Copper homeostasis networks in the bacterium Pseudomonas aeruginosa

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 CuSO 4 , 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 Cu 2 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-nodulationdivision (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 copR and cueR 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.