The two-component system CopRS maintains femtomolar levels of free copper in the periplasm of Pseudomonas aeruginosa using a phosphatase-based mechanism

Two component systems control periplasmic Cu homeostasis in Gram-negative bacteria. In characterized systems such as CusRS, upon Cu binding to the periplasmic sensing domain of CusS, a cytoplasmic phosphotransfer domain phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the two component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains a non-phosphorylated CopR when the periplasmic Cu levels are below its activation threshold. Upon Cu binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the Δ strain showed constitutive high expression of the CopRS regulon, lower intracellular Cu levels, and larger Cu tolerance when compared to wild type cells. The invariant phospho-acceptor residue His of CopS was not required for the phosphatase activity itself, but necessary for its Cu-dependency. To sense the metal, the periplasmic domain of CopS binds two Cu ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag binding sites) clearly explains the different binding stoichiometries in both systems. Interestingly, CopS binds Cu with 30 × 10 M affinities, pointing to the absence of free (hydrated) Cu in the periplasm.