FIG. 2.
FeSOD activity assay. Crude cell extracts of F. tularensis LVS and sodBFt grown on modified chocolate agar plates were prepared by resuspending 10 mg of the bacterial culture in 1.0 ml of lysis solution (50 mM Tris-HCl [pH 7.4], 0.1 mM EDTA, and 0.2 mg lysozyme per ml). The lysates were clarified, and the protein concentration of each lysate was determined with a bicinchoninic protein assay kit (Pierce, Rockford, IL). Protein (15 μg) was loaded on a 10% native polyacrylamide gel electrophoresis gel and run at 150 V for 90 min at 4°C. The gel was stained for SOD activity by the method of Beauchamp and Fridovich (3). (A) Zymogram demonstrating the expression of active FeSOD of F. tularensis LVS following 24, 48, and 72 h of growth on modified chocolate agar plates. (B) FeSOD activity of sodBFt mutants. Lane 1, F. tularensis LVS; lane 2, sodBFt mutant; lane 3, a fibrosarcoma cell line transfected with MnSOD was used as a positive control. Note the markedly reduced expression of FeSOD in the sodBFt mutant.