583 is also marked (box). The arrows approximate the locations representing specific primers used for PCR and for the primer extension experiment shown in Fig. 3. The locations of the −10 and −35 hexamers of the promoter, as well as the transcription start site (bent arrow) and the ribosome binding site (S. D.), are noted.

FIG. 2.

Amino acid sequence of Cad and hydrophilicity plot. (A and B) Sequences and comparison, respectively, of the 77-residue repeats. (C) Hydrophilicity plot. In panel A, large letters represent cAD1 and repeated sequences are underlined. In panel B, asterisks indicate where the amino acids differ.

FIG. 3.

Primer extension analysis of RNA representing cad. The primer used was rev-per-1. The asterisk marks the thymine corresponding to the 3′ end of the extended fragment; its complementary adenosine represents the 5′ transcriptional start site that is noted in Fig. 1.

FIG. 4.

Effects of the substitution of alanine in various positions within cAD1 on the clumping and mating response. E. faecalis strain DS16 was used as the responder for the clumping assays. The concentration of synthetic peptide used in each case was 1μg/ml. For the mating induction assays, the peptide concentration used was 50 ng/ml. The donor strain was FA2-2/pAM714, and the recipient was JH2SS. In each case, donors were exposed to the peptide for 45 min and then mixed with recipients for 10 min before being plated on selective medium. Note that a substitution at position 7 was not done because an alanine is already present at that position in cAD1. Solid bars, clumping titer; shaded bars, frequency per donor with induction; open bars, frequency per donor without induction.

FIG. 5.

View of how Eep and SPase II may act within the cytoplasmic membrane in the processing of the precursor (Cad) protein to generate cAD1 and the related lipoprotein. Note that the processing of the 21-residue inhibitor precursor pAD1-containing cells resembles that of Cad after removal of the lipoprotein moiety from the latter. Arrows point to cleavage sites.