Nitric Oxide Sensing and Response in Vibrio Cholerae

Nitric oxide (NO) is a radical capable of inhibiting bacterial growth. Bacteria in turn have multiple mechanisms of resisting the toxic effects of NO, usually encoded by genes under the control of NO-responsive transcription factors. However, our knowledge of the protein targets of NO is limited, as is the function of many NO-regulated genes. We studied two genes in V. cholerae, hmpA and nnrS, which encode a flavohemoglobin and a protein of unknown function, respectively, both predicted to be under control of the NO-responsive transcription factor NorR. We confirmed that both promoters were regulated by NorR and found that all three genes were important for growth in the presence of NO stress. We then performed a metabolomic study on multiple strains of V. cholerae, finding new potential metabolic targets of NO. In particular we found that substrates of iron-sulfur cluster-containing proteins accumulated in strains lacking nnrS, and that aconitase activity was decreased in cell-free extracts of nnrS mutants. Chelation of ferrous iron reversed the growth defect imposed by nnrS deletion; furthermore, strains lacking nnrS possessed lower ferrous iron concentrations. These data suggest that NnrS, a protein of previously unknown function, protects against the formation of NO-iron complexes. We also found that hmpA and norR are important for survival during colonization of the mouse intestines in response to host-generated NO, whereas nnrS is dispensable