Coy microoxic chamber

For in vitro growth assay, several types of E. coli strains (10³ CFU/100 μl) were mono-cultured aerobically in a minimal medium (MM) containing 0.1% glucose supplemented with a single 1 mM amino acid for 8 hours at 37°C in 20% O₂ and 5% CO₂. Bacterial growth was quantified by optical density measurement (O.D.600). High glucose DMEM (0.45% glucose, Gibco) was used as a positive control. For in vitro competition assay, MG1655 and LF82 were mixed at the ratio of 1:1 (10³ CFU each/100 μl) and cultured in MM supplemented with the indicated single amino acid (1 mM) for 8 hours at 37°C anaerobically. For in vitro tdcA activation assay, LF82 was cultured in a custom made DMEM (0% or 0.1% glucose). To examine the influence of O₂ concentration, E. coli was cultured in the Coy Microoxic Chamber (2% O₂, Coy Laboratory Products) or the Coy Anaerobic Chamber (0% O₂, Coy Laboratory Products) for 6 hours at 37°C. Bacterial RNA was extracted using the Trizol Max Bacterial RNA isolation kit (Ambion) and converted to cDNA using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). tdcA expression was quantified by qPCR (primers shown in Supplementary Table 4). For assessing the in vitro tdcA activation in response to NO, the NO-releasing compound 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamine (NOC-5, Calbiochem) was freshly prepared and added to LF82 culture.