FIG 3.

H₂S promotes H₂O₂ killing when added promptly after H₂O₂. (A) Synergistic inhibition of S. oneidensis growth by H₂S and H₂O₂ when added at the same time. Cells at an OD₆₀₀ of ∼0.01 were inoculated into LB in 24-well plates containing H₂S, H₂O₂, or both at the concentrations indicated and incubated statically at 30°C. The plates were photographed 24 h after inoculation. Experiments were performed five times, and similar results were obtained. (B) Mid-log-phase cells (OD₆₀₀ of ∼0.2) were treated for 20 min with H₂S, H₂O₂, or both at the concentrations indicated. The treated cultures were diluted, plated on LB, and incubated at 30°C. Survival was calculated as the ratio of the number of colonies in the treated cultures to that in the untreated control. Only plates containing 100 to 300 colonies were counted. (C) H₂S-H₂O₂ treatment generates extensive DNA damage, as determined by qPCR. Mid-log-phase cells were treated with 0.25 mM H₂S, 1.0 mM H₂O₂, or both. Total genomic DNA was extracted, and qPCR was performed with equivalent amounts of template DNA. The relative fluorescence was normalized to that of the untreated control. Data are reported as the mean ± SD (n = 4). (D) H₂S-H₂O₂ treatment generates extensive DNA damage, as determined by thyA mutation analysis. Mid-log-phase cells grown in LB were diluted 100-fold with fresh LB (--) or with LB containing 0.1 mM H₂S, 0.25 mM H₂O₂, or both. At each time point, CAT was added to degrade H₂O₂, and both total viability and the frequency of thyA mutants were determined. Data are reported as the mean ± SD (n = 4).