||Extraintestinal pathogenic Escherichia coli (ExPEC) is a subset of E. coli that has evolved the ability to successfully colonize multiple host niches. It spends most of its time as a commensal within the gut as a frequent member of the microbiota. When ExPEC disperses to extraintestinal sites, it can cause diseases such as urinary tract infections (UTIs), bacteremia, and neonatal meningitis. The cumulative impact of ExPEC infections is quite high given the sheer prevalence of UTIs and the severity of bacteremia and neonatal meningitis. A deeper understanding of ExPEC host colonization could lead to innovative therapies that would reduce the disease and financial burden imposed by this pathogen. Here, we investigate the bacterial genetics that underpin host colonization, searching for ExPEC genes that are required for persistence in the gut and infection of extraintestinal sites. We employed three separate approaches, each uncovering a bacterial function critical for host colonization. (1) We studied how rqlI contributes to bacterial fitness in vivo, and found that in the absence of RqlI, the activity of the helicase RqlH is toxic, creating DNA lesions that trigger the bacterial DNA repair response, inhibit growth, and induce mutations. We propose that RqlH and RqlI function together to modify or repair DNA. (2) A high-throughput screen was employed to find ExPEC genes necessary for metabolism of intestinal mucus, which is thought to provide nutrients to commensal E. coli in the gut. The beta-oxidation pathway, which breaks down fatty acids, was found to be an important aspect of mucus metabolism. The beta-oxidation pathway was disrupted by misregulation of the glycerol degradation pathway, as observed in the glpG mutant, a strain that exhibited a defect in gut colonization. (3) To see if canonical extraintestinal virulence factors are important for gut colonization, several mutants were created and tested in vivo. A mutant in type 1 pili had reduced fitness, which was only partly attributable to an increase in flagellar expression. Hopefully our work, and similar future endeavors to understand the mechanisms of ExPEC colonization, will improve current therapies and create new treatment strategies.