Unraveling the pathway of Copper Delivery to Cytochrome c oxidases in the Free-Living Bacterium Caulobacter vibrioides

Abstract Copper (Cu) is an essential micronutrient that serves as a cofactor for many enzymes but becomes toxic when present in excess. In most bacteria, CopA-like P1B-type ATPases mediate Cu detoxification by exporting cytoplasmic Cu to the periplasm or extracellular environment. In this study, we show that Caulobacter vibrioides lacks a canonical CopA-like ATPase but encodes a single FixI/CcoI-type Cu-transporting ATPase, previously implicated in Cu delivery to the cbb□-type cytochrome c oxidase (Cox) in species such as Rhodobacter capsulatus. C. vibrioides harbors two terminal cytochrome c oxidases in its cytoplasmic membrane: an aa□-type and a cbb□-type Cox. We also demonstrate that the activity of cbb□-Cox requires the FixI-type Cu transporter and the periplasmic Cu chaperone PccA. In contrast, aa□-Cox activity depends on PccA and the inner membrane-bound protein CtaG. Since the mechanism of Cu acquisition for aa□-Cox remains largely unknown, we conducted a genetic screen and identified a novel outer membrane TonB-dependent receptor (TccA) that is specifically required for aa□-Cox function. We also showed that cbb□-Cox is upregulated under microaerobic conditions, possibly such as those encountered on solid media where O2 diffusion is limited. Under normoxic conditions, the expression and the activity of cbb□-Cox decrease, and aa□-Cox becomes the predominant terminal oxidase. These findings demonstrate that C. vibrioides differentially utilizes its Cox enzymes in response to O2 availability and relies on a distinct Cu trafficking pathway for their maturation, including an outer membrane component that has not been previously described in bacterial Cu homeostasis. Competing Interest Statement The authors have declared no competing interest.