Enzychrom pyruvate assay kit

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.

NAD, Pyruvate and ATP levels were estimated in cell lysate using commercially available kits (EnzyChrom NAD+/NADH Assay Kit, BioAssay Systems; EnzyChrom Pyruvate Assay Kit, BioAssay Systems; ATP determination kit, Invitrogen) as per manufacturer’s procotol and were normalized to cellular protein.

Overnight cultures were back-diluted 1:100 in 96-well round-bottom plates containing 100 μl/well of growth medium consisting of 38% TSB (either with or without glucose) and 62% calprotectin buffer (20 mM Tris [pH 7.5], 100 mM NaCl, 3 mM CaCl₂, 10 mM β-mercaptoethanol) and were grown in the absence or presence of 240 μg/ml CP. Bacteria were harvested during logarithmic-phase growth at similar optical densities (OD₆₀₀ = 0.2 to 0.25). Bacterial culture (∼60 ml) was collected in a Millipore S-Pak membrane filter, washed twice with 0.5% NaCl, and stored at −80°C in 60% (vol/vol) high-performance liquid chromatography (HPLC)-grade ethanol. Prior to assay of pyruvate, cells were thawed, washed from the filter, and then lysed by mechanical disruption using a FastPrep-24 homogenizer (MP Biomedicals) for 45 s at 6.5 m/s. Cell lysates were clarified by centrifugation at 10,000 × g for 5 min at 4°C. The supernatants were collected and vacuum dried. The dehydrated pellets were resuspended in 100 μl of HPLC-grade water, and then pyruvate levels were measured using an EnzyChrom pyruvate assay kit (BioAssay Systems) as indicated by the manufacturer.

S. mutans UA159 wild type, Δpta, ΔackA and ΔptaΔackA mutants were grown in BHI medium at 37 °C in a 5% CO₂ atmosphere. For time course measurements of extracellular pyruvate, excreted during growth, samples (300 µL) were taken at 1–2 h intervals and 150 µL was used to measure the OD₆₀₀ in a spectrophotometer for monitoring growth. The rest of the sample (150 µL) was centrifuged for 2 min at 18,000× g to remove the cells, and pyruvate concentration of the supernatant was quantified with an EnzyChrom™ pyruvate assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions. The level of extracellular pyruvate levels at certain time points (i.e., during early exponential growth and stationary phase) are predictable without measurement [26 (link)]. Thus, the pyruvate quantification was largely carried out with a particular focus on the samples taken at late-exponential and early-stationary phases in which lrgAB induction and pyruvate uptake through LrgAB occur. The results are average or representative of two independent replicates, each performed in duplicate.

S. mutans UA159 wild-type and isogenic mutants (ΔlrgAB, ΔlytS, ΔcidB, or UA159/184-cidAB) strains were grown in chemically defined FMC medium, supplemented with either 11 mM or 45 mM glucose. For time course measurements of extracellular pyruvate during growth, samples (200 μl) were taken at 1–2 h intervals and half of this volume (100 μl) was used to measure the OD₆₀₀ in a spectrophotometer for monitoring growth. The other half (100 μl) was centrifuged for 2 min at 18,000 xg to remove the cells, and pyruvate and glucose concentrations of the supernatant were quantified with an EnzyChrom™ pyruvate assay kit (BioAssay Systems, Hayward, CA) or glucose (HK) assay kit (Sigma-Aldrich), respectively, according to the manufacturer’s instructions. The results are average or representative of at least two independent replicates, each performed in duplicate.

Streptococcus mutans strains were grown in a low‐glucose (11 mM) FMC medium. Time‐course measurements of extracellular pyruvate during growth were measured as described previously (Ahn et al., 2019). Briefly, for monitoring growth, samples (200 μl) were taken at 1‐ to 2‐hr intervals and half of this volume (100 μl) was used to measure the optical density at 600 nm in a spectrophotometer. The other half (100 μl) was used to quantify extracellular pyruvate concentrations in the supernatant with an EnzyChrom™ Pyruvate Assay Kit (BioAssay Systems). The results are an average of two independent replicates, each performed in duplicate.