Regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii by CydR (Fnr). Sensitivity to oxygen, reactive oxygen species, and nitric oxide.
Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant nitrogen fixation requires cytochrome bd. Regulation of cytochrome bd expression is achieved by CydR (an Fnr homologue), which represses transcription of the oxidase genes cydAB. cydAB mRNA was mapped by primer extension; the transcriptional start site was determined, and putative -10 and -35 regions were deduced. Two "CydR boxes," one at the +1 site and one upstream of the -35 region, were identified. Transcriptionally inactive, purified CydR was converted, by adding NifS, cysteine, and Fe(2+), into an active form possessing acid-labile sulfide and spectra suggesting a [4Fe-4S](2+) cluster. Reconstituted CydR specifically bound both CydR boxes cooperatively, with higher affinity for the nearer consensus +1 site. Low concentrations of O(2) or NO ([O(2)]/[[CydR] or [NO]/[CydR] = 0.1-0. 6) elicited loss of the 420 nm absorbance attributed to the [4Fe-4S](2+) cluster, formation of a 315 nm species, and loss of ability to retard DNA migration. Retardation by reconstituted CydR was enhanced by superoxide dismutase and/or catalase, suggesting a role for reactive oxygen species in CydR inactivation. The role of CydR in regulating cydAB expression in the supposedly anoxic cytoplasm of A. vinelandii and similarities to cydAB regulation by Fnr in Escherichia coli are discussed.
Wu
G
G
Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Cruz-Ramos
H
H
Hill
S
S
Green
J
J
Sawers
G
G
Poole
R K
RK
D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins; D001483:Base Sequence; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics*; D017931:DNA Primers; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C107798:CydR protein, Azotobacter vinelandii; D003573:Cytochrome b Group; D003580:Cytochromes; D017931:DNA Primers; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D009569:Nitric Oxide; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D010100:Oxygen