Colibactin induces a bacteria-specific mutational signature. (A) Overview of mutation accumulation experiment. Reporter cells were cocultured with pks⁺ cells in a pellet for 24 h before plating on selective agar. Single reporter colonies were selected and grown for subsequent exposure. Reporter cells were exposed 10 times before whole-genome sequencing. (B) Summary table of annotated mutations by mutation type. Bold mutations mark key differences between the pks⁺ and pks⁻ exposed populations. (C) Whole-genome sequencing coverage of colibactin/control conditions. The positions for all nine cryptic prophages are marked above. The gray-shaded region marks the e14 prophage region shown in more detail in the panel inset. (D) The A/T enriched motif found in the 13 bp region that surrounds positions of single-base substitutions (SBSs). The ring plots show positions of SBSs: outer ring in gray indicates SBSs matching the motif; inner ring in black, all other SBSs. The chromosome ring shows previously defined macrodomain regions. (E) SBS positions show genomic positional bias and are enriched near the terminus, increasingly so for mutations occurring in the identified motif. (F) Trinucleotide context of SBS mutations. The upper panel shows mutations identified after coculturing with pks⁺ bacteria. The middle panel shows mutations identified after coculturing with pks⁻ bacteria (control). The bottom panel shows mutations annotated in colon cells exposed to pks⁺ bacteria by Pleguezuelos-Manzano et al. (2020). Differential mutation signatures between our work and work in colon cells are highlighted in red.