Deshmukh M, Brasseur G, Daldal F
Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.
Mol Microbiol. 2000 Jan;35(1):123-38. doi: 10.1046/j.1365-2958.2000.01683.x.
Following chemical mutagenesis and screening for the inability to grow by photosynthesis and the absence of cyt cbb3 oxidase activity, two c-type cytochrome (cyt)-deficient mutants, 771 and K2, of Rhodobacter capsulatus were isolated. Both mutants were completely deficient in all known c-type cyts, and could not be complemented by the previously known cyt c biogenesis genes of R. capsulatus. Complementation of 771 and K2 with a wild-type chromosomal library led to the identification of two novel genes, cycJ and ccdA respectively. The cycJ is highly homologous to ccmE/cycJ, encountered in various Gram-negative species. Unlike in other species, where cycJ is a part of an operon essential for cyt c biogenesis, in R. capsulatus, it is located immediately downstream from argC, involved in arginine biosynthesis. Mutation of its universally conserved histidine residue, which is critical for its proposed haem chaperoning role, to an alanine led to loss of its function. All R. capsulatus cycJ mutants studied so far excrete copious amounts of coproporphyrin and protoporphyrin when grown on enriched media, suggesting that its product is also a component of the haem delivery branch of cyt c biogenesis in this species. In contrast, the R. capsulatus ccdA was homologous to the cyt c biogenesis gene ccdA, found in the gram-positive bacterium Bacillus subtilis, and to the central region of dipZ, encoding a protein disulphide reductase required for cyt c biogenesis in Escherichia coli. Membrane topology of CcdA was established in R. capsulatus using ccdA:phoA and ccdA :lacZ gene fusions. The deduced topology revealed that the two conserved cysteine residues of CcdA are, as predicted, membrane embedded. Mutagenesis of these cysteines showed that both are required for the function of CcdA in cyt c biogenesis. This study demonstrated for the first time that CcdA homologues are also required for cyt c biogenesis in some gram-negative bacteria such as R. capsulatus.
通过化学诱变并筛选不能通过光合作用生长且缺乏细胞色素cbb3氧化酶活性的菌株，分离出了深红螺菌（Rhodobacter capsulatus）的两个c型细胞色素（cyt）缺陷型突变体771和K2。这两个突变体在所有已知的c型细胞色素方面完全缺陷，并且不能被深红螺菌先前已知的细胞色素c生物合成基因互补。用野生型染色体文库对771和K2进行互补，分别鉴定出两个新基因cycJ和ccdA。cycJ与在各种革兰氏阴性菌中发现的ccmE/cycJ高度同源。与其他物种不同，在其他物种中cycJ是细胞色素c生物合成所必需的操纵子的一部分，而在深红螺菌中，它位于参与精氨酸生物合成的argC基因的紧邻下游。将其普遍保守的组氨酸残基（对其假定的血红素伴侣作用至关重要）突变为丙氨酸会导致其功能丧失。到目前为止，所研究的所有深红螺菌cycJ突变体在富集培养基上生长时都会大量分泌粪卟啉和原卟啉，这表明其产物也是该物种细胞色素c生物合成中血红素传递分支的一个组成部分。相比之下，深红螺菌的ccdA与在革兰氏阳性菌枯草芽孢杆菌中发现的细胞色素c生物合成基因ccdA以及dipZ的中央区域同源，dipZ编码大肠杆菌细胞色素c生物合成所需的一种蛋白质二硫键还原酶。利用ccdA:phoA和ccdA:lacZ基因融合在深红螺菌中确定了CcdA的膜拓扑结构。推导的拓扑结构表明，CcdA的两个保守半胱氨酸残基如预测的那样嵌入膜中。对这些半胱氨酸进行诱变表明，两者都是CcdA在细胞色素c生物合成中发挥功能所必需的。这项研究首次证明，在一些革兰氏阴性菌如深红螺菌中，CcdA同源物也是细胞色素c生物合成所必需的。
