Conjugative plasmids play an important role in transmissible antibiotic resistance, toxin production, and resistance to host defense mechanisms. The proposed research plan will contribute to an understanding of bacterial conjugation at the molecular level. The studies utilize the classic Escherichia coli sex factor F since it is genetically the best analyzed. Methods will be developed for the purification and detailed characterization of some of the most important proteins involved in conjugation, making extensive use of genetic analysis and mutant proteins. A detailed biochemial analysis of the F sex factor traT gene product, an outer membrane lipoprotein involved in both resistance to killing by serum complement and surface exclusion (a property whereby a donor bacterium is itself a poor recipient in conjugation) will be carried out. In order to define those portions of the protein that are involved in the specific contracts needed for surface exclusion or for resistance to serum complement, an analysis is proposed which includes cell surface labelling of exposed portions of TraTp, as well as the determination of altered amino acids in mutant traT proteins. A model is proposed for conjugative DNA metabolism and transport whereby the traD gene product serves as a membrane anchor for the DNA unwinding enzyme helicase I encoded by the traI gene. In this model, the energy associated with DNA unwiding and the processive translocation of helicase I with respect to DNA is directly converted into the motive force for trasnport of plasmid DNA into the recipient bacterum. Mutant studies will be used to demonstrate the functional significance of the TraDp-TraIp interaction. In vitro systems for nicking at the origin of DNA transfer and for transport of DNA into a membrane vesicle will be developed. Expression of the 20 or more F tra genes is subject to positive regulation by the traJ gene. A chimeric plasmid in which galK is expressed from the tra operon promoter subject to traJ control, will be used to isolate traJ-independent and traJ site-of-action mutants in order to elucidate the mechanism of action of TraJp.
