FIG. 5.

Restoration of ExoU expression in the ΔnirS mutant by supplementation with NO donors. (A) All strains were grown for 6 h in LB broth supplemented with 0.5 M EGTA to chelate calcium and induce the expression of T3SS exoproducts (23). Cultures were supplemented with either a NaNO₂ or a NO donor (DETA-NO, GSNO, or SNP) 1 h prior to supernatant harvesting, as indicated. Culture supernatant proteins were precipitated and analyzed by Western blotting with a mouse MAb against ExoU. T3SS exoproducts were not detected in supernatants from the ΔnirS mutant supplemented with NaNO₂. In contrast, exoproducts were detected in the supernatant of the ΔnirS mutant supplemented with all of the NO donors. (B) NO consumption by PA14 and the ΔnirS mutant in the presence of 3 mM SNP. The steady-state level of NO release by SNP occurred within 150 s, with a plateau at 1.6 μM (black line). The wild-type PA14 NO level peaked at 1.3 μM and reached a steady state at 200 nM (dark gray line). The ΔnirS mutant NO level peaked at 300 nM and reached a steady state at 100 nM.