Fig. 1. Identification and characterization of H₂O₂ sensitive mutants of F. tularensis LVS.

(A) H₂O₂ sensitive mutants identified by screening of transposon mutant library of F. tularensis LVS in the presence of 250μM of H₂O_2.

(B) The H₂O₂ sensitive mutants were grown in large culture volumes (25 ml) in the absence or presence of 250μM H₂O₂. The cultures were grown for 24 hrs and OD₆₀₀ readings were recorded every 4 hrs.

(C) Disc diffusion assay with superoxide generating compound pyrogallol (Concentration = 50μg/disc). The data are cumulative of two independent experiments. The data were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. *, p<0.05; **, p<0.01.

(D) MHS- cells were infected with the F. tularensis (Ft) LVS, or the H₂O₂ sensitive mutants at 100 MOI (n=4 biological replicates). The cells were lysed at 24 hrs and plated on MH-chocolate agar plates for enumeration of bacterial CFU. The data are representative of three independent experiments conducted with identical results and were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. Comparisons are shown with Ft LVS. **, p<0.01