cit-lacZ reporter assays in the wild-type and ccpA mutant strains verified CcpA-mediated regulation of cit. Western blot analysis showed that CcpA represses citCL and citHO expression. Visual inspection of the cit promoters identified three putative CcpA binding sites homologous to the E. faecalis CcpA consensus sequence. EMSA confirmed that CcpA bound specifically to the DNA probes containing the putative binding sites. The binding specificity was confirmed by showing that CcpA didn't bind to the probes containing mutated CcpA binding sites. LacZ reporter assays using cit promoters in which the binding sites were either mutated or deleted showed that CcpA-mediated repression was abolished in these promoters.
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|