Figure 2.

Mucoid P. aeruginosa FRD1 is selectively killed by NO₂^– in a pH-dependent manner. Anaerobic growth of P. aeruginosa strains at pH 6.5 using 15 mM NO₃^– or NO₂^– as a terminal electron acceptor. Aerobic overnight suspensions of PAO1, FRD1, and FRD1/pmucA were diluted 100-fold for the main anaerobic culture with NO₃^– (A) or NO₂^– (B). CFUs were enumerated each day and plotted as linear (A) and logarithmic (B) graphs. (C) NO₂^– sensitivity versus pH. FRD1 or FRD1/pmucA were seeded onto LB agar buffered at the indicated pH value. After placing a filter disk containing 10 μl of 1 M NO₂^–, the plates were incubated anaerobically for 48 hours and scanned for viewing the zone of killing. To support anaerobic growth, 15 mM NO₃^– was included in the media. (D) Mucoid strain FRD1 (gray bars) and nonmucoid FRD1/pmucA (black bars) were incubated together at the indicated ratios (FRD1/pmucA:FRD1) for 5 days in the presence of 15 mM NO₂^–, pH 6.5, after which CFUs were determined. *P < 0.001 versus CFU in the initial inoculum. (E) Dose-response killing of mucoid strain FRD1 by NO₂^–. Bacteria were suspended in LB (pH 6.5) with various amounts of NO₂^– for 24 hours under anaerobic conditions. Survival against NO₂^– is presented as the percentage of CFU relative to that in initial inoculum. (F) Long-term anaerobic exposure of mucoid strain FRD1 to 15 mM NO₂^– at pH 6.5. All experiments were performed in triplicate and presented as mean ± SEM.