title: NsrR-dependent method for detecting nitric oxide accumulation in the Escherichia coli cytoplasm and enzymes involved in NO production.
abstract: A β-galactosidase assay for detecting the accumulation of NO in the Escherichia coli cytoplasm has been developed based on the sensitive response of the transcription repressor, NsrR, to NO. The hcp promoter is repressed by NsrR in the absence of nitric oxide, but repression is relieved when NO accumulates in the cytoplasm. Most, but not all, of this NO is formed by the interaction of the membrane-associated nitrate reductase, NarG, with nitrite. External NO at physiologically relevant concentrations does not equilibrate across the E. coli membrane with NsrR in the cytoplasm. The periplasmic nitrite reductase, NrfAB, is not required to prevent equilibration of NO across the membrane. External NO supplied at the highest concentration reported to occur in vivo does not damage FNR sufficiently to affect transcription from the hcp or hmp promoters or from a synthetic promoter. We suggest that the capacity of E. coli to reduce NO is sufficient to prevent its accumulation from external sources in the cytoplasm.
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Vine
authors.forename: Sukhjit K
authors.initials: SK
authors.lastname: Purewal
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Cole
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D009154:Mutation; D050901:Nitrate Reductase / Q000235:genetics / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis* / Q000494:pharmacology; D009572:Nitrite Reductases / Q000235:genetics / Q000378:metabolism; D009573:Nitrites / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D013312:Stress, Physiological; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D009566:Nitrates; D009573:Nitrites; C529899:NsrR protein, E coli; D014157:Transcription Factors; D009569:Nitric Oxide; D009572:Nitrite Reductases; C079730:NirB protein, E coli; C508365:NarG protein, E coli; D050901:Nitrate Reductase; D001616:beta-Galactosidase