Soft sided vinyl chamber

We grew R. gnavus ATCC 29149 in BHI medium (37 g/L) containing: 5% sterile-filtered fetal bovine serum (Sigma-Aldrich), 1% vitamin K1-hemin solution (BD Biosciene), 1% trace mineral supplement (ATCC), 1% vitamin supplement (ATCC), 1 g/L D-(+)-cellobiose (Sigma-Aldrich), 1 g/L D-(+)-maltose (Sigma-Aldrich), 1 g/L D-(+)-fructose (Sigma-Aldrich) and 0.5 g/L L-cysteine (Sigma-Aldrich). Growth occurred under anaerobic conditions (atmosphere 5% H2, 20% CO2, 75% N2) in a soft-sided vinyl chamber (Coy Laboratory Products, Michigan, USA). We sterilized the media using a Corning filter unit (0.22 µm pore diameter). All metabolite standards (Sigma-Aldrich) were brought to 100 mM in DMSO (Sigma-Aldrich, D2438) prior to dilution for dose assays. Overnight bacterial cultures were diluted 100-fold in appropriate media and 40 µL were dispensed per well in 384-well plates (low evaporation lid, Costar 3680) containing metabolites or DMSO control. The plates were shaken to ensure homogeneity and bacterial growth was monitored anaerobically (absorbance at 600 nm) in a microplate reader (PowerWave HT Microplate Spectrophotometer, BioTek) for 24 hours at 37°C without shaking. Values recorded for DMSO controls and metabolite-treated triplicates were averaged.