Certain Escherichia coli strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin’s chemical structure and the molecular mechanism underlying its genotoxic effects have remained unknown for more than a decade. Here we combine an untargeted DNA adductomics approach with chemical synthesis to identify and characterize a covalent DNA modification from human cell lines treated with colibactin-producing E. coli. Our data establish that colibactin alkylates DNA with an unusual electrophilic cyclopropane. We show that this metabolite is formed in mice colonized by colibactin-producing E. coli and is likely derived from an initially formed, unstable colibactin-DNA adduct. Our findings reveal a potential biomarker for colibactin exposure and provide mechanistic insights into how a gut microbe may contribute to colorectal carcinogenesis.

Colibactin is a human gut bacterial genotoxin of unknown structure that has been linked to colon cancer. The biosynthesis of this elusive metabolite is directed by the pks gene cluster, which encodes a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line that is hypothesized to use the unusual polyketide building block aminomalonate. This biosynthetic pathway is thought to initially produce an inactive intermediate (precolibactin) that is processed to the active toxin. Here, we report the first in vitro biochemical characterization of the PKS components of the pks enzymatic assembly line. We evaluate PKS extender unit utilization and show that ClbG, a freestanding acyltransferase (AT) from the pks gene cluster, recognizes aminomalonyl-acyl carrier protein (AM-ACP) and transfers this building block to multiple PKS modules, including a cis-AT PKS ClbI. We also use genetics to explore the in vivo role of ClbG in colibactin and precolibactin biosynthesis. Unexpectedly, production of previously identified pks-associated metabolites is dramatically increased in a ΔclbP/ΔclbG mutant strain, enabling the first structure elucidation of a bithiazole-containing candidate precolibactin. This work provides new insights into the unusual biosynthetic capabilities of the pks gene cluster, offers further support for the hypothesis that colibactin directly damages DNA, and suggests that additional, uncharacterized pks-derived metabolites containing aminomalonate play critical roles in genotoxicity.

Colibactin is a structurally uncharacterized, genotoxic natural product produced by commensal and pathogenic strains of E. coli that harbor the pks island. A new metabolite has been isolated from a pks(+) E. coli mutant missing an essential biosynthetic enzyme. The unusual azaspiro[2.4] bicyclic ring system of this molecule provides new insights into colibactin biosynthesis and suggests a mechanism through which colibactin and other pks-derived metabolites may exert genotoxicity.

Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for non-ribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin’s genotoxicity.

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In this paper we show how graphical analysis of ligand effect data for families of ligands should be combined with regression analysis in order to gain a more self-consistent interpretation of regression results. If a steric threshold shows up in the graphical analysis of the data for the trialkyl ligands (AR 3 ), then the threshold must show up in regression analysis of the full set of data. Similarly, the dependence of the data for the isosteric ligands A(p-XC 6 H 4 ) 3 must have the same dependence on the electronic parameter χ that we find for the regression analysis for the full set of data. Finally, graphical analysis for both AR 3 and A(p-XC 6 H 4 ) 3 families shows vividly whether steric effects and/or aryl effects belong in the full analysis. Thus, for results of any regression analysis to be acceptable, they must be consistent with the results of graphical analysis.

Certain commensal and pathogenic bacteria produce colibactin, a small molecule genotoxin that causes interstrand cross-links in host cell DNA. Though colibactin alkylates DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and β-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line and incorporating two electrophilic cyclopropane warheads into the final natural product scaffold. This proposed transformation was supported by the detection of a colibactin-derived cross-linked DNA adduct. Overall, this work provides a biosynthetic explanation for colibactin's DNA cross-linking activity and paves the way for further study of its chemical structure and biological roles.

Through the quantitative analysis of ligand effects (QALE), we have probed π effects associated with Rh-P bond lengths, ν CO , and -∆H rx for Rh(CO)(Cl)(PX 3 ) 2 and ν CO and -∆H rx for Rh(acac)(CO)(PZ 3 ). It was found that π effects are complex and depend strongly on the nature of ancillary or other participatory groups. The results of the QALE analysis of -∆H rx point out the importance of synergistic interactions between PZ 3 and other ancillary ligands in accord with theoretical computations. Literature data for Rh(CO)(Cl)(PZ 3 ) 2 have been supplemented with new values of ∆H rx for PMe 3 (-75.6 ( 0.3 kcal/mol), P(i-Pr) 3 (-68.7 ( 0.3 kcal/mol), and PCy 3 (-66.4 ( 0.4 kcal/mol) as well as crystal structures for Rh(CO)(Cl)-(P(i-Pr 3 )) 2 (Rh-P ) 2.3488(3) Å) and Rh(CO)(Cl)(PCy 3 ) 2 (Rh-P ) 2.3508 (3)).