Fig. 2.

Coordinated regulation of cueP is required for optimal growth. (A) Comparative Cu MIC values in wild-type (W-t) Salmonella, ΔcueP, cpxR-box*, PcopA, and ΔcpxR mutant strains on LB plates containing increasing amounts of CuSO₄ in anaerobic conditions. (B) Detection of CueP in whole-cell extracts obtained from the cueP-3×FLAG (WT), the PcopA-cueP-3×FLAG mutant strain (PcopA), and the PcopA-cueP-3×FLAG in the ΔcpxR mutant strain (ΔcpxR PcopA), grown in LB without or with the addition of 1 mM CuSO₄. Here, 20 µg of total protein cell extracts was analyzed by SDS/PAGE, followed by transfer to nitrocellulose and development using monoclonal anti-FLAG antibodies. CueP relative levels were normalized to GroEL. (C) Mean value of three biological replicates analyzed in duplicate in each case. (D and E) Wild-type Salmonella and the PcopA-cueP mutant strain growth in static/microaerobic (D) or in aerobic conditions (E) in LB without or with the addition of sublethal concentrations of CuSO₄. The data correspond to mean values of at least four independent experiments performed in duplicate. *Significant differences in growth (P < 0.005) from the wild-type to the PcopA mutant strains grown aerobically in either the absence or the presence of Cu. (F) Wild-type Salmonella and the PcopA-cueP mutant strain growth in LB with sublethal concentrations of CuSO₄ and with the addition of 2.5 or 5 mM H₂O₂. The data correspond to mean values of at least four independent experiments performed in duplicate.