title: Redox-mediated interactions of VHb (Vitreoscilla haemoglobin) with OxyR: novel regulation of VHb biosynthesis under oxidative stress.
abstract: The bacterial haemoglobin from Vitreoscilla, VHb, displays several unusual properties that are unique among the globin family. When the gene encoding VHb, vgb, is expressed from its natural promoter in either Vitreoscilla or Escherichia coli, the level of VHb increases more than 50-fold under hypoxic conditions and decreases significantly during oxidative stress, suggesting similar functioning of the vgb promoter in both organisms. In the present study we show that expression of VHb in E. coli induced the antioxidant genes katG (catalase-peroxidase G) and sodA (superoxide dismutase A) and conferred significant protection from oxidative stress. In contrast, when vgb was expressed in an oxyR mutant of E. coli, VHb levels increased and the strain showed high sensitivity to oxidative stress without induction of antioxidant genes; this indicates the involvement of the oxidative stress regulator OxyR in mediating the protective effect of VHb under oxidative stress. A putative OxyR-binding site was identified within the vgb promoter and a gel-shift assay confirmed its interaction with oxidized OxyR, an interaction which was disrupted by the reduced form of the transcriptional activator Fnr (fumurate and nitrate reductase). This suggested that the redox state of OxyR and Fnr modulates their interaction with the vgb promoter. VHb associated with reduced OxyR in two-hybrid screen experiments and in vitro, converting it into an oxidized state in the presence of NADH, a condition where VHb is known to generate H2O2. These observations unveil a novel mechanism by which VHb may transmit signals to OxyR to autoregulate its own biosynthesis, simultaneously activating oxidative stress functions. The activation of OxyR via VHb, reported in the present paper for the first time, suggests the involvement of VHb in transcriptional control of many other genes as well.
authors.affiliation: Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India.
authors.forename: Arvind
authors.initials: A
authors.lastname: Anand
authors.forename: Brian T
authors.initials: BT
authors.lastname: Duk
authors.forename: Sandeep
authors.initials: S
authors.lastname: Singh
authors.forename: Meltem Y
authors.initials: MY
authors.lastname: Akbas
authors.forename: Dale A
authors.initials: DA
authors.lastname: Webster
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Stark
authors.forename: Kanak L
authors.initials: KL
authors.lastname: Dikshit
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D054793:Truncated Hemoglobins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D020589:Vitreoscilla / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D012097:Repressor Proteins; D054793:Truncated Hemoglobins; C499299:oxyR protein, E coli; C082613:hemoglobin protein, Vitreoscilla; D006861:Hydrogen Peroxide; C102664:hydroperoxidase II; D002374:Catalase; C051253:katG protein, E coli; C095500:SodA protein, Bacteria; D013482:Superoxide Dismutase