FIGURE 3.
WhiB3 regulates dynamic changes in intramycobacterial EMSH of M. tuberculosis in response to acid stress. WT M. tuberculosis, MtbΔwhiB3, and whiB3-comp strains expressing Mrx1-roGFP2 were grown in 7H9-tyloxapol medium at a pH of 7.0 (A), 6.2 (B), 5.5 (C), and 4.5 (D). At the indicated time points, EMSH of 30,000 bacterial cells was measured by flow cytometry as described under “Experimental Procedures.” At 72 h postincubation, 500 μm CCCP was added to dissipate the pH gradient, and intramycobacterial EMSH was measured 4 h post-CCCP treatment (i.e. 76 h). **, p < 0.01; ***, p < 0.005 (as compared with WT M. tuberculosis); +, p < 0.05; ++, p < 0.01; +++, p < 0.005 (as compared with whiB3-comp). Shown is the viability of WT M. tuberculosis, MtbΔwhiB3, and whiB3-comp upon CCCP treatment at pH 7.0 (E) or pH 4.5 (F and G) for 4 h, as determined by enumerating cfu. Data shown in each panel are the result of at least two independent experiments performed in triplicate. Error bars, S.D. **, p < 0.01; ***, p < 0.005.