Weaver 9723098 A small determinant, no larger than 404 bp, has been identified on the bacterium Enterococcus faecalis plasmid pAD1 that is essential for stable inheritance of the plasmid. This determinant, designated par, stabilizes the plasmid via a post-segregational killing mechanism. Stability determinants of this class stabilize plasmid inheritance in a bacterial population by killing host cells that lose the plasmid. Such systems are common on plasmids native to Gram-negative bacteria and generally encode a stable toxin and an unstable antidote. The antidote prohibits either toxin activity or translation. Par is the first such system identified on a plasmid native to a Gram-positive bacterium. Preliminary evidence suggests that par is regulated by a unique antisense RNA mechanism. The par "antidote" is a small RNA that contains segments complementary to both the 3' and 5' ends of the toxin-encoding RNA. The 5' complementary segments would be expected to sequester the translational start site for the proposed toxin of the system, but the 3' end may be necessary for initial interactions between the RNAs since it encodes the only complementary stem-loop structure present in both RNAs. Such stem-loops have been shown to be important for RNA-RNA interaction in other antisense systems. The putative par toxin is a small peptide of 33 amino acids. Evidence indicates that the toxin target is involved in chromosomal separation and/or cell division. The objectives of this project are to i) determine the molecular mechanisms of regulation of par function, ii) determine the nature of the toxin, and iii) identify the molecular target of the toxin. Three experimental approaches will be taken to address the first objective. First, par will be cloned on a conditional replicon and its effects on host viability assessed after a switch to non-permissive conditions. Plasmid loss should lead to host cell death. This experiment will verify the post-segregational nature of par-mediated killing. Second, relative stabilities of the antisense antidote and the toxin message will be determined in rifampicin-exposed cells. If par is regulated like other post-segregational systems, the antisense antidote should be less stable than the toxin message. Processing intermediates of the toxin message may also be identified by this approach. Third, the interaction between the par RNAs will be reproduced in vitro, where the specific elements involved in the interaction will be determined. RNAs will be produced in vitro using templates produced by PCR and linked to T7 RNA polymerase promoters. Interactions will be assessed by fractionation on various polyacrylamide gel formulations. Mutations of elements suspected of being required for interaction will be introduced by PCR-mediated mutagenesis. The second objective will be approached from both genetic and biochemical directions. In the genetic approach, the effects of mutations in the proposed toxin reading frame but not affecting the RNA structure and vice versa will be assessed. If the peptide is the toxin, mutations affecting its coding frame should result in loss of function regardless of effect on RNA structure. In the biochemical approach, production of the peptide will be assessed in vitro in the presence and absence of the antidote. In addition, antibody will be produced to peptides related to the putative toxin. These peptides will be produced either synthetically or using translational fusion vectors. The antibodies will be used to visualize the toxin in cell extracts by Western blot and may also be used for protein purification. The third objective will be addressed by isolating mutations in E. faecalis host cells resistant to the over-production of toxin. The affected gene will be identified by complementation analysis. Possible effects of toxin over-production on other bacterial hosts, such as Escherichia coli and Bacillus spp. will also be determined. This study should make a significant contribution to the understandi ng of the maintenance of extra-chromosomal genetic elements, particularly in Gram-positive hosts. Furthermore, the unique nature of the complementarity between the two par RNAs suggests that the study of their interaction may provide information on the variety of ways in which RNAs can interact, information that would be useful in fields ranging from evolution to the development of antisense therapeutics. Finally, the par toxin may provide a useful tool for the study of chromosomal separation and/or cell division in E. faecalis, a Gram-positive coccus. Very little data on these basic life processes are available for such organisms.
