In vivo quantification assay for (p)ppGpp synthesis.

M. smegmatis mc²155 strain was cultured in the presence of AB and AC compounds to an optical density at 600 nm (OD₆₀₀) of 0.2 in 5 ml of 1× morpholinepropanesulfonic acid (MOPS) defined medium supplemented with 80 μg/ml Casamino Acids, 0.05% Tween 80, and 2% glucose at 37°C with agitation. Cultures were radiolabeled by adding [o-³²P]phosphoric acid (specific activity, >3,000 mCi/mmol; Board of Radiation and Isotope Technology, India) directly to the growth medium to a final concentration of 100 μCi/ml. The cells were harvested at 72 h of growth, followed by lyophilization. Equal amounts of cells were suspended in 20 μl of 1× MOPS solution. The cells were lysed by the addition of 12 N formic acid and stored on ice for 20 min. The sample was subjected to centrifugation at 13,000 rpm and 4°C for 10 min. Then, 2 μl of supernatant, normalized to an OD₂₆₀ of 2.0, was spotted onto a polyethylenimine-cellulose sheet (Merck) for TLC analysis (1.5 M KH₂PO₄ [pH 3.4]) (27). The TLC sheets were air dried and phosphorimaged, and the (p)ppGpp spots were examined by densitometry as described previously. The identity of the spots was further confirmed by MALDI-TOF mass spectrometry.