The two-component system CopRS maintains femtomolar levels of free copper in the periplasm ofPseudomonas aeruginosausing a phosphatase-based mechanism

Two component systems control periplasmic Cu+homeostasis in Gram-negative bacteria. In characterized systems such asEscherichia coliCusRS, 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 thePseudomonas aeruginosatwo 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 ΔcopSstrain 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 His235of 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+/2+with 30 × 10−15M affinities, pointing to the absence of free (hydrated) Cu+/2+in the periplasm.IMPORTANCECopper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins, and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two component systems control the periplasmic response to metal overload. This manuscript shows that the copper sensing two component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of the response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the sensor is activated by copper levels compatible with the absence of free copper in the cell periplasm. This emphasizes the diversity of molecular mechanisms that have evolved in various bacteria to manage the copper cellular distribution.