Genes Linking Copper Trafficking and Homeostasis to the Biogenesis and Activity of the -Type Cytochrome Oxidase in the Enteric Pathogen .

Bacterial C-type haem-copper oxidases in the 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 -oxidase (Cco) assembly and activity in the Gram-negative pathogen , the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes were shown to be essential ( and ) or important ( and ) 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 . 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 () and () abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCu C (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the mutant by exogenous copper. Phenotypic analyses of gene deletions in the cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of (), or () 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.