Figure 2.

c-di-GMP enhances the binding of Ethr to the etha promoter both in vitro and in vivo. (a). Graphical schematic of the regulatory pathway of Ethr in mycobacteria. (b). EMSA assay to assess the effect of c-di-GMP to the DNA binding activity of Ethr on etha promoter. Biotinylated etha promoter was co-incubated with Ethr in presence of serially diluted c-di-GMP from 200~0 μM. High close (200 μM) of cGMP and c-di-AMP were also included as negative control. (c). Assess the effect of c-di-GMP to Ethr’s DNA binding activity on etha promoter by BLI assay. Biotinylated etha promoter DNA was immobilized on streptavidin coated biosensors and incubated with Ethr in the presence of 50 μM (10-fold) concentration of c-di-GMP. The cGMP was included as control. (d). qRT-PCR assay for determining the relative transcription level of the dgc genes. Transcription level of gene was normalized using the sigA gene as an invariant transcript. Means ± standard deviation (S.D.) represent the variant range of the data derived from three biological replicates. (e,f). qRT-PCR assay for determining the relative transcription level of the etha and ethr genes. The experiment was performed and analyzed same as that of (d). The P-values of the relative expression data were calculated by unpaired two-tailed Student’s t-test using GraphPad Prism 5. The P-values of the results (<0.05 or <0.01 or <0.001) are indicated by an asterisk (* or ** or ***).