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Shiga toxin-producing Escherichia coli (STEC) 091:H21 strain B2F1, an isolate from a patient with the hemolytic uremic syndrome (HUS), produces elastase-activatable Shiga toxin (Stx) type 2d and adheres well to human colonic epithelial T84 cells. This adherence phenotype occurs even though B2F1 does not contain the locus of enterocyte effacement (LEE) that encodes the primary adhesin for E. coli O157:H7. To attempt to identify genes involved in binding of B2F1 to T84 cells a bank of mini-Tn5phoACmr transposon mutants of this strain was generated. Several of these mutants exhibited a reduced adherence phenotype, but none of the insertions in these mutants were within putative adhesin genes. Rather, insertional mutations within hns resulted in the loss of adherence. Moreover, the hns mutant also displayed an increase in the production of hemolysin and alkaline phosphatase and a loss of motility with no change in Stx2d-activatable expression levels. When B2F1 was cured of the large plasmid that encodes the hemolysin, the resulting strain adhered well to T84 cells. However, an hns mutant of the plasmid-cured B2F1 strain exhibited a reduction in adherence to T84 cells. Taken together, these results indicate that H-NS regulates the expression of several genes and some potential virulence factors in the intimin-negative B2F1 STEC strain and that the large plasmid is not required for T84 cell colonization.