Fig. 4.

Colibactin induces SOS response and ROS accumulation in V. cholerae. (A) O₂ affects EMS1 inhibition of V. cholerae growth. V. cholerae alone or cocultured with EMS1 was grown aerobically or microaerobically in M9 medium for 24 h. Viable V. cholerae CFU values were then determined. The mean of three independent assays is shown, and error bars represent the SD; *P < 0.05 (t test). (B) clb expression. EMS1 harboring a chromosomal clbB–lacZ transcriptional fusion was grown aerobically and microaerobically. At the growth phase indicated, samples were withdrawn, and β-galactosidase activity was measured. The mean of three independent assays is shown, and error bars represent the SD; ****P < 0.0001 (one-way ANOVA). (C) Induction of SOS response. V. cholerae harboring chromosomal PrecA-gfp and Ptet-mCherry reporters was inoculated alone or with either EMS1 or ΔclbN mutants into M9 medium. The cultures were grown for 16 h, and then green fluorescent protein (GFP) and mCherry intensity of each V. cholerae cell was measured using a Nikon NiU fluorescence microscope. For each condition, 200 cells were analyzed; ****P < 0.0001 (two-way ANOVA). (D) ROS accumulation staining assay of V. cholerae when cocultured with EMS1 and colibactin-deficient clbN mutant. V. cholerae harboring a chromosomal Ptet-mCherry reporter was inoculated alone and with EMS1 or ΔclbN mutants in M9 medium. The cultures were grown for 24 h, and DCFDA staining was performed and visualized using the GFP filter and normalized against mCherry intensity. For each condition, 100 cells were analyzed; ****P < 0.0001 (two-way ANOVA).