Central metabolite extraction

Mycobacteria were grown to an OD₆₀₀ of 1.0. Approximately 1 × 10⁸ cells were collected by filtration on 0.22-µm nylon/polyvinylidene fluoride filters (Millipore GVWP02500) and transferred to 7H11 supplemented with 0.4% glycerol, sodium propionate, cholesterol, sodium pyruvate, or oleic acid as the sole carbon source. Plates were incubated for 6 days at 30°C or 37°C for bacterial replication and biomass production. The polar metabolites were extracted in prechilled (−40°C) 2:2:1 methanol/acetonitrile/water, and cells were lysed six times by bead-beating for 30s with incubation on ice for 30s between pulses. Soluble extracts were filtered (Spin-X filter tubes) at 5,000 × g for 5 min at 4°C and then stored at −80°C for liquid chromatography–mass spectrometry. The bacterial biomass of each sample was determined by measuring the residual protein content in the metabolite extracts.