M. bovis BCG cultures were grown to mid-log phase in 1 L roller bottles (Corning) at 37 °C and pelleted in 50 mL tubes at 3200 rpm for 10 min. Pellets were resuspended in the specific growth medium after adjusting to an OD600 of 0.2 and incubated with or without POA, nicotinic acid, or benzoic acid. At each time point for each treatment, the samples were plated on 7H10 to determine CFU, and an equivalent of 2 × 109 CFU was pelleted by centrifugation. The pellet was resuspended in 500 μL of phosphate-buffered saline (PBS) and homogenized by bead beating (Precellys 24 homogenizer) at 6500 rpm three times for 30 s each. The lysate was pelleted by centrifugation, and the supernatant was subsequently used for analysis by a CoA assay kit (Sigma-Aldrich MAK034) per the manufacturer’s instructions. The fluorescence intensity was read on a Tecan Infinite M200 plate reader (λex = 535 nm/λem = 587 nm), and the CoA quantity was determined in nanomoles from a standard curve that was obtained for each experiment. These experiments were performed in technical replicates of independent biological replicates and normalized per CFU.
The lysate supernatant was also used for mass spectrometric quantification of CoA and pantothenate, which was achieved by using liquid chromatography coupled to mass spectrometry (LC-MS). (Details are described inSupporting Information , Supplemental methods .)
The lysate supernatant was also used for mass spectrometric quantification of CoA and pantothenate, which was achieved by using liquid chromatography coupled to mass spectrometry (LC-MS). (Details are described in