PHENOTYPIC AND GENETIC-CHARACTERIZATION OF CYTOCHROME-C2 DEFICIENT MUTANTS OF RHODOBACTER-SPHAEROIDES
PHENOTYPIC AND GENETIC-CHARACTERIZATION OF CYTOCHROME-C2 DEFICIENT MUTANTS OF RHODOBACTER-SPHAEROIDES
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DOI：
10.1021/bi00406a018
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发表时间：
1988-03-22
期刊：
影响因子：
2.9
通讯作者：
KAPLAN, S
中科院分区：
文献类型：
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作者：
DONOHUE, TJ;MCEWAN, AG;KAPLAN, S
球形红细菌细胞色素c2（cyt c2）缺失突变体是通过野生型基因组cyt c2结构基因（cycA）和含有缺陷型cyc操纵子的自杀质粒之间的位点特异性重组构建的，其中cycA序列的缺失伴随着KnR基因的插入。Southern印迹分析证实，野生型cyc操纵子被交换为失活的cycA基因，推测是通过双互惠重组。光谱和免疫化学测量，加上遗传互补，确定这些突变体不能在光合条件下生长是由于缺乏细胞色素c2。细胞色素c2缺陷菌株减少光氧化反应中心复合物约4个数量级更慢于亲本菌株。当在稳定的低拷贝质粒上引入野生型cyc操纵子时，这些突变体的表型和特征得以恢复。这些实验为cyt c2在野生型光合电子传递中的专性作用提供了第一个遗传学证据。sphaeroides我们还观察到，R.类球孢菌CytC2缺陷型菌株自发地以一定的频率产生具有光合能力的假回复突变体，该频率表明抑制CytC2缺陷型菌株表型的不依赖于CytC2的光合电子传递是基因组中其它地方的单一突变的结果。
Rhodobacter sphaeroides mutants lacking cytochrome c2 (cyt c2) have been constructed by site-specific recombination between the wild-type genomic cyt c2 structural gene (cycA) and a suicide plasmid containing a defective cyc operon where deletion of cycA sequences was accompanied by insertion of a KnR gene. Southern blot analysis confirmed that the wild-type cyc operon was exchanged for the inactivated cycA gene, presumably by double-reciprocal recombination. Spectroscopic and immunochemical measurements, together with genetic complementation, established that the inability of these mutants to grow under photosynthetic conditions was due to the lack of cyt c2. The cyt c2 deficient strains reduced photooxidized reaction center complexes approximately 4 orders of magnitude more slowly than the parent strain. The phenotype and characteristics of these mutants were restored when a wild-type cyc operon was introduced on a stable low copy number plasmid. These experiments provide the first genetic evidence for the obligatory role of cyt c2 in wild-type cyclic photosynthetic electron transport in R. sphaeroides. We have also observed that the R. sphaeroides cyt c2 deficient strains spontaneously gave rise to photosynthetically competent pseudorevertants at a frequency which suggests that the cyt c2 independent photosynthetic electron transport which suppresses the phenotype of the cyt c2 deficient strains was the result of a single mutation elsewhere in the genome.