Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus

Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood, We performed TnphoA mutagenesis on E. coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression, Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U, H, Stroeher, L. E, Karageorgos, R Morona, and P. A. Manning, Proc, Natl, Acad, Sci, USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase. This open reading frame was designated rfbE(EcO157:H7). The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E. coli. rfbE(EcO157:H7) is conserved in nontoxigenic E, coli O157 strains expressing a variety of other flagellar antigens but is not found in E. coli O55:H7 strains, which are more closely related to E, coli O157:H7, Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E, coli O157:H7 parent, but they did not differ in other phenotypes, Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12. We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E. coli rfb genes occurred independently in E, coli O157:H7 and unrelated O157 strains, and that the O side chain of E, coli O157:H7 lipopolysaccharide interferes with the adherence of E. coli O157:H7 to epithelial cells.