ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in EnterohemorrhagicEscherichia coli

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Abstract
Expression of the type III protein secretion system (TTSS), encoded in the locus of enterocyte effacement (LEE) of enterohemorrhagicEscherichia coli(EHEC), has been shown to be controlled by various regulators. In a search for additional regulatory genes, we identified a DNA fragment containingclpXandclpPthat has a positive regulatory effect on LEE expression in EHEC O157. The expression of LEE-encoded Esp proteins was significantly reduced in aclpXPdeletion mutant. Deletion ofgrlR, a negative regulatory gene within LEE, markedly increased LEE expression even in theclpXPmutant. To verify the regulatory mechanism of GrlR expression, a chromosomal epitope-tagged allele ofgrlR(grlR-FLAG) was constructed. GrlR-FLAG expression was increased significantly in theclpXPdeletion mutant, suggesting that the GrlR level is under the control of ClpXP, and this regulation is critical for the ClpXP-dependent expression of LEE in EHEC. Deletion ofrpoS, the gene encoding a stationary-phase-inducing sigma factor that is a substrate for ClpXP protease, partially restored LEE expression in theclpXPmutant. A multicopy plasmid carryingrpoSstrongly repressed expression of Esp proteins, suggesting that positive regulation by ClpXP is partially mediated through a negative effect of RpoS on LEE expression. We also found thatrpoSdeletion induces transcription ofpchA, which encodes one of the positive regulators for LEE expression in EHEC. These results suggest that ClpXP controls expression of LEE through the regulation of RpoS and GrlR levels in EHEC.

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