Genetic Functions of an Enterococcal R Factor

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DESCRIPTION (Verbatim from Applicant's Abstract): The enterococci have become prevalent as causes of nosocomial infections. The high incidence of resistance of these organisms to the most efficacious antibiotics (e.g., vancomycin) causes major problems in treating enterococcal infections, and the tremendous reservoir of enterococcal resistance determinants serves as a vector for the spread of these genes to other, more pathogenic bacterial genera. This research is focused on dissection of a mechanism of horizontal genetic transfer of the antibiotic resistance plasmid pCF10 in Enterococcus faecalis. The most novel feature of this transfer system is that the pCF10-containing donor cell perceives the presence of potential recipients in its vicinity by sensing a small peptide signal (a sex pheromone called cCF10) excreted by the recipients. The donor cell only expresses genes required for plasmid transfer when exogenous cCF10 is detected in the growth medium. Both the donor cells response to exogenous pheromone, and the negative control system that prevents expression of transfer functions in the absence of exogenous pheromone, are complex processes that have been studied in detail. Enterococci produce a variety of peptide pheromones, consisting of hydrophobic peptides 7-8 amino acids in length. Different families of plasmids each encode a highly specific response to a single cognate pheromone. When a single cell carrying multiple pheromone plasmids is exposed to one pheromone, only the corresponding plasmid is transferred, even though the sensing systems are quite similar for all the plasmids examined to date. In the next funding period, the focus of the experiments will be on the molecular and genetic basis for the specificity of the pheromone response. The specific aims are: 1) Determine the molecular basis for specificity of pheromone cCF10 interactions with PrgZ (the extracellular pCF10-encoded pheromone binding protein), and with PrgX (the putative intracellular receptor for cCF10 believed to comprise the molecular switch involved in the intracellular phase of pheromone induction. 2) Determine the molecular basis for specific abolition of endogenous cCF10 activity in pCF10-containing donor cells by PrgY (a pCF10-encoded membrane protein), and by iCF10 (a plasmid-encoded peptide inhibitor of cCF10). 3) Determine the molecular basis for the activity and specificity of a novel regulatory RNA, Qa in blocking expression of conjugation in uninduced cells via its interaction with PrgX and with the Qs RNA encoded in the positive control region of pCF10. 4) Begin an experimental analysis of the genetic and molecular basis for specificity of the downstream steps in pheromone induction. These steps include post-transcriptional activation of transfer gene expression, and conjugative DNA processing.