FIG 4.

Cation supplementation restores anaerobic viability of ypfP mutants undergoing respiration arrest. (A) Membrane potential of the WT and ypfP mutant. The y axis shows the quotient of the 610 nm and 530 nm emission spectra. Data are means from three independently performed experiments performed in technical triplicate. Error bars represent one standard deviation from the mean. Significance was determined using two-way ANOVA. ****, P < 0.0001. (B) The antimicrobial activity of the potassium-specific ionophore valinomycin was determined for WT and ypfP mutant cells. The WT and the ypfP mutant were normalized to an OD₆₀₀ of 0.5 and were allowed to grow in liquid medium in the presence of various concentrations of valinomycin anaerobically for 16 h. Cells were suspended by pipetting, and the OD₆₀₀ was measured. Results are percent OD₆₀₀ compared to that of the untreated control. The mean for five independent experiments performed in triplicate is shown. The regression line was mapped using GraphPad Prism. Error bars represent one standard deviation from the mean. (C) Anaerobic viability of the WT and the ypfP mutant on medium supplemented with various concentrations of KCl, NaCl, or the osmoprotectant GB. CFU were enumerated after 24 h of growth at 37°C. Data are means from three independent experiments performed in triplicate. Error bars represent one standard deviation from the mean. (D) Anaerobic viability of the WT or the ypfP mutant on medium buffered to a pH of 7 or 6. CFU were enumerated after 48 h of growth at 37°C. Data are means from three independent experiments performed in triplicate. Error bars represent one standard deviation from the mean. (E) The WT and the ypfP mutant were subcultured 1:100 in TSB, and the OD₆₀₀ and pH were measured at the indicated time points. The data are averages from three independent experiments. Error bars represent one standard deviation from the mean. (B and C) Significance was determined using one-way ANOVA with multiple comparisons. ****, P < 0.0001; **, P <0.005.