Bacterial copper (Cu⁺) homeostasis enables both precise metallation of diverse cuproproteins and control of variable metal levels. To this end, protein networks mobilize Cu⁺ to cellular targets with remarkable specificity. However, the understanding of these processes is rather fragmented. Here, we use genome-wide transcriptomic analysis by RNA-Seq to characterize the response of Pseudomonas aeruginosa to external 0.5 mm CuSO₄, a condition that did not generate pleiotropic effects. Pre-steady-state (5-min) and steady-state (2-h) Cu⁺ fluxes resulted in distinct transcriptome landscapes. Cells quickly responded to Cu²⁺ stress by slowing down metabolism. This was restored once steady state was reached. Specific Cu⁺ homeostasis genes were strongly regulated in both conditions. Our system-wide analysis revealed induction of three Cu⁺ efflux systems (a P_1B-ATPase, a porin, and a resistance-nodulation-division (RND) system) and of a putative Cu⁺-binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Both CopZ chaperones could bind Cu⁺ with high affinity. Importantly, novel transmembrane transporters probably mediating Cu⁺ influx were among those largely repressed upon Cu⁺ stress. Compartmental Cu⁺ levels appear independently controlled; the cytoplasmic Cu⁺ sensor CueR controls cytoplasmic chaperones and plasma membrane transporters, whereas CopR/S responds to periplasmic Cu⁺. Analysis of ΔcopR and ΔcueR mutant strains revealed a CopR regulon composed of genes involved in periplasmic Cu⁺ homeostasis and its putative DNA recognition sequence. In conclusion, our study establishes a system-wide model of a network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu⁺ levels in P. aeruginosa compartments.

Copper homeostasis in pathogenic bacteria is critical for cuproprotein assembly and virulence. However, in vivo 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 Pseudomonas aeruginosa 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 P. aeruginosa duplication time. Therefore, they appeared as two separate cellular Cu⁺ pools. Although both chaperones transferred Cu⁺ to CueR in vitro, experiments in vivo 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), in vitro and in silico 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 Cu2+ stress. Under resting conditions, CopZ2 remained largely in its apo form. Metal stress quickly induced CopZ2 expression, and its holo 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.

Copper is essential in cells as a cofactor for key redox enzymes. Bacteria have acquired molecular components that sense, uptake, distribute, and expel copper ensuring that cuproenzymes are metallated and steady-state metal levels are maintained. Toward preventing deleterious reactions, proteins bind copper ions with high affinities and transfer the metal via ligand exchange, warranting that copper ions are always complexed. Consequently, the directional copper distribution within cell compartments and across cell membranes requires specific dynamic interactions and metal exchange between cognate holo-apo protein partners. These metal exchange reactions are determined by thermodynamic and kinetics parameters and influenced by mass action. Then, copper distribution can be conceptualized as a molecular system of singular interacting elements that maintain a physiological copper homeostasis. This review focuses on the impact of copper high-affinity binding and exchange reactions on the homeostatic mechanisms, the conceptual models to describe the cell as a homeostatic system, the various molecule functions that contribute to copper homeostasis, and the alternative system architectures responsible for copper homeostasis in model bacteria. Graphical

Tuberculosis (TB) has been the biggest killer in the human history; currently,Mycobacterium tuberculosis(Mtb) kills nearly 2 million people each year worldwide. The high prevalence of TB obligates the identification of new therapeutic targets and the development of anti-TB vaccines that can control multidrug resistance and latent TB infections. Membrane proteins have recently been suggested as key targets for bacterial viability. Current studies have shown that mycobacteria P-type ATPases may play critical roles in ion homeostasis and in the response of mycobacteria to toxic substances in the intraphagosomal environment. In this review, we bring together the genomic, transcriptomic, and structural aspects of the P-type ATPases that are relevant during active and latentMtbinfections, which can be useful in determining the potential of these ATPases as drug targets and in uncovering their possible roles in the development of new anti-TB attenuated vaccines.