Escherichia coli strains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coli K1 neonatal isolates carried clbA and clbP, genes contained within the pks pathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbA and clbP in E. coli A192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coli K1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate.E scherichia coli strains belonging to phylogenetic group B2 are found with increasing frequency in the feces of healthy individuals from high-income countries (1) and are responsible for a range of extraintestinal diseases that include urinary tract infections, sepsis, pneumonia, and neonatal meningitis (2). These extraintestinal pathogenic E. coli (ExPEC) strains carry genes encoding a diverse range of virulence determinants that promote colonization, invasion, survival in the blood compartment, and the capacity to evade host defenses and damage the host (3). These virulence-enhancing genes tend to cluster on pathogenicity islands, genomic elements that facilitate dissemination by horizontal gene transfer (4). The pks genomic island is found in 30% to 40% of E. coli B2 strains and codes for the production of colibactin, a polyketide-peptide genotoxin of as-yet-unknown chemical structure that causes doublestrand (ds) breaks in DNA, leading to cell cycle arrest, chromosome instability, and increased lymphopenia in septic rodents (5-8). Carriage of the pks island is linked to long-term persistence in the gastrointestinal (GI) tract (9), and pks-bearing strains represent a high-virulence subset within the B2 group (6). The 54-kb pks island encodes nonribosomal peptide synthases, polyketide synthases, and hybrid synthases, in addition to accessory, tailoring, and editing enzymes; the clbA gene encodes a phosphopantetheinyl transferase required for colibactin synthesis, and clbP specifies a D-amino peptidase involved in colibactin maturation (10, 11). The pks island also contributes to the synthesis of siderophore...