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Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

机译:铜绿假单胞菌对氰化物不敏感的呼吸途径有缺陷的突变体的分离和鉴定。

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摘要

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 microM. 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.
机译:铜绿假单胞菌的分支呼吸链包含至少两种在正常生理条件下有活性的末端氧化酶。其中一种是细胞色素c,是一种细胞色素c氧化酶,其被呼吸抑制剂氰化钾的浓度低至100 microM完全抑制。第二种氧化酶,对氰化物不敏感的氧化酶,对超过1 mM的氰化物以及叠氮化钠具有抗性。在这项工作中,我们描述了对氰化物不敏感的呼吸缺陷的铜绿假单胞菌突变体的分离和表征。用mini-D171(铜绿假单胞菌噬菌体D3112的复制缺陷型衍生物)作为诱变剂,并通过筛选所得的四环素抗性转导子在1 mM叠氮化钠存在下生长能力的丧失,来分离该插入突变体。 。对NADH介导的突变体呼吸速率的极谱研究表明,该活性大约损失了50%,针对增加的氰化物浓度滴定该活性可得到单相曲线,清楚地表明了对氰化物不敏感的呼吸作用的完全丧失。受氰化物不敏感,氧化酶终止的呼吸途径影响的突变体的突变基因已指定为cio。通过在体内用质粒pADD386上的另一个mini-D元素mini-D386进行体内克隆,我们用该基因的野生型拷贝补充了该突变体的叠氮化物敏感表型。互补的cio突变体恢复了在含有1 mM叠氮化物的培养基上生长的能力,用氰化物滴定其NA​​DH氧化酶活性产生了类似于野生型生物的双相曲线,并且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的膜的细胞色素含量进行光谱分析表明,cio突变体在任何与膜结合的细胞色素中均无缺陷,并且互补基因不编码血红素蛋白。

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