The long-term goal of this research is to develop a detailed, integrated view of how Sinorhizobium meliloti establishes the chronic intracellular infection that underlies symbiosis and to use these findings to gain new insights into molecular mechanisms used by chronic intracellular pathogens of mammals. This research is suggesting possible new drug targets for Brucella, a serious, hard-to-treat pathogen and a bioterrorism threat, offering detailed insights into the roles of lipopolysaccharides and exopolysaccharides in bacterial interactions with their eukaryotic hosts, and also helping elucidate the unknown part of vitamin B12 biosynthesis. We will continue to investigate the biosynthesis and function of vitamin B12 in S. meliloti by analyzing the 5,6-dimethylbenzimidazole biosynthetic pathway and investigating which B12-dependent enzymes are of symbiotic importantance in S. meliloti. We will continue to investigate CbrA, a previously unrecognized master regulator of symbiosis, including identifying regulatory targets through microarray analysis, determining the basis of detergent-sensitivity of cbrA mutants, and determining the role of the Brucella CbrA homolog in pathogenesis. We will determine the molecular basis of the interactions between symbiotically active rhizobial exopolysaccharides and the plant host by utilizing M. truncatula microarrays to identify plant genes regulated in response to succinoglycan and then dissecting the function of these genes using RNAi knockdowns and other approaches. We will continue to investigate the importance of lipopolysaccharide modifications for symbiosis and with a focus on BacA, including comparing plant responses to bacA mutants to that of a succinoglycan-deficient mutant, determining the topology of BacA protein, and screening for proteins that interact with BacA. We will continue to investigate the role of manganese in oxidative stress protection and symbiosis by further characterizing control of superoxide dismutase levels by manganese, investigating additional factors in superoxide sensitivity, testing the possible symbiotic importance of other Mn2+dependent enzymes, and testing the importance of non-homologous end-joining for symbiosis. We will determine the biochemical and physiological function of pmh, a highly conserved bacterial gene and a possible new drug target whose inactivation greatly sensitizes cells to numerous antibiotics that are no longer clinically useful because of acquired drug resistance.