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

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.

The novel ECF56 SigG1-RsfG system modulates morphological differentiation and metal-ion homeostasis in Streptomyces tsukubaensis

Extracytoplasmic function (ECF) sigma factors are key transcriptional regulators that prokaryotes have evolved to respond to environmental challenges. Streptomyces tsukubaensis harbours 42 ECFs to reprogram stress-responsive gene expression. Among them, SigG1 features a minimal conserved ECF σ2–σ4 architecture and an additional C-terminal extension that encodes a SnoaL_2 domain, which is characteristic for ECF σ factors of group ECF56. Although proteins with such domain organisation are widely found among Actinobacteria, the functional role of ECFs with a fused SnoaL_2 domain remains unknown. Our results show that in addition to predicted self-regulatory intramolecular amino acid interactions between the SnoaL_2 domain and the ECF core, SigG1 activity is controlled by the cognate anti-sigma protein RsfG, encoded by a co-transcribed sigG1-neighbouring gene. Characterisation of ∆sigG1 and ∆rsfG strains combined with RNA-seq and ChIP-seq experiments, suggests the involvement of SigG1 in the morphological differentiation programme of S. tsukubaensis. SigG1 regulates the expression of alanine dehydrogenase, ald and the WhiB-like regulator, wblC required for differentiation, in addition to iron and copper trafficking systems. Overall, our work establishes a model in which the activity of a σ factor of group ECF56, regulates morphogenesis and metal-ions homeostasis during development to ensure the timely progression of multicellular differentiation.

PSVII-21 The impact of cell culture and copper dose on copper trafficking genes in bovine liver

The objective of the current experiment was to investigate the influence of Cu dose on the relative abundance of Cu trafficking genes in cultured bovine hepatocytes. A liver sample was obtained immediately post-mortem from one healthy Angus steer. Hepatocytes were isolated, counted, and seeded at equal density into 15 separate wells, and incubated for 1 hour in culture media containing: 0.0, 0.10, 1.0, 10.0, or 100 mg Cu/L (3 replicates per Cu dose). Following incubation, cells were collected and total RNA was isolated. Quantitative RT-PCR was used to determine the abundance of transcripts for proteins involved in Cu homeostasis. The identified targets were: ALDH2, APOA1, ATOX1, ATP7A, ATP7B, BHMT, BLVRB, CA2, CCS, COX17, CTR1, ELN, GAPDH, GLUD1, GSS, LOXL1, PDIA3, SOD1, SOD3. β-Actin (ACTB) served as the endogenous control. Significant linear responses existed for ALDH2 (P < 0.001), ATOX1 (P < 0.01), PDIA3 (P < 0.05). As Cu dose increased, the relative abundance of ALDH2 increased, and ATOX1 and PDIA3 decreased. Significant quadratic responses existed for ATP7B (P < 0.001), COX17 (P < 0.05), and SOD1 (P < 0.05). The relative abundance of COX17 was lesser at 0.1 and 1.0 mg Cu/L when compared to 0.0, 10, and 100 mg Cu/L. Transcript abundance for ATP7B and SOD1 was lower at 0, 1, and 100 mg Cu/L when compared to 0.1 and 10 mg Cu/L. These data indicate that certain transcripts are differentially expressed in cultured bovine hepatocytes in response to increasing Cu dose.

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone

Copper’s essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

l-Cysteine-mediated modulation of copper trafficking in prostate cancer cells: an in vitro and in vivo investigation with 64Cu and 64Cu-PET

This article reports a novel role of thiol amino acids in modulating intracellular retention of copper in cancer cells and employs PET imaging with 64Cu to investigate this effect on the whole body level.

Restoration of myocellular copper-trafficking proteins and mitochondrial copper enzymes repairs cardiac function in rats with diabetes-evoked heart failure

Diabetes impairs systemic copper regulation, and acts as a major independent risk factor for heart failure (HF) wherein mitochondrial dysfunction is a key pathogenic process.