FIG 4.

(A) Acid survival assay. WT, ΔsigB, ΔrsbX, ΔrsbX + rsbX, and ΔsigB ΔrsbX strains were grown overnight and resuspended in BHI of pH 2.5 and incubated at 37°C. Samples were taken at indicated time points and spread on agar plates to determine survival rate (CFU per milliliter). The graphic shows the average values of 3 biological replicates. A 2-way ANOVA with multiple comparisons was used for statistical analysis comparing all strains at the different time points with WT. *, P < 0.05. (B) Acid adaptation assay at 37°C. WT and the ΔrsbX mutant were grown until OD₆₀₀ of ∼0.4 was reached when the cultures were either acidified (+) or not (−) at pH 5.0. (B) After 15 min, the cultures were acidified to pH 2.5 and the CFU per milliliter was counted at indicated time points. (C and D) The total RNA was extracted after 20 min of mild acidification, and the levels of transcripts of lmo2230 (C) and lmo0596 (D) were measured using RT-qPCR and normalized against 16S rRNA. Student's t test was used for statistical analysis. **, P < 0.01; **, P < 0.001 (n = 3).