Intellectual merit: The long-term goal of this research is to elucidate the structural biology and mechanism of horizontal DNA transfer mediated by bacterial type IV secretion systems (TFSS's). These systems comprise a broadly distributed group of molecular nano-machines that function to secrete macromolecules from gram-negative bacteria to other bacterial or eukaryotic cells. Several TFSS classes are distinguishable by sequence comparisons, and these systems may have arisen more than once during bacterial evolution. Type IV secretion systems play a crucial role in interaction networks within bacterial communities and between bacteria and other organisms, including plants and animals. With their capacity to transfer DNA as well as proteins, type IV systems as a group are directly involved in such phenomena as bacterial pathogenesis in plants and animals and biofilm formation, in addition to the dissemination of antibiotic resistance. Given their ubiquity and importance in the natural history of bacterial communities, detailed analysis of these systems is clearly important.
This renewal project addresses an archetypical TFSS, that which is encoded by the F plasmid of Escherichia coli. TFSS-F includes about 14 tra genes whose cognate Tra (DNA transfer) proteins are required to form conjugative (in this case F-) pili. Conjugative pili are surface filaments, approximately 9 nanometers in outer diameter and several micrometers long, that mediate early events required for horizontal DNA transfer among gram-negative bacteria. Dr. Silverman's previous NSF-supported work has defined Tra protein interaction groups that function in the assembly of F-pili. This project extends that work, focusing on a stable subassembly of Tra proteins that is believed to span the cell envelope, from the inner membrane (TraB) through the periplasm (TraB/TraK) to the outer membrane (TraK/TraV). Similar proteins in other TFSS's have been described as components of a core complex that functions as a translocation channel, or translocon. Based on the working hypothesis that the TraB/K/V subassembly is likewise part of a trans-envelope translocon, the specific aims incorporate biochemical as well as genetic strategies to address three issues: what Tra proteins comprise the core complex? what are the Tra protein requirements for complex formation or stability in vivo? and how do individual complex components interact with the others to yield the functional assembly? The specific aims are: 1, to establish the localization and orientation of TraB in Tra+ cells and its relation to the TraV lipoprotein, isolate and characterize the TraB/K/V complex, and determine the Tra protein composition of purified complexes by Western blot and mass spectometry; 2, to determine if the TraB/K/V complex requires other Tra proteins for stability or assembly in vivo and, if so, identify them, and 3, to identify protein-protein interaction domains among complex components, map interaction domains, and isolate complexes that form between the Tra segments and intact Tra proteins in vivo. By focusing on F-pilin secretion, this project complements efforts in numerous laboratories to establish the mechanism of DNA transfer mediated by type IV secretion systems. It is anticipated that the work will elucidate how conjugative pilin translocation and translocation of DNA-protein complexes are mechanistically related and guide development of in vitro assays necessary to understand the function of Tra proteins and protein assemblies that affect conjugative pili.
Broader impacts: The project includes a collaboration with Dr. Jim Bidlack, Professor of Biology at the University of Central Oklahoma, a predominantly undergraduate institution, and will provide research opportunities for undergraduate students from that institution. Dr. Silverman has recently been appointed "Leader in Residence" at the University of Central Oklahoma, whereby he will serve as a role model and mentor for the faculty during the 06-07 academic year.
