Figure 6

Experimental verification of the predicted transcriptional regulation of the murMN- SMU_718c operon by the response regulator MbrC. The predicted transcriptional regulatory relationship was based on a well correlated (A) expression profile between mbrC and the murMN-SMU_718 operon as well as the presence of a (B) putative MbrC binding site (TTACAA-AT-TTCTAC) in the upstream regulatory regions of the murMN-SMU_718 operon. The alignment among the MbrC binding sites in other experimentally verified targets (black) reported by Ouyang et al. [50] and the putative site upstream of the predicted target (red) murMN-SMU_718 operon is shown. The signature repeats of the MbrC binding motif are italicized, underlined and shown in bold. (C) Binding of MbrC to the promoter region of the gene SMU_1006 (positive control) was verified using Electro Mobility Shift Assays (EMSA), as already reported by Ouyang et al.[50]. (D) EMSA also provided the verification of the in-vitro binding of the MbrC protein to the promoter region of the predicted target murMN-SMU_718c operon via the putative binding site thus confirming that the latter is a transcriptional regulatory target of MbrC. The triangles indicate increasing concentrations of MbrC in the binding reactions. Black triangles followed by IR indicate target DNA fragments lacking the MbrC binding site.