The gene function of the locus of enterocyte effacement (LEE) is essential for full virulence of enterohemorrhagic Escherichia coli (EHEC). Strict control of LEE gene expression is mediated by the coordinated activities of several regulatory elements. We previously reported that the ClpX/ClpP protease positively controls LEE expression by down-regulating intracellular levels of GrlR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrlR on LEE expression was mediated through GrlA, a positive regulator of LEE expression. In this study, we found that the FliC protein, a major component of flagellar filament, was overproduced in clpXP mutant EHEC, as previously reported for Salmonella. We further found that FliC expression was reduced in a clpXP grlR double mutant. To determine the mediators of this phenotype, FliC protein levels in wild-type, grlR, grlA, and grlR grlA strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrlR is mediated by GrlA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grlA or grlR grlA mutant. Because overexpression of grlA from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrlA acts as a negative regulator of flagellar-gene expression. The fact that an EHEC strain constitutively expressing FlhD/FlhC cannot adhere to HeLa cells leads us to hypothesize that GrlA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.

Shiga toxin-producing Escherichia coli (STEC) are important enteropathogens causing severe diseases such as hemorrhagic colitis and hemolytic-uremic syndrome in humans. The majority of STEC strains of serogroups O157, O26, or O111 associated with severe cases of these diseases possess a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE, which is responsible for the formation of attaching-and-effacing lesions on intestinal epithelial cells, is important for the full virulence of STEC. Nonetheless, LEE-negative STEC strains have repeatedly been reported to be associated with severe diseases in humans. In this study, we characterized adhesion to cultured epithelial cells of certain LEE-negative STEC isolated from humans with or without bloody diarrhea. Several LEE-negative STEC belonging to serogroup O91 showed an unusual, chain-like adhesion pattern to HEp-2 cells. Using Tn5-based transposon mutagenesis, we identified the gene essential for the chain-like adhesion phenotype of this O91 STEC strain. Sequence analysis of the Tn5-inserted allele identified a novel chromosomal open reading frame (ORF) encoding a polypeptide with a high degree of similarity to the E. coli immunoglobulin-binding (Eib) proteins EibA, -C, -D, -E, and -F. Therefore, the ORF was designated EibG. Laboratory E. coli strain MC4100 transformed with a multicopy plasmid carrying eibG showed chain-like adhesion to HEp-2 cells, and whole-cell lysates of the strain bound to human-derived immunoglobulin G (IgG) Fc and IgA. These results indicate that EibG acts as an IgG Fc-and IgA-binding protein, as well as an adhesin of LEE-negative STEC.