Identification and characterization of a determinant (eep) on the Enterococcus faecalis chromosome that is involved in production of the peptide sex pheromone cAD1

Abstract

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.

FIG. 1

Map of the pAD1 regulatory region with features relating to control of the pheromone response. Regulation is geared to controlling transcriptional readthrough of the two transcription terminator sites t1 and t2 from the iad promoter. The pheromone (cAD1) is imported into the cell via a host-encoded uptake system, a process aided by the plasmid-encoded surface protein TraC. The negatively regulating (neg. reg.) TraA binds directly to cAD1, which results in an upregulation of transcription from the iad promoter, some of which passes through the terminators to ultimately generate TraE1. The transcript mD is present at high levels in the uninduced state and plays a significant role in preventing transcriptional readthrough of t1; it becomes greatly reduced upon induction. The traB product is involved in shutdown of endogenous cAD1 in plasmid-containing cells. traC encodes a surface protein that binds to exogenous pheromone (43). Arrows below the map indicate relative degrees of transcription in various regions; horizontal arrows above the map represent orientations of the indicated determinants.

FIG. 2

Nucleotide and protein sequences of eep. Segments corresponding to specific primers used for generating PCR products as well as for the primer extension study are underlined, as are regions corresponding to possible Shine-Dalgarno (SD), −10, and −35 sequences. The beginning of a downstream open reading frame corresponding to what is likely for a prolyl-tRNA synthetase is also shown.

FIG. 3

Southern blot hybridization of E. faecalis chromosomal DNA digested with EcoRI. The probe corresponded to pAM3329 carrying an internal segment of eep. Lane 1, OG1X; lane 2, FA3328 (eep mutant); lane 3, FA3329 (eep mutant); lane 4, FA3328R; lane 5, FA3329R; lane 6, pAM3329.

FIG. 4

Primer extension analysis. Lanes 1 and 2 represent two different extractions of RNA from E. faecalis OG1X cells. The sequence data relate to use of the same primer, using as a template pAM3327. The asterisks 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. 2.