Pathways of copper import and utilization that support respiration in Bacillus subtilis

Copper is an enzyme cofactor that is essential for cellular respiration in most aerobic organisms but can be toxic in excess. Although most copper enzymes are localized within or external to the cell membrane, proper copper loading often relies on copper import into the cytosol. In Bacillus subtilis, the YcnJ membrane protein has been proposed to serve as a copper importer, but its biological function remains poorly characterized. Here we demonstrate that under copper starvation, YcnJ is important for aerobic growth by supporting the proper function of Qox, the major respiratory menaquinol oxidase (cytochrome aa₃). YcnJ function requires a Cu²⁺-binding site in an extracellular CopC domain and a second putative copper-binding site in its transmembrane CopD domain. YcnJ additionally contains a carboxy-terminal YtkA domain with homology to the membrane-anchored lipoprotein CtaK that has been implicated in the assembly of the Cu_A center in the caa₃-type cytochrome oxidase Cta. The function of Cta is compromised in a ctaK mutant, and this effect is exacerbated if YcnJ or its C-terminal YtkA domain is absent. We further show that during copper starvation, inactive heme-containing Qox and CtaA accumulate in the membrane and that under copper excess, CtaM is involved in copper resistance and oxidase assembly. These findings refine current models of copper import and trafficking in B. subtilis as it supports the function of respiratory heme-copper oxidases.

Copper is a critical micronutrient for bacteria; however, it is toxic in excess. This leads to the cell having tightly regulated import and export systems to prevent intoxication while having sufficient copper for cellular needs. Copper export has been well studied, but copper import and the effects of copper starvation are less defined. In this article, we characterize the Bacillus subtilis high-affinity copper importer YcnJ. We show that under copper limitation, the cell is deficient in copper-containing oxidases, which leads to respiratory defects. Copper imported by YcnJ is proposed to function in a pathway with other proteins implicated in the assembly of respiratory oxidases. More generally, this work contributes to the underexplored area of copper import and trafficking in bacteria.