M. tuberculosis H37RvMA and its coaBC Tet-OFF conditional knockdown derivative12 (link) were grown in Difco Middlebrook 7H9 broth (BD) supplemented with Middlebrook albumin-dextrose-catalase (ADC) enrichment (BD), 0.2% glycerol (Sigma-Aldrich) and 0.05% Tween-80, unless otherwise indicated. Hygromycin and kanamycin were used at final concentrations of 50 μg/mL and 25 μg/mL, respectively, and pantethine (Sigma-Aldrich) supplementation was included at 2.5 mg/mL where required. The anhydrotetracycline (ATc) inducer was used at concentrations up to 200 ng/mL in order to transcriptionally silence coaBC in the Tet-OFF hypomorph.
The minimum inhibitory concentrations (MIC99) of the compounds were determined by measuring fluorescence output using Alamar Blue, as previously described (Singh et al.56 (link)). Briefly, 2-fold serial dilutions of compound were inoculated with a suspension of M. tuberculosis at a cell density of ~105 CFU/mL in a 96-well microtiter plate and incubated at 37 °C for 10 days, following which 10 μL Alamar Blue solution was added and the plates were incubated for a further 24 h. Fluorescence as an indication of growth was measured using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices) in bottom-reading mode with excitation at 544 nm and emission at 590 nm.
The minimum inhibitory concentrations (MIC99) of the compounds were determined by measuring fluorescence output using Alamar Blue, as previously described (Singh et al.56 (link)). Briefly, 2-fold serial dilutions of compound were inoculated with a suspension of M. tuberculosis at a cell density of ~105 CFU/mL in a 96-well microtiter plate and incubated at 37 °C for 10 days, following which 10 μL Alamar Blue solution was added and the plates were incubated for a further 24 h. Fluorescence as an indication of growth was measured using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices) in bottom-reading mode with excitation at 544 nm and emission at 590 nm.
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