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

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 cbb₃-type cytochrome c oxidase (Cox) in species such as Rhodobacter capsulatus. C. vibrioides harbors two terminal cytochrome c oxidases in its cytoplasmic membrane: an aa₃-type and a cbb₃-type Cox. We also demonstrate that the activity of cbb₃-Cox requires the FixI-type Cu transporter and the periplasmic Cu chaperone PccA. In contrast, aa₃-Cox activity depends on PccA and the inner membrane-bound protein CtaG. Since the mechanism of Cu acquisition for aa₃-Cox remains largely unknown, we conducted a genetic screen and identified a novel outer membrane TonB-dependent receptor (TccA) that is specifically required for aa₃-Cox function. We also showed that cbb₃-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 cbb₃-Cox decrease, and aa₃-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.