Characterization of the pheromone response of the Enterococcus faecalis conjugative plasmid pCF10: complete sequence and comparative analysis of the transcriptional and phenotypic responses of pCF10-containing cells to pheromone induction

Abstract

The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10(-1) transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.

FIG. 1.

Physical map of plasmid pCF10. Putative ORFs and their orientations are shown. The colors indicate the closest homology of the protein products to E. faecalis plasmids pAD1 (red), pAM373 (yellow), pPD1 (magenta), pRE25 (orange), and pTEF2 (blue). ORFs found only on pCF10 are purple. Regions with homology to chromosomal regions of S. agalactiae NEM316 are indicated in green, and similarities to L. lactis are shown in white. The blue inner circle indicates the region of 95 to 97% homology on the nucleotide level between the plasmids pCF10 and pTEF2.

FIG. 2.

Phylogenetic analysis of pheromone plasmids and selected plasmid-encoded proteins. (A and B) Pustell DNA matrix comparing pAD1 with pAM373 (A) or pCF10 (B). pAM373 is more closely related to pAD1 than pCF10, despite the lack of a surface exclusion protein and a nonhomologous AS. Tn925 was omitted from pCF10 for the purpose of this comparison. (C) Comparison of the prgC gene product and its homologues situated downstream of AS. (D) Comparison of described AS on pheromone plasmids and the chromosome of E. faecalis V583. The AS of pCF10/pTEF2 and pAD1/pTEF1 are identical. (E) Comparison of the pheromone plasmid negative regulator family TraA/PrgX. TraA of the plasmids pAD1/pTEF1 is identical, in contrast to PrgX on pCF10 and pTEF2. Methods used include neighbor joining and best tree; distance was determined by Poisson correction, and gaps are distributed proportionally.

FIG.3.

Spotfire analysis of cCF10 induction of plasmid pCF10 in E. faecalis. (A) Gene expression at 15, 30, 60, 120, and 480 min is shown in comparison to that in the uninduced control after induction of strain OG1RF(pCF10) with 100 pg of cCF10/ml. The color bar on the right indicates expression of background. An increase of 1.5-fold is considered significant. (B) Expression profile of the reading frames prgY, prgQ, prgA, and prgB. prgQ and prgA showed their highest expression 30 min after induction and prgB peaked at the 60-min time point, while prgY as a gene in the negative regulatory region remained unaltered during the time course of cCF10 induction. (C) Expression profile of reading frames prgE, prgJ, prgL, and pcfA. The prgB downstream reading frames showed no significant expression at the 30-min time point and reached their peak at 60 min, with prgL and pcfA at higher expression levels than prgB at that time point. The expression of all selected reading frames returned to uninduced levels at the 120-min time point. (D) No significant induction of transcription can be seen in the Tn925 upstream region and the reading frames pcfS, pcfT, pcfY, and pcfZ.

FIG. 4.

Phenotypic effects of pheromone induction. (A) Plasmid transfer. Donor OG1RF(pCF10) was induced with 100 pg of cCF10/ml. At the indicated time points, donor cells were added to the recipient OG1SSp. After a 10-min mating, donor and transconjugants were enumerated as described in Materials and Methods. The graph represents the T/D frequency. (B) AS expression. OG1RF(pCF10) was induced with 100 pg of cCF10/ml. Surface extracts were prepared at the indicated time points. Protein extracts were separated on an 8% polyacrylamide gel electrophoresis gel, and AS was detected by Western blot analysis with AS-specific antibodies. Equivalent amounts of protein were loaded in each lane. The 156- and 78-kDa markers represent the full-length and amino-terminal segment of AS.