AUTHOR=Garg Nitanshu , Taylor Aidan J. , Pastorelli Federica , Flannery Sarah E. , Jackson Phillip J. , Johnson Matthew P. , Kelly David J. 

TITLE=Genes Linking Copper Trafficking and Homeostasis to the Biogenesis and Activity of the cbb3-Type Cytochrome c Oxidase in the Enteric Pathogen Campylobacter jejuni

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.683260

DOI=10.3389/fmicb.2021.683260

ISSN=1664-302X

ABSTRACT=<p>Bacterial C-type haem-copper oxidases in the <italic>cbb</italic><sub>3</sub> 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 <italic>cbb</italic><sub>3</sub>-oxidase (Cco) assembly and activity in the Gram-negative pathogen <italic>Campylobacter jejuni</italic>, the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes <italic>ccoNOQP</italic> were shown to be essential (<italic>cj1483c</italic> and <italic>cj1486c</italic>) or important (<italic>cj1484c</italic> and <italic>cj1485c</italic>) 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 <italic>Mycobacterium smegmatis</italic>. 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 <italic>ccoI</italic> (<italic>cj1155c</italic>) and <italic>ccoS</italic> (<italic>cj1154c</italic>) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of <italic>cj0369c</italic> encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCu<sub><italic>A</italic></sub>C (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the <italic>cj0911</italic> mutant by exogenous copper. Phenotypic analyses of gene deletions in the <italic>cj1161c–1166c</italic> cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of <italic>copA</italic> (<italic>cj1161c</italic>), or <italic>copZ</italic> (<italic>cj1162c</italic>) 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.</p>