Figure 4.

NO is involved in killing of mucoid mucA mutant bacteria. (A) Anaerobic sensitivity of P. aeruginosa under conditions allowing for the formation of identical NO levels. Bacteria were incubated anaerobically for 4 days with or without 15 mM and 1.5 mM NO₂^– at pH 6.5 and 5.5, respectively. To enumerate viable cells, 10 μl of serial dilutions were spotted on LB agar plates and incubated at 37°C for 15 hours. The left and right side of each plate represent control and NO₂^–-exposed bacteria. (B) NO levels generated by 15 mM NO₂^– (pH 6.5) over time. Carboxy-PTIO (200 nM) was added, and the stoichiometric decrease in NO signal is shown. The coefficient of determination (r²) between NO concentration and electric current (pA) was 0.9997. (C) Effects of NO scavengers on protection of FRD1 from acidified NO₂^–. Carboxy-PTIO (5 mM) or deoxyhemoglobin (0.5 mM) was added in the initial media (LB, pH 6.5, 15 mM NO₂^–). (D) Toxicity of NO toward FRD1 and FRD1/pmucA in LB at pH 6.5. NO (333 parts per million) balanced with argon was continuously bubbled for 24 hours anaerobically and viable bacteria were subsequently enumerated. Results of 4 experiments are shown. In NO-treated FRD1, differences in CFU between 2 time points were statistically significant (*P < 0.01). (E) P. aeruginosa PAO1 (circles) and isogenic sodAsodB double mutants (diamonds) were grown in LB, pH 6.5, containing 15 mM NO₂^– under aerobic (2 days, dotted lines) and anaerobic (4 days, solid lines) conditions. CFUs were measured daily and plotted in logarithmic scale. For aerobic samples, strains were grown with vigorous shaking (300 rpm).