GdhR directly represses lctP transcription.

A. In vitro transcription was conducted in the presence of increasing concentrations of purified GdhR and 50 nM RNAPσ70 using as template a DNA fragment encoding lctP promoter from -192 to +381 relative to TSS (lctP WT) and the same fragment harboring a 21 bp deletion in the FadR consensus DNA-binding motif (lctPΔFadR). The size of the lctP transcript peaks is given at the top x axis. Purified MtrR protein was used as specificity control of the transcription inhibition reaction. B. Transcription inhibition curves were generated by plotting the height of the peaks (considering the 0 μM protein reaction as the 100% transcription point) vs. transcription factor molar concentration. (C) Binding of GdhR to WT lctP promoter from -192 to +99 relative to the TSS (lctP WT) and to the same promoter fragment harboring the 21 bp deletion in the FadR DNA-binding motif (lctPΔFadR) was compared by EMSA. (D) GdhR binding curves to the WT and mutant lctP probes and the dissociation constant (Kd) were determined by densitometry from the EMSA gels adjusting the data to a nonlinear regression analysis using the Hill equation [69].