Extended Data Fig. 2. Inflammation-associated milieu may regulate the expression of tdcA in E. coli.

(a) Mucin broth was prepared by dissolving 0.5 % mucin in None-Carbon E medium (NCE) supplemented with trace elements. Sodium nitrate, dimethyl sulfoxide (DMSO), and trimethylamine N-oxide (TMAO) were added to a final concentration of 40 mM. Mucin broth without any supplementation (None) was used as a control. Each E. coli strain (MG1655 (MG), LF82 (LF), and LF82ΔtdcΔsda double mutant (TS)) was inoculated (1 x 10⁴ CFU/ml) in the medium and incubated anaerobically for 24 h at 37°C. Bacterial numbers were determined by spreading dilutions on selective LB agar plates. The fold increase was calculated by normalizing the CFU at 24 hrs to the respective CFU at 0 hrs. Data represent geometric mean ± s.d. (N=3, biologically independent samples). N.S.; not significant by 1-Way ANOVA followed by Bonferroni post-hoc test. (b) AIEC LF82 was cultured in vitro for 8 hrs at 37°C. The expression of tdcA mRNA was assessed by qPCR. Fold expression of tdcA in steady state-like conditions (DMEM supplemented with 0.1% glucose, cultured in 0% oxygen) is shown. Different concentrations of glucose (0.1% glucose or 0% glucose) and oxygen (0% oxygen or 2% oxygen) were tested. 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamine (NOC-5) (10 mM) was added to mimic the presence of nitric oxide (NO). Data are represented as mean ± s.d.. Dots indicate individual biological replicates. ***; P < 0.001, ****; P < 0.0001 by Dunnett test. (c) SPF C57BL/6 mice WT (Ctrl) and Cybb^−/− mice were treated with 3% DSS for 5 days. At day 5 post DSS treatment, mice were co-inoculated with LF82 WT and ΔtdcΔsda (ΔTS) mutant (1 x 10⁹ CFU each/mouse). Fecal samples were collected 24 hrs post E. coli inoculation. Fecal lipocalin-2 (Lcn2) levels (a) and the competitive index (LF82 WT/LF82 ΔTS) (d) are shown. Bars represent geometric mean ± s.d. Dots indicate individual mice. (N=4-5, biologically independent animals). N.S.; not significant, * P < 0.05 by Man-Whitney U test (two-sided).