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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028925-07
Application #
3276297
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-08-01
Project End
1988-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
7
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Carnegie-Mellon University
Department
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Panicker, M M; Minkley Jr, E G (1992) Purification and properties of the F sex factor TraD protein, an inner membrane conjugal transfer protein. J Biol Chem 267:12761-6
Dash, P K; Traxler, B A; Panicker, M M et al. (1992) Biochemical characterization of Escherichia coli DNA helicase I. Mol Microbiol 6:1163-72
Bradshaw Jr, H D; Traxler, B A; Minkley Jr, E G et al. (1990) Nucleotide sequence of the traI (helicase I) gene from the sex factor F. J Bacteriol 172:4127-31
Traxler, B A; Minkley Jr, E G (1988) Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein. J Mol Biol 204:205-9
Traxler, B A; Minkley Jr, E G (1987) Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport. J Bacteriol 169:3251-9
Ippen-Ihler, K A; Minkley Jr, E G (1986) The conjugation system of F, the fertility factor of Escherichia coli. Annu Rev Genet 20:593-624
Panicker, M M; Minkley Jr, E G (1985) DNA transfer occurs during a cell surface contact stage of F sex factor-mediated bacterial conjugation. J Bacteriol 162:584-90