Cytochrome bd Biosynthesis in Bacillus subtilis: Characterization of the cydABCD Operon

Under aerobic conditions Bacillus subtilis utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in B. subtilis: a caa(3)-, an aa(3)-, and a bd-type oxidase. We report here the cloning of the structural genes (cydA and cydB) encoding the cytochrome bd complex. Downstream of the structural genes, cydC and cydD are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of cydA or deletion of cydABCD resulted in the loss of spectral features associated with cytochrome bd. Gene disruption experiments and complementation analysis showed that the cydC and cydD gene products are required for the expression of a functional cytochrome bd complex. Disruption of the cyd genes had no apparent effect on the growth of cells in broth or defined media. The expression of the cydABCD operon was investigated by Northern blot analysis and by transcriptional and translational cyd-lacZ fusions. Northern blot analysis confirmed that cydABCD is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.

在有氧条件下，枯草芽孢杆菌（Bacillus subtilis）利用由多种细胞色素（cytochromes）与末端氧化酶（terminal oxidases）组成的分支电子传递链（branched electron transport chain）。目前已有研究证实，枯草芽孢杆菌中存在三类末端氧化酶：caa₃型、aa₃型与bd型氧化酶。本研究报道了编码细胞色素bd复合物（cytochrome bd complex）的结构基因cydA与cydB的克隆工作。在上述结构基因的下游区域，存在cydC与cydD基因，二者编码的蛋白与细菌ATP结合盒（ATP-binding cassette, ABC）型转运蛋白具有序列相似性。对分离得到的细胞膜进行分析后发现，敲除cydA基因或缺失cydABCD基因簇，均会导致与细胞色素bd复合物相关的光谱特征消失。基因敲除实验与互补验证实验结果表明，cydC与cydD的基因产物对于功能性细胞色素bd复合物的表达不可或缺。破坏cyd基因对肉汤培养基或限定培养基中的细胞生长无显著影响。本研究通过Northern印迹分析（Northern blot analysis）以及转录与翻译水平的cyd-lacZ融合系统，探究了cydABCD操纵子（operon）的表达模式。Northern印迹分析证实，cydABCD以多顺反子转录本的形式被转录。研究发现，该操纵子在低氧张力条件下的表达量达到峰值。