The locus of enterocyte effacement (LEE) encoded operons (LEE1 - LEE5) enable enterohemorrhagic Escherichia coli (EHEC) O157:H7 to produce attaching and effacing lesions on intestinal cells (A/E). We have previously reported that hha-deleted EHEC O157:H7 up-regulated LEE4 through increased expression of ler, the regulatory gene encoded by the LEE1 operon. In the present study, we demonstrate that a transpositional insertion in clpXP abrogated the increased expression of the LEE4 in the hha-deleted strain. The loss of LEE4 up-regulation in an EHEC O157:H7 [Delta]hha, [Delta]clpXP mutant was restored by transposon insertions immediately upstream of grlR of the grlR-grlA operon, suggesting that grIR represses the expression of LEE4 in the absence of Hha and ClpXP. RT-PCR analysis revealed that grlR transcription increased by about 100-fold in strains lacking hha but remained the same in the presence or absence of clpXP thus indicating that hha acts as a negative regulator of grlR transcription. RT-PCR data also showed a 10-fold reduction in LEE4 transcription for EHEC O157:H7 [Delta]clpXP thus suggesting that the absence of clpXP resulted in an increased accumulation of GrlR and presumably Hha thereby causing an additional reduction in LEE4 transcription compared to that observed for LEE4 in EHEC O157:H7. Western blot analysis of the cell-free extracts of EHEC O157:H7 [Delta]clpXP revealed increased accumulation of Hha. Altogether, the results described in this report indicate that ClpXP is important for maintaining a critical intracellular pool of Hha, which in turn modulates transcription of grlR-grlA so that a basal level of transcription could continue through LEE4 and the other LEE operons.