Colibactin inflicts self-damage. (A) Schematic of the fluorescent DNA damage reporter plasmid used to quantify self-damage. CFP is expressed under a constitutive promoter, and YFP is expressed under a DNA damage–inducible promoter. Representative microscopy images of the engineered (BAC) pks⁺ and pks⁻ colonies (B) or Nissle 1917 pks⁺ (EcN) and pks⁻ (EcN ΔclbN) expressing our recA reporter plasmid (C). Images show YFP expression, which represents recA activation in the colonies owing to colibactin-induced self-damage. The fluorescence intensity range was set according to the intensity observed in the colibactin-producer field of view per strains. Violin plots display the median YFP signal intensity per colony for each strain. Background YFP and CFP autofluorescence of the colonies was subtracted from each channel before YFP was normalized to CFP per colony (***) P < 0.001, two-sample t-test. (D) Schematic of our analysis of 9089 genomes to calculate the trinucleotide skew toward ATA/TAT over AAA/TTT sequences. Genomes are broken down by phylogroup, and the total number of genomes per phylogroup, as well as the number of pks⁺ genomes, included in our analysis is reported. The pie chart shows the total percentage of genomes each phylogroup represented. (E, left) Violin plots of trinucleotide skewness by E. coli phylogroup. Median skew is marked by the dashed line. (Right) The B2 phylogroup skewness is further divided into genomes with colibactin and genomes without colibactin. The number of genomes in each condition is labeled in this panel.