Fig. S5.

Better fitness of the ΔhflX mutant under heat stress. (A) Acute thermal killing assay. The ΔhflX mutant is at least sixfold more resistant to heat killing. Stationary phase TSB cultures were diluted with fresh TSB to an OD₆₀₀ of 0.4, split into two portions and incubated at 37 °C and 62 °C in a thermomixer shaker at 300 rpm. After 30 min of incubation, the cells were subjected to 10-fold serial dilutions with sterile 1× PBS buffer. Five microliters of each dilution was spotted on TSB agar plates and incubated for 18 h at 37 °C. The results are representative of three independent biological replicates. (B) A temperature downshift from 47 °C to a permissive temperature of 37 °C restores the growth of the WT and ΔhflX complementing strains. The cells were grown at 47 °C for 3 h, during which the OD₆₀₀ of the ΔhflX mutant peaked at 2 h and gradually decreased (yellow box, Inset), whereas the WT and complementing strains were less viable (see also Fig. 4A). Upon heat removal, the WT and complementing strains regrew to levels comparable to the ΔhflX mutant. The error bars are SEs from four independent experiments.