One common mechanism for acquisition of new traits in bacteria is by the transmission of DNA between a donor and a recipient cell via conjugation. This project will characterize the activities of a protein, TraD, essential for conjugation of the well-characterized F (fertility) plasmid of Escherichia coli. TraD is a member of a conserved and broadly distributed family of cytoplasmic membrane proteins that act during the DNA transfer step of conjugation. Preliminary studies have suggested that TraD is a DNA-dependent ATPase, possibly involved in energizing DNA transfer through a conjugation-specific pore in the cell envelope. In addition, TraD probably interacts with two other transfer (Tra) proteins necessary for DNA processing during conjugation, the cytoplasmic TraI (oriT nickase and DNA helicase) and TraM proteins. This work will characterize wild type and mutant forms of TraD in a variety of in vitro and in vivo assays, focusing on characterizing the enzymatic parameters of the TraD ATPase activity and determining its requirement in conjugation; defining interactions between TraD and TraM/TraI; and probing contacts between extra-cytoplasmic domains of TraD and other cell envelope proteins required to signal the initiation of DNA metabolism. TraD homologues are expressed by other conjugal plasmids, thus the study of the F plasmid TraD protein may have profound implications for understanding the transfer of genetic information via conjugation among a wide variety of bacterial genera.
