ISOLATION AND CHARACTERIZATION OF MUTANTS DEFECTIVE IN THE CYANIDE-INSENSITIVE RESPIRATORY PATHWAY OF PSEUDOMONAS-AERUGINOSA
ISOLATION AND CHARACTERIZATION OF MUTANTS DEFECTIVE IN THE CYANIDE-INSENSITIVE RESPIRATORY PATHWAY OF PSEUDOMONAS-AERUGINOSA
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DOI：
10.1128/jb.177.2.432-438.1995
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发表时间：
1995-01-01
影响因子：
3.2
通讯作者：
WILLIAMS, HD
中科院分区：
文献类型：
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作者：
CUNNINGHAM, L;WILLIAMS, HD
铜绿假单胞菌的支链呼吸链中至少含有两种末端氧化酶，在正常生理条件下具有活性。其中一种是细胞色素co，它是一种细胞色素c氧化酶，呼吸抑制剂氰化钾的浓度低至100 μ m就能完全抑制它。第二种氧化酶是对氰化物不敏感的氧化酶，它对超过1 μ m的氰化物浓度和叠氮化钠都有抵抗力。在这项工作中，我们描述了在氰化物不敏感呼吸缺陷铜绿假单胞菌突变体的分离和表征。该插入突变体是用mini-D171 （P. aeruginosa噬菌体D3112的复制缺陷衍生物）作为诱变原分离出来的，并通过筛选产生的四环素抗性转导剂，使其在1mm氮化钠存在下失去生长能力。对nadh介导的突变体呼吸速率的极谱研究表明，该突变体的活性损失了大约50%，对氰化物浓度增加的活性滴定得出的单相曲线清楚地表明，该突变体完全丧失了对氰化物不敏感的呼吸。在氰化物不敏感，氧化酶终止呼吸途径影响突变基因的突变已被指定cio。我们通过在体内克隆另一个携带在质粒pADD386上的mini-D元件mini-D386，将该突变体的叠氮化物敏感表型与该基因的野生型拷贝进行了互补。补充后的cio突变体在含1 mM叠氮化物的培养基上恢复了生长能力，用氰化物滴定其NADH氧化酶活性得到与野生型相似的双相曲线，呼吸速率恢复到正常水平。对野生型、cio突变体和互补突变体细胞膜细胞色素含量的光谱分析表明，cio突变体没有任何膜结合细胞色素缺陷，并且互补基因不编码血红素蛋白。
The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases,which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 mu M. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.