Nitric oxide (NO) is an important signaling molecule in higher eukaryotes, and is a member of the arsenal of toxic molecules deployed against invading pathogens. NO is also generated as a byproduct of nitrate and nitrite respiration, so nitrate-respiring bacteria such as Escherichia coli are exposed to endogenously-generated NO. Thus, biological responses to NO are widespread in both eukaryotes and prokaryotes, and attract considerable interest. In E. coli and related enteric bacteria, a major regulator of the response to NO is the recently described transcriptional activator designated NorR. NorR activates the expression of a flavorubredoxin (that reduces NO to nitrous oxide) encoded by the norV gene, by binding to sites in the promoter region of norV and activating transcription catalyzed by RNA polymerase containing the alternative sigma 54 factor. In the denitrifying bacterium Ralstonia eutropha, NorR regulates expression of the norB gene encoding NO reductase, as well as a gene designated norA that is of unknown function but is widespread in the genomes of nitrate respiring organisms. The overall goals of the research are to further understanding of the mechanism by which NorR responds to NO and activates norV expression, to identify new members of the NorR regulon, and to explore the role of the norA (ytfE) gene in E. coli. Specific objectives are: (1) to identify residues in NorR that are involved in sensing NO; (2) to identify additional NorR-regulated genes in the E. coli genome, making use of an unbiased genome-wide search; (3) to evaluate the individual roles of the three NorR binding sites and the two IHF binding sites in the norV promoter region, and (4) to test the hypothesis that the product of the NO-inducible ytfE gene has a physiological role during nitrate and nitrite respiration, and to identify the regulator of ytfE expression. The broader impact of this research will involve the training and teaching of post-doctoral, graduate and undergraduate researchers, and outreach to promote the science eduction of the broader community.
