Schildbach 9733655 F factor, like all conjugative plasmids, directs its transfer from one bacterium to another, even between species. While the genetics of F factor have been studied extensively, little is known about the structural basis of conjugal transfer. The investigator is mounting a long-term effort to describe F factor conjugation in molecular detail. The initial research will concentrate on a complex of three proteins and plasmid DNA that facilitates DNA cleavage with strand and sequence specificity. Of these proteins, only plasmid-encoded TraI possesses nucleolytic activity. Integration host factor (IHF) and plasmid-encoded TraY are required for efficient TraI activity, although their roles are unknown. To better define TraY activity, the investigator will identify TraY amino acids involved in DNA recognition through site-directed mutagenesis, and will characterize the F plasmid DNA sequence bound by TraY. The binding of IHF and TraY to DNA will be assessed for cooperativity, and for its effect on DNA conformation. Crystallization of TraY will also be attempted, in preparation for eventual structure determination by X-ray crystallography. This research, coupled with teaching in lecture and seminar courses, is intended to demonstrate to graduate and undergraduate students the value of examining biological systems using interdisciplinary studies that include structural biology. Conjugation is an efficient mechanism for transferring DNA between even unrelated bacterial species. Consequently, acquisition of antibiotic resistance genes by pathogenic bacteria frequently occurs through conjugal transfer. Drugs that inhibit conjugation, given in concert with regular antibiotic treatment, may render antibiotics more effective and may reduce the incidence of antibiotic resistance. Design of such inhibitors requires an understanding of the structures and mechanisms of the proteins and DNA involved in conjugation. The proposed activities will provide understanding of one step i n bacterial conjugation, as well as instill in students an appreciation of the value and design of such studies.
