Summary/Abstract Nitric oxide (NO·) is a radical molecule produced by cells of the mammalian innate immune system as a defense against pathogens. While replication of enteric pathogens such as the Gram-negative bacterium Salmonella is inhibited by NO·, the mechanisms by which NO· exerts bacteriostatic effects are only partly understood. Salmonella encodes a flavohemoglobin, Hmp, as a defense against NO· but other pathways and cellular processes are also involved in the nitric oxide stress response. The goal of this proposal is to characterize the role that the transition metal ion manganese plays in promoting resistance to, and recovery from, nitrosative stress in Salmonella. Aim 1 seeks to investigate possible mechanisms underlying the prolonged period of bacteriostasis observed in manganese-limited Salmonella. Electrode-based probes and molecular assays will be used to monitor respiratory activity and cellular ATP levels. This aim will also investigate whether the ability to acquire manganese protects against DNA damage by determining the frequency of replication blocking-lesions and mutation rates. A role for manganese-requiring peptidases in promoting turnover of damaged proteins will be studied using genetic mutants. Aims 2&3 will use growth assays and enzyme activity assays in various genetic backgrounds. Aim 2 experiments will determine the activities of metalloenzymes that are putative targets of NO· inhibition and the repair rates of these enzymes following NO· exposure under manganese-replete and manganese-limited conditions. Aim 3 will investigate the requirements for efflux of manganese and import of iron and magnesium during late stages of recovery from nitrosative stress. Aim 4 will use RNA isolation and qPCR to investigate the expression and activity of alternative manganese-dependent enzymes during the nitrosative stress response. Characterizing the molecular targets of NO· will improve understanding of how this innate immune defense molecule exerts its bacteriostatic effects as well as how Salmonella can circumvent these actions. The long-term objective of this work is to identify novel candidates for inhibition by future antimicrobial therapies.