STABILITY GENES OF E FAECALIS PLASMID PAD1

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The broad, long-term objective of this proposal is to define the molecular mechanisms of action and regulation of the par stability determinant encoded on the Enterococcus faecalis plasmid pAD1. par is no larger than 404 nucleotides and encodes 2 small RNAs essential for its function. Evidence indicates that par functions as a post-segregational killing system. The larger of the 2 RNAs, RNA1, is the toxic component of the system and encodes and 33 amino acid peptide. The smaller RNA, RNA, RNAII, regulates RNAI activity. It is hypothesized that RNAII inhibits peptide translation as an antisense RNA and that the RNAI- encoded peptide inhibits some essential host function. Four specific aims will be pursued to test this hypothesis. First, detailed structural studies on both par RNAs and their interaction complex will be performed in vitro and in vivo. Second, potential autoregulatory effects on par RNA production will be examined by a variety of genetic approaches. Third, the toxin target will be determined by identifying host proteins that interact with the RNAI-encoded peptide. Fourth, other plasmid- encoded stability determinants that interact with par will be identified by conducting competition experiments with various constructs and by characterizing a potential active partition system located adjacent to par. It is hoped that the par system could be manipulated in some way to provide a useful antibacterial treatment for enterococci infection. Alternatively, identification of the par toxin target could provide a useful target for the design of new antibiotics effective against the enterococci.