Analysis of the amino acid sequence specificity determinants of the enterococcal cCF10 sex pheromone in interactions with the pheromone-sensing machinery

Abstract

The level of expression of conjugation genes in Enterococcus faecalis strains carrying the pheromone-responsive transferable plasmid pCF10 is determined by the ratio in the culture medium of two types of signaling peptides, a pheromone (cCF10) and an inhibitor (iCF10). Recent data have demonstrated that both peptides target the cytoplasmic receptor protein PrgX. However, the relative importance of the interaction of these peptides with the pCF10 protein PrgZ (which enhances import of cCF10) versus PrgX is not fully understood, and there is relatively little information about specific amino acid sequence determinants affecting the functional interactions of cCF10 with these proteins in vivo. To address these issues, we used a pheromone-inducible reporter gene system where various combinations of PrgX and PrgZ could be expressed in an isogenic host background to examine the biological activities of cCF10, iCF10, and variants of cCF10 isolated in a genetic screen. The results suggest that most of the amino acid sequence determinants of cCF10 pheromone activity affect interactions between the peptide and PrgX, although some sequence variants that affected peptide/PrgZ interactions were also identified. The results provide functional data to complement ongoing structural studies of PrgX and increase our understanding of the functional interactions of cCF10 and iCF10 with the pheromone-sensing machinery of pCF10.

FIG. 1.

Extracellular (A) and intracellular (B) peptide-mediated control circuits in the pCF10 system. (A) Pheromone cCF10 (LVTLVFV) is produced (double arrows) from the chromosome (thin line with broken end) by proteolytic processing of the chromosomal ccfA gene product (3), whereas inhibitor iCF10 (AITLIFI) is produced by processing of the polypeptide encoded by the prgQ gene (38) of pCF10 (thin circle); prgQ is actually the first gene in a long, pheromone-inducible conjugation operon. Either endogenously produced or exogenous cCF10 pheromone can induce expression of Asc10 and other pCF10-encoded conjugation gene products. Pheromone activity of cCF10 is inhibited by iCF10, but previous work has not fully elucidated whether this occurs inside the cell, outside the cell, or in both locations. In a plasmid-containing donor cell, the pCF10-encoded PrgY protein reduces the amount of cCF10 produced, and the cell produces enough iCF10 to neutralize the residual cCF10 that escapes PrgY control (9, 10, 25); this precise balance in opposing activities can be shifted by addition of exogenous cCF10 from recipient cells. (B) Working model for the function of PrgX as the intracellular switch controlling pheromone-inducible conjugation. The protein consists of an N-terminal DNA binding domain (oval) connected by a flexible linker to a central dimerization domain (filled box), which also contains a peptide binding pocket (39). When cCF10 pheromone occupies this pocket, the PrgX C-terminal regulatory domain (represented by a thin, curved line) undergoes a marked shift to allow for interactions of amino acids between positions 295 and 300 with the bound peptide; this displaces a loop in this domain (residues 287 to 294) that is important for PrgX tetramer formation (39). In complexes with iCF10, PrgX amino acids between residues 311 and 315 interact with the bound peptide; this actually stabilizes the position of the tetramer-promoting loop. Tetramers of PrgX are predicted to simultaneously bind two operator sites in pCF10 DNA more stably than a pair of unlinked dimers. One of these operators overlaps the prgQ promoter, such that conjugation is repressed when it is occupied by PrgX (6, 7).

FIG. 2.

Diagram of pheromone-coding sequence (66 bp) and primers used for oligonucleotide-directed random mutagenesis of plasmid pPCR-4. The coding sequence is derived from the prgQ open reading frame in pCF10 and includes the prgQ promoter (P_Q) and altered coding sequence so that cCF10 is expressed instead of the native iCF10 (indicated in checked region of the arrow); the complete DNA sequence for the start codon, the pheromone-coding region, and the four codons immediately upstream is shown. Primers used for random PCR mutagenesis are indicated below; the bracketed region of the prgQ-ccfA degenerate primer indicates the region directed for degenerate base incorporation (see Materials and Methods).

FIG. 3.

Importance of PrgZ and PrgX in cCF10 response. (A) PrgX requirement for cCF10 response as shown by measuring β-galactosidase induction of plasmid p043lacZ carrying prgX (see text) or a an isogenic plasmid with a deletion in prgX, p043lacZ dX (dX), harbored by either a wild-type strain (OG1Sp) or an isogenic strain with a chromosomal copy of prgX (100-5). Strains grown overnight in M9 medium were diluted 1:5 with no peptides added (black bars), or peptides were added as indicated. cCF10 was added at 10 ng/ml (dark gray bars), and in some cases iCF10 was also added at 10 times (100 ng/ml, light gray bars) or 100 times (1,000 ng/ml, white bars) the cCF10 concentration. (B and C) Response to exogenously added cCF10 in the absence (vector control, gray bars) or presence (pMSP6043-1, white bars) of PrgZ. Cultures grown overnight in M9 medium were diluted 1:5, and cCF10 was added in increasing amounts and harvested at 90 min (B) or added at 5 ng/ml and harvested at the indicated time points (C). Data shown are representative of at least two independent experiments done in duplicate; error bars represent one standard deviation of the mean.

FIG. 4.

Activity of cCF10 and variant peptides to induce clumping of OG1RF(pCF10) cultures. Activity was measured as the reciprocal of the highest dilution of E. coli-produced or synthetically derived cCF10 or mutant derivatives to aggregate an OG1RF(pCF10) indicator strain. The relative amounts of pheromone activity are representative of at least two independent experiments. The peptide LVTLVF- (V7Δ) was not found in the original assay but was synthesized and tested in the clumping assay. Supernatants from E. coli strains expressing wild-type (WT) cCF10 (LVTLVFV) from pPCR-4 or each of the mutant peptides were assayed for activity (light bars) following overnight growth with aeration in LB medium. Activities of synthesized peptides (dark bars) were assayed from twofold serial dilutions of a 50-ng/ml stock solution.

FIG. 5.

PrgZ effects on p043lacZ induction ability of peptides. Synthetic cCF10 variant peptides were added at 5 ng/ml to cCF10-deficient strain JRC104 harboring p043lacZ with a second plasmid expressing PrgZ from pMSP6043-1 (Z⁺, dark bars) or a vector control, pMSP6043-2 (Z⁻, light bars). Peptides were added immediately after a 1:5 dilution from stationary-phase cultures, and β-galactosidase induction levels were measured after 90 min incubations. Data shown are representative of at least two independent experiments done in duplicate; error bars represent one standard deviation of the mean.