Similarities in multidomain signatures support post-assembly-line transformations. (A) The multidomain signatures of reactive precolibactins, linear (7) and lactam (8), en route to precolibactin C (5) (top, bottom, and Figure 3, bottom left, respectively) show similar multidomain dependencies, indicating that the chemical transformations occur off the assembly line. (B) New precolibactin metabolites were confirmed via comparisons with synthetic standards. (C) ClbP cleaves supplemented 7 to form lactam 10. Pyridone 9 was also detected. Coinjections with synthetic standards via the intermediacy of 12 confirmed the production of these two colibactin metabolites. (D) Proposed biosynthetic fate of reactive precolibactin 7 metabolite in the presence and absence of peptidase ClbP. ClbP is required in order to access lactam 10, a known genotoxic scaffold.