ABSTRACTEscherichia colipersistently colonizes the mammalian intestine by mechanisms that are not fully understood. Previously, we found when streptomycin-treated mice were fedE. coliMG1655, the intestine selected forenvZmissense mutants that outcompeted the wild type. The better-colonizingenvZmutants had a higher level of OmpC and reduced OmpF. This suggested the EnvZ/OmpR two-component system and outer membrane proteins play a role in colonization. In this study, we show that wild-typeE. coliMG1655 outcompetes anenvZ-ompRknockout mutant. Moreover,ompAandompCknockout mutants are outcompeted by the wild type, while anompFknockout mutant colonizes better than the wild type. Outer membrane protein gels show theompFmutant overproduces OmpC. AnompCmutant is more sensitive to bile salts than the wild type andompFmutant. TheompCmutant initiates colonization slowly because it is sensitive to physiological concentrations of bile salts in the intestine. Overexpression ofompCunder the control of a constitutive promoter confers a colonization advantage only whenompFis deleted. These results indicate that fine-tuning of OmpC and OmpF levels is needed to maximize competitive fitness in the intestine. RNA sequencing reveals the EnvZ/OmpR two-component system is active in the intestine:ompCis upregulated andompFis downregulated. While other factors could also contribute to the advantage provided by OmpC, we provide evidence that OmpC is important forE. colito colonize the intestine because its smaller pore size excludes bile salts or other unknown toxic substances, while OmpF is deleterious because its larger pore size allows bile salts or other unknown toxic substances to enter the periplasm.IMPORTANCEEvery mammalian intestine is colonized withEscherichia coli. AlthoughE. coliis one of the most studied model organisms, how it colonizes the intestine is not fully understood. Here, we investigated the role of the EnvZ/OmpR two-component system and outer membrane proteins in colonization of the mouse intestine byE. coli. We report that anompCmutant is a poor colonizer, while anompFmutant, which overproduces OmpC, outcompetes the wild type. OmpF has a larger pore size that allows toxic bile salts or other toxic compounds into the cell and is deleterious for colonization of the intestine. OmpC has a smaller pore size and excludes bile salts. Our findings provide insights into whyE. colifine-tunes the levels of OmpC and OmpF during colonization via the EnvZ/OmpR two-component system.