Northern blot hybridization in wild-type DC3000 and rhpS mutant showed that rhpR was expressed at a much higher level in the rhpS– mutant than in the WT strain. rhpR-luc reporter assay showed that activity was 10-fold higher in the rhpS– mutant than in the WT, indicating an autoactivation of rhpR expression by RhpR. Promoter deletion analysis indicated the presence of an RhprR-dependent promoter element in the -170 to -120 region in the rhpR promoter. rhpR transcriptional start site was identified by 5'RACE reaction. Site-directed mutagenesis of the putative RhpR binding site in conjunction with rhpR-luc reporter assay showed that the IR element in the rhpR promoter is important in regulating the promoter activity. Also, it was shown that the 6 bp length of the spacer is required for the activity of the IR element. Additionally, the DC3000 genome was searched for the IR sequence with upto one mismatch. The RhpR-dependent expression of the genes identified by this search was confirmed using RNA blotting, ChIP, and qRT-PCR.
Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.
|Site sequence||Regulated genes||Gene diagram||Experimental techniques||TF function||TF type|