The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli

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 GrIR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrIR on LEE expression was mediated through GrIA, 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, grL4, and grlR grL4 strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrIR is mediated by GrIA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grL4 or grlR grL4 mutant. Because overexpression of grL4 from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrIA 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 GrIA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.