Quantification of Extracellular Pyruvate in S. mutans

S. mutans UA159 wildtype, SAB161, and SAB163 were grown in FMC medium containing 11 mM glucose. Measurements of extracellular pyruvate during growth were taken from 250 μl samples at 1–2 h intervals and 100 μl was used to monitor growth (OD₆₀₀). Extracellular pyruvate levels during early-exponential growth and stationary phase were predicted based on previously published data (Ahn et al., 2019 (link)). Thus, pyruvate quantification in this study was focused on samples taken at late-exponential and early-stationary phases, time points at which lrgAB induction and LrgAB-dependent pyruvate uptake occur. The remaining 150 μl of each sample was centrifuged for 2 min at 18,000 × g to remove cells, and pyruvate concentrations of supernatants quantified with an EnzyChrom^TM pyruvate assay kit (BioAssay Systems, Hayward, CA, United States), according to the manufacturer’s instructions.