The O-antigen moiety of the LPS is one of the most variable cell surface components of the Gram-negative bacterial outer membrane. Variation is due to the presence of different sugars and sugar linkages. Here, it is reported that a group of Escherichia coli O serogroups (O17, O44, O73, O77 and O106), and the Salmonella enterica serogroup O : 6,14 (H), share a common four-sugar backbone O-subunit structure, and possess almost identical O-antigen gene clusters. Whereas the E. coli O77 antigen does not have any substitutions, the other O antigens in this group differ by the addition of one or two glucose side branches at various positions of the backbone. The O-antigen gene clusters for all members of the group encode only the proteins required for biosynthesis of the common four-sugar backbone. The identification of three genes within a putative prophage in the E. coli O44 genome is also reported; these genes are presumably involved in the glucosylation of the basic tetrasaccharide unit. This was confirmed by deletion of one of the genes, which encodes a putative glucosyltransferase. Structural analysis of the O antigen produced by the mutant strain demonstrated the absence of glucosylation. An O-antigen structure shared by five E. coli and one S. enterica serogroups, all of which have a long evolutionary history, suggests that the common backbone may be important for the survival of E. coli strains in the environment, or for their pathogenicity.