Garg Nitanshu, Taylor Aidan J, Pastorelli Federica, Flannery Sarah E, Jackson Phillip J, Johnson Matthew P, Kelly David J
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, United Kingdom.
Front Microbiol. 2021 Jun 25;12:683260. doi: 10.3389/fmicb.2021.683260. eCollection 2021.
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.
该家族中的细菌C型血红素-铜氧化酶广泛存在于微需氧菌中，这些微需氧菌利用其高氧结合亲和力在微氧生态位中生长。在微需氧病原体中，C型氧化酶对于感染可能至关重要，但与模式细菌相比，对其生物发生的了解却很少。在这里，我们已经在革兰氏阴性病原体中鉴定出参与Cco氧化酶（Cco）组装和活性的基因，该病原体是人类食源性细菌性肠胃炎最常见的病因。氧化酶结构基因下游的几个功能未知的基因被证明对Cco活性至关重要（ 和 ）或很重要（ 和 ）；Cj1483是CcoH的同源物，但Cj1484（命名为CcoZ）与MSMEG_4692具有结构相似性，后者参与结核分枝杆菌中Qcr-Cco氧化酶超复合物的形成。去垢剂溶解膜的蓝色天然聚丙烯酰胺凝胶电泳显示出三条主要条带，其中一条包含CcoZ以及Qcr和氧化酶亚基。推定的铜转运基因 和 的缺失消除了Cco活性，在生长过程中添加铜可部分恢复该活性，而编码CcoG同源物的 的失活导致Cco活性部分降低。编码PCuC（Cj0909）和Sco（Cj0911）周质铜伴侣同源物的操纵子的缺失降低了Cco活性，在 突变体中外源铜可部分恢复该活性。对编码参与细胞内金属稳态的几个基因的 基因簇中的基因缺失进行的表型分析表明， 的失活（ ）或 的失活（ ）导致细胞内铜升高和Cco活性降低，在高外部铜浓度下这种影响会加剧。因此，我们的工作已经鉴定出（i）额外的Cco亚基，（ii）一组以前未表征的连接铜转运和Cco活性的基因，以及（iii）在这种重要病原体中与铜稳态的联系。
