Fig. 2

a To test if induction at pH 4.2 is reversible, M. smegmatis containing pBEN, pMPR, or pFPV27 were grown to mid-logarithmic phase at pH 7.0, then exposed to pH 4.2, 5.0, or 7.0 for 3 h. An additional culture had been previously grown at pH 7.0 to mid-logarithmic phase, shifted to pH 4.2 for 3 h, and then shifted to pH 5.0 for an addition 3 h. AFUs were determined as previously described. The data are presented as the mean ± the standard deviation. *P < 0.04, significantly different from pH 4.2 3 h of exposure. The sample size was 3 and represents biological replicates. b To determined viability of mycobacteria exposed to acidity, the M. smegmatis containing pMPR were exposed to pH 7.0, 5.0, or 4.2, diluted to 1:10⁵, and plated onto 7H10 plates. Bacterial colony forming units (CFU)/ml of the undiluted sample were quantitated from the bacterial plates and were normalized by dividing by the OD₆₀₀ of the bacterial undiluted samples. Exposure to 3 h of acidity did not reduce mycobacterial viability in any of the conditions tested including 3 h of exposure to pH 4.2. As a control M. smegmatis was grown to mid-logarithmic phase and then exposed to pH 4.5 or 7.0 for 24 h. The bacteria were then diluted and plated onto agar plates and the CFU/ml normalized for OD₆₀₀ as before. As expected exposure to 24 h at pH 4.5 resulted in a 75% reduction of viability of the mycobacteria whereas exposure to pH 7.0 did not. The data are presented as the mean ± the standard deviation. *P < 0.05, significantly different from pH 4.5 24 h exposure; ^#P < 0.03, significantly different from pH 4.5 24 h exposure; ⁺P  < 0.02, significantly different from pH 4.5 24 h exposure; ^oP < 0.02, significantly different from pH 7.0 24 h exposure. There was no statistical difference for pH 4.2 or 5.0 3 h of exposure compared to pH 7.0 3 h of exposure. There was also no statistical difference between pH 4.2, 5.0, or 7.0 3 h of exposure compared to pH 7.0 24 h of exposure. Statistical difference was defined as a P < 0.05, the sample size was 3 and represents biological replicates. c M. smegmatis containing pMPR was grown to mid-logarithmic phase and exposed to pH 4.2, 4.3, 5.0, or 7.0 for 3 h, or 4.2 3 h and then 5.0 3 h. Individual M. smegmatis bacilli were visualized via differential interference contrast microscopy (DICM) and fluorescence microscopy at ×40 magnification. This experiment was repeated three times with similar results. Approximately 7 images were sampled for each condition