During conjugal plasmid transfer the movement of plasmid DNA across the donor cell membrane and wall systems is accomplished through the activities of Type IV transport systems. The VirB complex of Agrobacterium tumefaciens is the prototypical membrane localized Type IV transport complex. In addition to its role in plant transformation, the VirB complex can support the conjugal transfer of plasmid RSF1010 between agrobacteria. The goals of this project were developed in response to an unexpected finding: expression of specific subsets of the VirB proteins in a recipient Agrobacterium strain increases the frequency of virB-mediated conjugal RSF1010 transfer into it by over three orders of magnitude. This is intriguing because the mechanism by which Type IV transport substrates cross the recipient cell envelope are not clear. The specific aims of the work are to identify genes in A. tumefaciens that are, along with the VirB proteins, required for the increase in the capacity to serve as a conjugal recipient. Additionally, cellular and molecular analysis of the mutant recipients will be carried out, focusing on novel strategies to analyze mating pair formation as well as experiments to examine the expression, localization and role of the identified proteins in the recipient phenotype. In relation to the broader impact of the research, plasmid conjugal transfer is the most prevalent means of antibiotic resistance spread in a wide variety of environments ranging from soil to hospital intensive care units. Yet the role of the recipient cell is poorly understood, particularly in systems that require solid surfaces for successful mating. Defining how the recipient affects the efficiency of transfer has implications for microbial ecology and population biology as well as the more medically oriented problem of antibiotic resistance.
