ClpXP protease controls expression of the type III protein secretion system through regulation of RpoS and GrlR levels in enterohemorrhagic Escherichia coli

Expression of the type III protein secretion system (TTSS), encoded in the locus of enterocyte effacement (LEE) of enterohemorrhagic Escherichia coli (EHEC), has been shown to be controlled by various regulators. In a search for additional regulatory genes, we identified a DNA fragment containing clpX and r1pP that has a positive regulatory effect on LEE expression in EHEC O157. The expression of LEE-encoded Esp proteins was significantly reduced in a clpYP deletion mutant. Deletion of grlR, a negative regulatory gene within LEE, markedly increased LEE expression even in the clpXP mutant. To verily the regulatory mechanism of GrlR expression, a chromosomal epitope-tagged allele of grlR (grlR-FLAG) was constructed. GrlR-FLAG expression was increased significantly in the clpXP deletion 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 of rpoS. the gene encoding a stationary-phase-inducing sigma factor that is a substrate for ClpXP protease, partially restored LEE expression in the clpXP mutant. A multicopy plasmid carrying rpoS strongly 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 that rpoS deletion induces transcription of pch4, 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.