title: Nitric oxide reactivities of the two globins of the foodborne pathogen Campylobacter jejuni: roles in protection from nitrosative stress and analysis of potential reductants.
abstract: BACKGROUND
  During infection and pathogenesis, Campylobacter, the leading cause of gastroenteritis, encounters NO and reactive nitrogen species (RNS) derived from the host. To combat these species, Campylobacter jejuni expresses two haemoglobins: the single domain haemoglobin (Cgb) detoxifies NO but the role of the truncated globin (Ctb) is unclear. Confirmation of Cgb activity and more extensive exploration of Ctb function(s) in vivo are restricted due to difficulties in expressing proteins in Campylobacter and our lack of understanding of how the globin haems are re-reduced after ligand reactions.
  
  
  METHODS
  The cgb and ctb genes were cloned under the control of arabinose-inducible promoters and the globins expressed in an Escherichia coli mutant lacking the main NO detoxification mechanisms (Hmp and the Nor system comprising the transcription regulator NorR, the flavorubredoxin and its reductase (NorVW)); cellular responses under oxidative and nitrosative stress conditions were assessed. Spectroscopic changes of the Cgb and Ctb haems in soluble fractions after oxidation by NO were evaluated. Construction of E. coli nor mutants and a ubiquinone-defective strain allowed the exploration of the flavorubredoxin reductase and the aerobic respiratory chain as candidates for Cgb electron donors in E. coli mutants.
  
  
  RESULTS
  Cgb, but not Ctb, complements the NO- and RNS-sensitive phenotype of an E. coli hmp mutant in aerobic conditions; however, Cgb fails to protect an hmp norR mutant in the absence of oxygen. Reduction of Cgb and Ctb in E. coli and C. jejuni soluble extracts and turnover after NO oxidation is demonstrated. Finally, we report a minor role for NorW as a Cgb reductase partner in E. coli but no role for respiratory electron flux in globin redox cycling.
  
  
  CONCLUSIONS
  The NO detoxification capacity of Cgb is confirmed by heterologous expression in E. coli. The reducibility of Cgb and Ctb in E. coli and C. jejuni extracts and the lack of dependence of reduction upon flavorubredoxin reductase and the respiratory chain in E. coli argue in favor of a non-specific reductase system.
  
  
  CONCLUSIONS
  We present the most persuasive evidence to date that Cgb, but not Ctb, confers tolerance to NO and RNS by reaction with NO. Since certain hypotheses for the mechanism of haem re-reduction in E. coli following the reaction with NO are not proven, the mechanisms of reduction in C. jejuni now require challenging experimental evaluation.
authors.affiliation: Department of Molecular Biology & Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK. Electronic address: mbp09mt@sheffield.ac.uk.
authors.forename: Mariana
authors.initials: M
authors.lastname: Tinajero-Trejo
authors.forename: Angie
authors.initials: A
authors.lastname: Vreugdenhil
authors.forename: Svetlana E
authors.initials: SE
authors.lastname: Sedelnikova
authors.forename: Kelly S
authors.initials: KS
authors.lastname: Davidge
authors.forename: Robert K
authors.initials: RK
authors.lastname: Poole
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000378:metabolism*; D004093:Dihydropteridine Reductase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D015538:Nitrosation / Q000502:physiology*; D010084:Oxidation-Reduction; D026422:S-Nitrosoglutathione / Q000378:metabolism; D013312:Stress, Physiological / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism; D054793:Truncated Hemoglobins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C489659:Cgb protein, Campylobacter jejuni; D029968:Escherichia coli Proteins; D006420:Hemeproteins; C504602:NorR protein, E coli; D015534:Trans-Activators; D054793:Truncated Hemoglobins; D009243:NAD; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D004093:Dihydropteridine Reductase; C076538:hmp protein, E coli; D009247:NADH, NADPH Oxidoreductases
keywords: (Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; 1-(hydroxyl-NNO-azoxy)-L-proline; Bacterial haemoglobin; Campylobacter jejuni; Campylobacter single domain haemoglobin; Campylobacter truncated haemoglobin; Cgb; Ctb; DETA NONOate; E. coli flavorubredoxin protein; E. coli flavorubredoxin reductase; Escherichia coli; GSNO; Heterologous expression; Hmp; NOS; Nitrosative stress; NorR; NorV; NorW; NssR; PROLI NONOate; RNS; ROS; Reductase; S-nitrosoglutathione; flavohaemoglobin; nitric oxide synthase; nitrosative stress sensing Regulator; reactive nitrogen species; reactive oxygen species; transcriptional regulator of norVW genes in E. coli