Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides. Characterization of fbc deletion mutants and complementation by a site-specific mutational variant.
Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides. Characterization of fbc deletion mutants and complementation by a site-specific mutational variant.
复制标题
编码球形红杆菌细胞色素 bc1 复合物的 fbc 操纵子的克隆和 DNA 测序。
DOI：
10.1111/j.1432-1033.1990.tb15633.x
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Gennis,RB
中科院分区：
文献类型：
--
作者：
Yun,CH;Beci,R;Crofts,AR;Kaplan,S;Gennis,RB
泛醇：细胞色素-环氧还原酶（cytochromebc 1 complex）是线粒体呼吸链以及许多原核生物的呼吸和/或光合系统的中心组分。在类球红细菌中，bc 1复合物具有双重功能。当细胞以光合方式生长时，bc 1复合物存在于胞质内膜中，是循环电子传递系统的关键组成部分。当细胞在有氧存在的黑暗中生长时，samebc 1复合物是细胞色素c2依赖性呼吸链的必要组成部分。bc 1复合物是由R.类球蛋白已被广泛研究，再加上这种生物体的遗传操作能力，使其成为一个理想的系统，使用定点突变来解决与bc 1复合物的结构和功能有关的问题。sphaeroidesis报道。与其他细菌一样，该操纵子包含三个基因，分别编码Rieske 2Fe-2S亚基、细胞色素b亚基和细胞色素1亚基。使用纯化技术从染色体上删除整个refbcoperon。所得到的菌株不能光合生长，但可以利用醌醇氧化酶有氧生长。光合生长恢复提供fbcoperon的质粒，并再现的蛋白质亚基和光谱特征，由于thebc 1复合物也被证明。最后，在编码细胞色素b亚基的基因内引入突变，该突变被预测为赋予对抑制剂myxothiazol的抗性。结果表明，所得到的菌株含有一个功能性的bc 1复合物，正如预期的那样，是耐抑制剂。因此，该系统是适合的详细表征的bc 1复合物，结合定点诱变与生物化学和生物物理技术，已开发的光合细菌的研究。
The ubiquinol: cytochrome‐coxidoreductase (cytochromebc1complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. InRhodobacter sphaeroides, thebc1complex has a dual function. When the cells are grown photosynthetically, thebc1complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the samebc1complex is a necessary component of the cytochrome‐c2‐dependent respiratory chain. The fact that thebc1complex fromR. sphaeroideshas been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site‐directed mutagenesis to address questions relating to the structure and function of thebc1complex.In the current work, the cloning and complete sequence of thefbcoperon fromR. sphaeroidesis reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe–2S subunit, the cytochromebsubunit, and the cytochromec1subunit. Recombination techniques were used to delete the entirefbcoperon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providingfbcoperon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to thebc1complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochromebsubunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functionalbc1complex which, as expected, is resistant to the inhibitor.Hence, this system is suitable for the detailed characterization of thebc1complex, combining site‐directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.