Establishing the risk of human infection is one of the goals of public health. For bacterial pathogens, the virulence and zoonotic potential can often be related to their host source. Escherichia coli bacteria are common contaminants of water associated with human recreation and consumption, and many strains are pathogenic. In this study, we analyzed three promoter-containing intergenic regions from 284 diverse E. coli isolates in an attempt to identify molecular signatures associated with specific host types. Promoter sequences controlling production of curli fimbriae, flagella, and nutrient import yielded a phylogenetic tree with isolates clustered by established phylogenetic grouping (A, B1, B2, and D) but not by host source. Virulence genes were more prevalent in groups B2 and D isolates and in human isolates. Group B1 isolates, primarily from nonhuman sources, were the most genetically similar, indicating that they lacked molecular adaptations to specific host environments and were likely host generalists. Conversely, B2 isolates, primarily from human sources, displayed greater genetic distances and were more likely to be host adapted. In agreement with these hypotheses, prevalence of S activity and the rdar morphotype, phenotypes associated with environmental survival, were significantly higher in B1 isolates than in B2 isolates. Based on our findings, we speculate that E. coli host specificity is not defined by genome-wide sequence changes but, rather, by the presence or absence of specific genes and associated promoter elements. Furthermore, the requirements for colonization of the human gastrointestinal tract may lead to E. coli lifestyle changes along with selection for increased virulence.Pathogenic strains of Escherichia coli cause millions of cases of human infection each year (52) as well as severe problems in the livestock industry (78). Yet in other situations, many E. coli strains coexist peacefully as commensals in the intestinal tract of their warm-blooded hosts. A significant amount of research has sought to understand the relationship between pathogenic and commensal E. coli strains and determine if they can be distinguished from each other (18,55,72,95). E. coli is also a common contaminant of water and various food sources (63). From a public health standpoint, it is important to establish the zoonotic and virulence potential of strains, which may be related to their natural lifestyle and host source.E. coli has been proposed to have two principal habitats, the primary being the intestinal tract of mammals and birds and the secondary being water, sediment, and soil (58, 81). Survival and adaptation in both habitats are necessary for continued evolutionary success. For isolates that have evolved toward commensalism, signs of adaptation should be apparent, be it through altered regulatory sequences, gene expression, or metabolism (29). E. coli strains can be differentiated into four main phylogenetic groups (A, B1, B2, and D) along with two minor groups (C and E) (41). In general, these grou...