FIG. 4.
Effect of sodB mutation on sensitivity to paraquat. (A) The left and right panels represent F. tularensis LVS and sodBFt mutant strains, respectively. Briefly, 1 × 109 F. tularensis LVS or sodBFt bacteria were plated on modified chocolate agar plates. Sterile filter paper discs were placed on each plate, and 5 μl of 1.25, 2.5, 5.0, and 10 mg/ml concentrations of freshly prepared paraquat was added to individual discs. The plates were incubated for 48 to 72 h at 37°C, and the zones of growth inhibition were observed. Insets show the zone of growth inhibition observed with kanamycin, which was used as an experimental control. (B) Quantitation of the zone of growth inhibition. The zone of growth inhibition around the discs impregnated with a 10 mg/ml concentration of paraquat was quantitated using AlphaEase FC software (Alpha Innotech Inc., San Leandro, CA). Data represent the means ± standard errors of the means for the zone of inhibition determined for each strain (n = 5 plates per strain) and the cumulative results of two independent experiments. Comparisons between the strains were made using a nonparametric Mann-Whitney test (P < 0.0001). (C) F. tularensis LVS and the sodBFt mutant strain were grown in MHB to an OD600 of 0.2. Paraquat (1 mM) was then added to the cultures, and cultures were allowed to grow at 37°C with constant shaking. Aliquots were removed at the times indicated, diluted 10-fold, and plated on modified chocolate agar plates to determine the number of CFU. Data are representative of two independent experiments.