Abstract
Bacterial C-type haem-copper oxidases in the cbb3 family are widespread in microaerophiles, which exploit their high oxygen-binding affinity for growth in microoxic niches. In microaerophilic pathogens, C-type oxidases can be essential for infection, yet little is known about their biogenesis compared to model bacteria. Here, we have identified genes involved in cbb3-oxidase (Cco) assembly and activity in the Gram-negative pathogen Campylobacter jejuni, the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes ccoNOQP were shown to be essential (cj1483c and cj1486c) or important (cj1484c and cj1485c) for Cco activity; Cj1483 is a CcoH homologue, but Cj1484 (designated CcoZ) has structural similarity to MSMEG_4692, involved in Qcr-oxidase supercomplex formation in Mycobacterium smegmatis. Blue-native polyacrylamide gel electrophoresis of detergent solubilised membranes revealed three major bands, one of which contained CcoZ along with Qcr and oxidase subunits. Deletion of putative copper trafficking genes ccoI (cj1155c) and ccoS (cj1154c) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of cj0369c encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCuAC (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the cj0911 mutant by exogenous copper. Phenotypic analyses of gene deletions in the cj1161c–1166c cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of copA (cj1161c), or copZ (cj1162c) led to both elevated intracellular Cu and reduced Cco activity, effects exacerbated at high external Cu. Our work has therefore identified (i) additional Cco subunits, (ii) a previously uncharacterized set of genes linking copper trafficking and Cco activity, and (iii) connections with Cu homeostasis in this important pathogen.
Highlights
Haem-copper oxidases (HCOs) are proton-translocating respiratory complexes widely distributed in both prokaryotes and eukaryotes that contain a bi-nuclear haem-copper site at which oxygen reduction occurs
The intestine is a sulphide rich environment and we confirmed from the results of our mutant studies the sulphide sensitivity of the C. jejuni Cco complex and sulphide resistance of CioAB seen in other bacteria (Forte et al, 2016; Korshunov et al, 2016)
A continued supply of copper is clearly essential for Cco assembly and activity, but how copper is imported into C. jejuni is unknown
Summary
Haem-copper oxidases (HCOs) are proton-translocating respiratory complexes widely distributed in both prokaryotes and eukaryotes that contain a bi-nuclear haem-copper site at which oxygen reduction occurs. Type A include the cytochrome aa3-type oxidases found in a range of aerobic bacteria and mitochondria, type B includes the ba oxidases only found in bacteria, and type C contains the cbb3-type oxidases, which have an integral c-type cytochrome subunit (Pereira et al, 2001). This type of enzyme has a high oxygen affinity and is typically found in a range of microaerophilic bacteria (Pitcher and Watmough, 2004). All HCOs operate using a redox loop mechanism for proton translocation across the membrane as well as pumping protons directly. Working in combination with the quinol-cytochrome c reductase (Qcr) complex, the redox loop mechanism means that a higher overall H+/e ratio can be achieved during electron transfer from quinol to oxygen
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