The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of Pseudomonas aeruginosa Using a Phosphatase-Based Mechanism

Cu tolerance of ΔcopR and ΔcopS mutant strains. Growth rate of WT, ΔcopR, ΔcopS (PW5705 and PW5706), ΔcopA1, and CopR and CopS complemented strains in the absence or the presence of increasing (0 to 4 mM) concentrations of CuSO₄. Data are the mean ± SEM from at least three independent experiments.

Whole-cell Cu levels in WT, ΔcopR, ΔcopS, ΔcopA1, and CopR and CopS complemented strains under normal growth conditions (i.e., no additional CuSO₄ added) (A) and after 10 min exposure to 2 mM CuSO₄ (B) or 4 mM CuSO₄ (C). Data are the mean ± SEM from three independent experiments. Significant differences from values with the WT strain as determined by unpaired two-tailed Student’s t test are *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Expression of genes in the CopRS regulon in WT, ΔcopR, ΔcopS, and corresponding complemented strains in the absence (white) and the presence (black) of 0.5 mM CuSO₄ (5-min treatment). Transcript levels of pcoA, pcoB, PA2807, ptrA, and queF genes are plotted relative to that of the housekeeping gene PA4268. Data are the mean ± SEM from three independent experiments.

Cu tolerance of ΔcopR and ΔcopS mutant strains complemented with CopR and CopS mutant proteins lacking the phosphorylatable residues. (A) Growth rate of the ΔcopR mutant complemented with copR_D51A or copR_D51E in the absence or the presence of increasing (0 to 4 mM) concentrations of CuSO₄. (B) Growth rate of the ΔcopS mutant complemented with copS_H235A in the presence of 0 to 4 mM CuSO₄. Data are the mean ± SEM from three independent experiments.

Expression of pcoB in ΔcopR and ΔcopS mutant strains complemented with CopR and CopS lacking the phosphorylatable residues. pcoB expression was determined in the absence (white) and the presence (black) of 2 mM CuSO₄ (5-min treatment) in the indicated strains. The ΔcopR mutant was complemented with copR coding for substitutions Asp₅₁Ala and Asp₅₁Glu. The ΔcopS mutant was complemented with the copS gene coding for substitution His₂₃₅Ala. Transcript levels of pcoB are plotted relative to the housekeeping gene PA4268. Data are the mean ± SEM from three independent experiments.

Structural superposition of the periplasmic Cu⁺ binding loop of P. aeruginosa CopS (gray) and E. coli CusS (yellow). The structure of CopS was modeled using the CusS structure as the template (PDB ID: 5KU5 [43]). An overall root mean square deviation of 0.791 Å (Cα atoms) was calculated for the superposition of CopS and CusS structures. Conserved Cu binding sites at the dimeric interface (His₄₁, Phe₄₂, and His₁₄₀) are shown as sticks in the structural model and highlighted in yellow in the sequence alignment. The Cu⁺ binding sites within the CusS orange loops (framed in rectangle in the alignment) are not conserved in CopS.

Determination of the dissociation constants K_D of the periplasmic Cu binding loop of CopS_(34–151). (A) Spectrophotometric titration of 100 μM BCA and 18.7 μM Cu⁺ with 10 to 50 μM His-tagged CopS_(34–151). The arrow indicates the decrease in absorbance at 562 nm upon protein addition. The inset shows the fitting of the data set to equation 2 with a K_D of (2.77 ± 0.07) × 10⁻¹⁴ M (R² 0.992). Two Cu sites per CopS dimer are assumed. (B) Spectrophotometric titration of 10 μM PAR and 4 μM Cu²⁺ with 2 to 20 μM Strep-tagged CopS_(34–151). The arrows indicate the increase in absorbance at 415 nm and the decrease at 562 nm upon protein addition. The inset shows the fitting of the data set to equation 4 with a K_D of (3.3 ± 0.1) × 10⁻¹⁴ M (R² 0.984).