The interplay of the metallosensor CueR with two distinct CopZ chaperones defines copper homeostasis in .

Copper homeostasis in pathogenic bacteria is critical for cuproprotein assembly and virulence. However, biochemical analyses of these processes are challenging, which has prevented defining and quantifying the homeostatic interplay between Cu-sensing transcriptional regulators, chaperones, and sequestering molecules. The cytoplasm of contains a Cu-sensing transcriptional regulator, CueR, and two homologous metal chaperones, CopZ1 and CopZ2, forming a unique system for studying Cu homeostasis. We found here that both chaperones exchange Cu, albeit at a slow rate, reaching equilibrium after 3 h, a time much longer than duplication time. Therefore, they appeared as two separate cellular Cu pools. Although both chaperones transferred Cu to CueR , experiments indicated that CopZ1 metallates CueR, eliciting the translation of Cu efflux transporters involved in metal tolerance. Although this observation was consistent with the relative Cu affinities of the three proteins (CopZ1 < CueR < CopZ2), and analyses also indicated a stronger interaction between CopZ1 and CueR that was independent of Cu In contrast, CopZ2 function was defined by its distinctly high abundance during Cu stress. Under resting conditions, CopZ2 remained largely in its form. Metal stress quickly induced CopZ2 expression, and its form predominated, reaching levels commensurate with the cytoplasmic Cu levels. In summary, these results show that CopZ1 acts as chaperone delivering Cu to the CueR sensor, whereas CopZ2 functions as a fast-response Cu-sequestering storage protein. We propose that equivalent proteins likely play similar roles in most bacterial systems.