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 sequenc e of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromos omal DNA by PCR, was 57% identical to part, of the Escherichia coli cy oB gene, encoding subunit I of the cytochrome bo-type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resista nce cassette and introduced into the chromosome of A. vinelandii by ho mologous 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 th e wild-type strain. Respiration of the mutant was insensitive to chlor promazine, an inhibitor thought to act specifically on cytochrome o. C ytochrome o-deficient mutants fixed nitrogen in air, clearly distingui shing the role of this oxidase from that of cytochrome bd, which is re quired for respiratory protection of oxygen-labile nitrogenase.