Leung D, van der Oost J, Kelly M, Saraste M, Hill S, Poole R K
Division of Life Sciences, Kings College London, UK.
FEMS Microbiol Lett. 1994 Jun 15;119(3):351-7. doi: 10.1111/j.1574-6968.1994.tb06912.x.
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.
通过PCR从维涅兰德固氮菌染色体DNA扩增得到的一个0.55 kb片段，其核苷酸序列经翻译后得到的氨基酸序列与大肠杆菌cyoB基因（编码细胞色素bo型喹啉氧化酶的亚基I）的一部分有57%的同源性。该片段经体外插入卡那霉素抗性盒而发生突变，并通过同源重组导入维涅兰德固氮菌的染色体。该突变体中没有光谱可检测到的细胞色素o。然而，在生长稳定期，交替氧化酶（细胞色素bd）的水平比野生型菌株高11倍。该突变体的呼吸作用对氯丙嗪不敏感，氯丙嗪是一种被认为特异性作用于细胞色素o的抑制剂。缺乏细胞色素o的突变体在空气中能固定氮，这清楚地将这种氧化酶的作用与细胞色素bd的作用区分开来，细胞色素bd是对氧敏感的固氮酶进行呼吸保护所必需的。
