Mutagenesis of a gene encoding a cytochrome o-like terminal oxidase of Azotobacter vinelandii: a cytochrome o mutant is aero-tolerant during nitrogen fixation.
The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo-type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A. vinelandii by homologous recombination. The mutant contained no spectrally detectable cytochrome o. However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd) was 11-fold higher than in the wild-type strain. Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o. Cytochrome o-deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd, which is required for respiratory protection of oxygen-labile nitrogenase.
Leung
D
D
Division of Life Sciences, Kings College London, UK.
van der Oost
J
J
Kelly
M
M
Saraste
M
M
Hill
S
S
Poole
R K
RK
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D001483:Base Sequence; D002746:Chlorpromazine / Q000494:pharmacology; D003001:Cloning, Molecular; D003576:Electron Transport Complex IV / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009586:Nitrogen Fixation / Q000502:physiology; D010101:Oxygen Consumption / Q000187:drug effects
C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; D002746:Chlorpromazine