NSF Award
9900533
Silverman, Philip M.<br/>MCB- 9900553<br/>Abstract<br/><br/> The long-term goal of this research is to elucidate the structural biology of conjugal DNA transfer between F+ and F- strains of Escherichia coli. Among several considerations that make this research both timely and important are the facts that conjugal DNA transfer is inextricably connected to the dissemination of antibiotic resistance and one of a growing number of mechanisms by which bacterial cells secrete macromolecules across their surface barriers. This research seeks to define how the DNA transfer (Tra) proteins encoded by the F plasmid are organized at the bacterial cell surface to create the organelle(s) that actually mediate DNA transfer. To simplify the project initially, the focus will be on the 15 or so Tra proteins (of about 30) that are required by F+ cells to elaborate F-pili. F-pili are filaments extending from the donor cell surface and required for the earliest, cell-cell contact stages of DNA transfer. They are built from a single subunit, F-pilin, though more than a dozen other Tra proteins are required for F-pilus biogenesis or affect F-pilus structure or function without abolishing filament assembly. The organization and functions of these Tra proteins are not at all understood. The first specific aim of the research is to identify protein-protein interactions among these Tra proteins by yeast two-hybrid assay, with the goal of creating a Tra protein linkage map. The second aim is to establish which of the interactions identified in yeast also occur in E. coli and are required for F-pilus biogenesis, with special attention paid to sets of Tra proteins suggested by two-hybrid assay to span the bacterial cell envelope. A combination of conditional (temperature-sensitive) interaction mutants, biochemical assays, and immunoelectron microscopy will be used to provide a secondary confirmation of the interactions predicted to occur by the linkage map in E. coli. The third specific aim of the research is to characterize selected Tra proteins, particularly those with purine nucleotide binding motifs, since these suggest specific biochemical functions. Biochemical and genetic approaches will be used to determine which, if either, of two Tra proteins with Walker box A motifs is an ATPase and, if so, whether or not this activity is essential for F-pilus biogenesis and function. <br/> This research will address the structural aspects of conjugation, which is a fundamental process in bacteria. The outcomes of this research may increase our understanding of the transfer of genetic material, including antibiotic resistance genes, in nature and should provide insight into the protein interactions that are essential for F-pilus formation and function.
