citZ-lacZ fusion in wild type and ccpA mutant strains showed that citZ repression was reduced in the ccpA mutant compared to the wild type strain. Primer extension analysis further verified the role of CcpA in repression of citZ expression. Visual inspection of the citZ promoter region identified a putative CcpA binding site (cre site). Site-directed mutagenesis was used to introduce mutations into the cre site. LacZ reporter assays showed that certain mutations in the putative binding site resulted in higher expression of citZ than in the wild type B.subtilis SMY strain. EMSA was used to compare binding of CcpA to wild type and mutated citZ promoters. The results confirmed that CcpA bound specifically to the citZ promoter. DNase I footprinting showed that cre sequence was protected from DNase I digestion.
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|