Conjugation is the primary vehicle for the horizontal transfer virulence factor genes, such as antibiotic resistance, within and between bacterial strains. In certain epicenters, such as hospitals in less developed parts of the world, immune-compromised patients and misuse of antibiotics combine to select for the development and dissemination of these pathogenicity factors via conjugation. Inhibition of conjugation would prove a boon for curbing the creation and spread of new virulent or multi-drug resistant strains. DNA relaxases are the keystone proteins of each conjugative system. TraI is the relaxase of the F plasmid, the archetypal model system for conjugation. Toward revelation and inhibition of relaxase mechanisms, I used bioinformatics and limited proteolysis to find and identify new domains on the F TraI enzyme. I then solved TraI/DNA co-crystal structures that showed a novel DNA binding mode. Based on structure comparisons, sequence conservation, and mutant activity studies, I proposed a mechanism for TraI activity that required the existence of a dual phosphotyrosine intermediate, which has since been observed. I then demonstrated that bisphosphonate compounds mimicking this intermediate are nanomolar in vitro TraI inhibitors. I determined the relaxase structure in complex with one such inhibitor, bound as predicted. We showed that several of these compounds are potent in cell inhibitors of conjugation that often selectively kill conjugation-capable cells, a novel antibiotic paradigm. We showed that oral treatment of gnotobiotic mice with two inhibitors, clodronate and etidronate, decreased the gastrointestinal F+ bacterial load twentyfold without apparent side effects. Beyond the model system, etidronate showed selective in cell potency versus cells harboring a known clinical resistance-bearing R100 plasmid. Etidronate and clodronate are already clinically approved for the treatment of bone loss in humans. Toward more general relaxase inhibition phylogenetic analyses and studies with other medically relevant relaxases, including viral replicative relaxases, are ongoing. Advisors/Committee Members: Lujan, Scott, Redinbo, Matthew R., Wolfenden, Richard, Pielak, Gary J., Matson, Steven, Temple, Brenda.