Genetic characterization of factors influencing expression of the conjugation genes of the Enterococcus faecalis plasmid pAD1.

Abstract

The Enterococcus faecalis plasmid pAD1 conjugatively transfers in response to a sex pheromone, cAD1, excreted by potential recipient cells. pAD1 genes responsible for regulation of pAD1 transfer include traA, which encodes a negative regulator of the pheromone response, and the E region, which is believed to encode at least one positive regulator. The nucleotide sequence and transcription of traA and a portion of the E region were analyzed. traA was deduced to encode a protein of 319 amino acids with a molecular weight of 37,856. The amino acid sequence showed limited homology to several DNA binding proteins. An ORF within the E region, designated traE1, was also identified. Insertional mutagenesis of this ORF resulted in complete loss of plasmid transfer capability. Analysis of Tn917-lac insertions giving rise to transcriptional lacZ fusions provided some insight into the roles of potential regulatory signals within and around the nucleotide sequences reported here. A regulatory role for certain key transcription termination signals seems evident. Phase variation, affecting pAD1 transfer was also characterized. Spontaneous dry colony (dry$\sp{\rm c}$) variants of E. faecalis (pAD1) were found to arise at a frequency of $10\sp{-4}-10\sp{-2}$. Reversion of dry$\sp{\rm c}$ variants to a cAD1-inducible phenotype (dry$\sp+$) occurred at a similar frequency. Dry$\sp{\rm c}$ phase variants constitutively expression aggregation and plasmid transfer functions. Results obtained from LacZ fusion studies and complementation tests suggest that the site of mutation(s) responsible for phase variation is within the regulation-related region of pAD1. Phase variation affecting mating functions is an alternative (pheromone independent) method of regulating pAD1 transfer.