FIG 6.

FhtR controls intracellular utilization of heme. (A) fhtR deletion limits heme-dependent KatA activity. KatA enzymatic activity in E. faecalis was assessed on WT, ΔfhtR, and ΔfhtR(pfhtR) grown overnight (ON) with 1 μM hemin. Catalase activity was determined on an equivalent number of bacteria incubated with 100 μM H₂O₂ for 1 h with the spectrophotometric FOX1 method based on ferrous oxidation in xylenol orange. Results are expressed as the percentage of H₂O₂ metabolized in respective strains grown without hemin. Results represent the means plus standard deviations from three biological replicates. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test comparing each strain to the WT strain control with significance indicated as follows: ns, P > 0.05; **, P = 0.008. (B) Expression of KatA is impaired in the ΔfhtR mutant. Equivalent amounts of protein (20 μg) from lysates of WT, ΔfhtR, and ΔfhtR(pfhtR) strains as described for panel A were separated by SDS-PAGE, and immunoblots were probed with an anti-KatA polyclonal antibody. The presented results are representative of three independent experiments. (C and D) The ΔfhtR mutant is hypersensitive to H₂O₂. ON cultures of WT, ΔfhtR, and ΔfhtR(pfhtR) strains were diluted to an OD₆₀₀ of 0.01 and grown to an OD₆₀₀ of 0.5 in the absence of hemin (C) or in the presence of 1 μM hemin (D). Cultures were distributed in wells on a 96-well plate and supplemented with 2.5 mM H₂O₂ or not supplemented with H₂O₂. OD₆₀₀ was monitored every 20 min in a microplate spectrophotometer (Spark; Tecan). OD₆₀₀ at time zero was normalized to 0. Results represent the means ± standard deviations from three biological replicates. (E) KatA limits heme toxicity. ON cultures of WT and katA::tetR strains were diluted to an OD₆₀₀ of 0.01 and grown in 96-well plate in the presence of the indicated concentration of hemin as described for panel D. Results represent the means ± standard deviations from three biological replicates.