Analysis of DNA Transfer During Conjugation

University Of Washington, Seattle WA

Investigators

Abstract

Conjugation is an efficient means for transfer of genetic information among diverse bacterial species and leads to the dissemination of a variety of traits, including antibiotic resistance determinants and catabolic pathways. Conjugation relies on a donor cell engaging a recipient cell to form a mating pair, assembling an apparatus in the cell envelope through which DNA can be transferred, and processing DNA to produce the transferred substrate. In a broader context, the process of conjugation is related to a mechanism that pathogens use to export virulence determinants into eukaryotic cells. This project focuses on the characterization of the DNA transfer stage of bacterial conjugation for the F plasmid of Escherichia coli, using both genetic and biochemical approaches. Experiments will explore the nature of the apparatus that mediates the passage of the transferred DNA molecule through the membrane and how this apparatus contacts the DNA processing machinery. The studies will define important domains for different transfer (Tra) proteins involved in these processes (specifically, TraI, TraD, and TraM) and test interactions between those proteins. A collection of TraD and TraI mutants will enable analysis of relevant protein structure and protein-protein interactions. Comparisons of the behaviors of wildtype and mutant proteins will test for the importance of observed interactions to the conjugation process.