Study of stability genes of E. faecalis plasmid pAD1

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Enterococcus faecalis is a Gram-positive bacterium and a member of the normal flora of the intestinal tract. It is also capable of causing opportunistic infection and has gained notoriety recently because of a propensity to rapidly accumulate antibiotic resistance genes. These genes are commonly located on mobile genetic elements that facilitate their dissemination to other bacterial species. The objective of this proposal is to determine the molecular mechanism of action of a genetic locus, par, required for the stable inheritance of the pheromone-responsive conjugative plasmid pAD1. Par operates via a post-segregationat killing mechanism, programming for death any cell that loses the plasmid. Par is regulated by a unique mechanism involving inhibition of toxin translation by interaction of its message with an antisense RNA at multiple dispersed sites and the formation of a stable complex. The goal of Aim 1 of this proposal is to determine the mechanism by which the complex is activated for translation in plasmid-free segregants. This will involve transcriptional analysis of the par RNAs, identification of sequence elements and RNases involved in complex degradation, and evaluation of sequence elements required for stability of the toxin mRNA. Expression of the par toxin, Fst, disrupts the integrity of the host cell membrane. The goal of Aim 2 of this proposal is to determine the mechanism of action of Fst. Two models of Fst function, one involving processing by cellular proteases and one involving selective action at the internal surface of the cytoplasmic membrane, will be tesed. In addition, the characteristics of Fst-resistant mutants will be determined in an effort to identify the specific target of Fst. Finally, saturation mutagenesis will be performed on Fst in an effort to identify which amino acids are essential for its activity.