title: Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemostat conditions reveal major changes in methionine biosynthesis.
abstract: Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction. Nitric oxide and S-nitrosothiols can be metabolized by bacteria, but only a few enzymes have been shown to be important in responses to such stresses. Glycerol-limited chemostat cultures in defined medium of Escherichia coli MG1655 were used to provide bacteria in defined physiological states before applying nitrosative stress by addition of S-nitrosoglutathione (GSNO). Exposure to 200 microm GSNO for 5 min was sufficient to elicit an adaptive response as judged by the development of NO-insensitive respiration. Transcriptome profiling experiments were used to investigate the transcriptional basis of the observed adaptation to the presence of GSNO. In aerobic cultures, only 17 genes were significantly up-regulated, including genes known to be involved in NO tolerance, particularly hmp (encoding the NO-consuming flavohemoglobin Hmp) and norV (encoding flavorubredoxin). Significantly, none of the up-regulated genes were members of the Fur regulon. Six genes involved in methionine biosynthesis or regulation were significantly up-regulated; metN, metI, and metR were shown to be important for GSNO tolerance, because mutants in these genes exhibited GSNO growth sensitivity. Furthermore, exogenous methionine abrogated the toxicity of GSNO supporting the hypothesis that GSNO nitrosates homocysteine, thereby withdrawing this intermediate from the methionine biosynthetic pathway. Anaerobically, 10 genes showed significant up-regulation, of which norV, hcp, metR, and metB were also up-regulated aerobically. The data presented here reveal new genes important for nitrosative stress tolerance and demonstrate that methionine biosynthesis is a casualty of nitrosative stress.
authors.affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Janet
authors.initials: J
authors.lastname: Flatley
authors.forename: Jason
authors.initials: J
authors.lastname: Barrett
authors.forename: Steven T
authors.initials: ST
authors.lastname: Pullan
authors.forename: Martin N
authors.initials: MN
authors.lastname: Hughes
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.forename: Robert K
authors.initials: RK
authors.lastname: Poole
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004093:Dihydropteridine Reductase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005990:Glycerol / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism; D006710:Homocysteine / Q000378:metabolism; D008715:Methionine / Q000378:metabolism*; D008954:Models, Biological; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026422:S-Nitrosoglutathione / Q000378:metabolism*; D015398:Signal Transduction; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D012333:RNA, Messenger; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D006710:Homocysteine; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D008715:Methionine; D013385:Succinate Dehydrogenase; D004093:Dihydropteridine Reductase; C076538:hmp protein, E coli; D009247:NADH, NADPH Oxidoreductases; D009584:Nitrogen; D005990:Glycerol