Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis.
Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis.
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ctaA 的分离和序列，ctaA 是枯草芽孢杆菌细胞色素 aa3 生物合成和孢子形成所需的基因。
DOI：
10.1128/jb.171.9.4967-4978.1989
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发表时间：
1989
影响因子：
3.2
通讯作者：
Taber,HW
中科院分区：
文献类型：
--
作者：
Mueller,JP;Taber,HW
细胞色素aa 3是枯草芽孢杆菌电子传递链中的两个末端氧化酶复合物之一。设计了一种新的遗传策略，该策略允许分离B。通过选择不能氧化人工电子供体N，N，N ′，N ′-四甲基-对苯二胺的链霉素抗性克隆，获得缺乏细胞色素aa 3的枯草杆菌突变体。两个突变进行了深入研究。光谱检测表明，每个突变体都缺乏细胞色素aa 3;它们也是无孢子的，不能以乳酸作为唯一的碳源和能源。这些突变被定位到指定为ctaA的基因座，位于B上pyrD和metC之间的127度处。枯草染色体两种ctaA突变都通过转化到pycA基因座而密切相关。从B克隆ctaA基因座和pycA基因座的一部分。在大肠杆菌中构建枯草杆菌整合文库。含有B的4.0-内切酶插入片段的重组质粒。枯草杆菌DNA能将两种ctaA突变体转化为CtaA+。用亚克隆片段进行的基因破坏和互补实验表明ctaA位点由一个大小约为1.35个酶对的单个转录单位组成。ctaA转录单位的核苷酸序列含有能够编码预测分子量为34，065的蛋白质的单个开放阅读框。预测的蛋白质是非常疏水的，有几个可能的跨膜结构域。ctaA和高度保守的原核和线粒体氧化酶多肽之间没有序列相似性。两个ctaA突变的克隆和序列分析显示，一个等位基因是在羧基末端的无义突变，另一个是在氨基末端的错义突变;这表明，由每个突变赋予的多效性表型是由ctaA或其活性的丧失引起的。遗传学证据表明，ctaA基因产物是必需的作为辅助蛋白质的遗传表达，翻译后生物合成，或两者，细胞色素aa 3复合物和孢子发生的早期阶段。
Cytochrome aa3 is one of two terminal oxidase complexes in the Bacillus subtilis electron transport chain. A novel genetic strategy was devised which permitted the isolation of B. subtilis mutants lacking cytochrome aa3 by selection for streptomycin-resistant clones which failed to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Two mutations were studied intensively. Spectroscopic examination showed that each mutant lacked cytochrome aa3; they were also asporogenous and unable to grow on lactate as the sole carbon and energy source. These mutations were mapped to a locus designated ctaA, located at 127 degrees between pyrD and metC on the B. subtilis chromosome. Both ctaA mutations were closely linked by transformation to the pycA locus. The ctaA locus and a portion of the pycA locus were cloned from a B. subtilis integration library constructed in Escherichia coli. A recombinant plasmid containing a 4.0-kilobase insert of B. subtilis DNA could transform both ctaA mutants to CtaA+. Gene disruption and complementation experiments with subcloned fragments revealed that the ctaA locus consisted of a single transcriptional unit about 1.35 kilobase pairs in size. The nucleotide sequence of the ctaA transcriptional unit contains a single open reading frame capable of coding for a protein with a predicted molecular weight of 34,065. The predicted protein is extremely hydrophobic, with several probable membrane-spanning domains. No sequence similiarity was found between ctaA and the highly conserved procaryotic and mitochondrial oxidase polypeptides. Cloning and sequence analysis of two ctaA mutations revealed that one allele is a nonsense mutation in the carboxy terminus and the other is a missense mutation in the amino terminus; this indicates that the pleiotropic phenotype conferred by each mutation was caused by loss of CtaA or of its activity. Genetic evidence suggests that the ctaA gene product is required as an accessory protein in the genetic expression, posttranslational biogenesis, or both, of the cytochrome aa3 complex and during an early stage of sporogenesis.