Abstract
Aerobic respiration in Corynebacterium glutamicum involves a cytochrome bc(1)-aa(3) supercomplex with a diheme cytochrome c(1), which is the only c-type cytochrome in this species. This organization is considered as typical for aerobic Actinobacteria. Whereas the biogenesis of heme-copper type oxidases like cytochrome aa(3) has been studied extensively in α-proteobacteria, yeast, and mammals, nothing is known about this process in Actinobacteria. Here, we searched for assembly proteins of the supercomplex by identifying the copper-deprivation stimulon, which might include proteins that insert copper into cytochrome aa(3) Using gene expression profiling, we found two copper starvation-induced proteins for supercomplex formation. The Cg2699 protein, named CtiP, contained 16 predicted transmembrane helices, and its sequence was similar to that of the copper importer CopD of Pseudomonas syringae in the N-terminal half and to the cytochrome oxidase maturation protein CtaG of Bacillus subtilis in its C-terminal half. CtiP deletion caused a growth defect similar to that produced by deletion of subunit I of cytochrome aa(3), increased copper tolerance, triggered expression of the copper-deprivation stimulon under copper sufficiency, and prevented co-purification of the supercomplex subunits. The secreted Cg1884 protein, named CopC, had a C-terminal transmembrane helix and contained a Cu(II)-binding motif. Its absence caused a conditional growth defect, increased copper tolerance, and also prevented co-purification of the supercomplex subunits. CtiP and CopC are conserved among aerobic Actinobacteria, and we propose a model of their functions in cytochrome aa(3) biogenesis. Furthermore, we found that the copper-deprivation response involves additional regulators besides the ECF sigma factor SigC.