Genetic screen reveals key role of homologous recombination in colibactin response. (A) Overview of the reduced viability assay in response to colibactin. Reporter cells were cocultured with pks⁺ cells in a pellet and plated on selective agar to determine the number of viable reporter cells. The colony images on the right show representative results of a spotting assay with reduced viability after pks⁺ cocultures. (B) Colibactin toxicity correlates with coculture period. A reporter strain was cocultured in pellets with pks⁺ (light gray) or pks⁻ (dark gray) strains at a 10:1 ratio in M9. The bar graphs show the mean CFUs back-calculated from the spotting assay. Error bars show standard deviation of triplicates. (**) P < 0.01, (***) P < 0.001, (n.s.) not significant, two-sample t-test. (C) Colibactin toxicity is influenced by coculture conditions. All conditions were evaluated at 24 h. (Left) Toxicity is impacted by growth media: Cocultures were conducted in either nutrient-poor (M9) or -rich media (LB) at a 10:1 ratio and pelleted. (Middle) Toxicity is impacted by growth conditions: Cocultures were in nutrient-poor media at a 10:1 ratio either pelleted or in suspension. (Right) Toxicity is impacted by incubation ratio: Cocultures were in nutrient-poor media at either a 10:1 or 1:1 ratio of pks⁺ to reporter strain and pelleted. Error bars show standard deviation of triplicates. (*) P < 0.05, (**) P < 0.01, (n.s.) not significant, two-sample t-test. (D) Overview of barcoded genetic screen approach. (E) Volcano plots of screen results. Resistant hits are colored in green, and sensitive hits are colored in blue. The number of hits for each direction are reported in colored circles on each volcano plot. Vertical gray lines represent the fold-change cutoff. (F) Venn diagram of shared sensitive hits between 24 h 10:1, 48 h 1:1, and 48 h 10:1 screens.