Abstract
Transcriptional control is a finely tuned process, crucial for adaptation, differentiation, and survival. However, deciphering the contribution of promoter elements to overall promoter strength is tedious. We present the novel plasmid-based screening system pHIS (pHigh Screen) as a tool for easy and quantitative monitoring of promoter activity via fluorescence reporter gene expression for a reliable in vivo promoter analysis in real time. As a proof of principle, we first validated the functionality of our pHIS system by analyzing the known Spx operator of a SigA-type promoter in Bacillus subtilis. Then, the pHIS plasmid was used to characterize a new operator element, which was predicted by in silico analysis of SigB-type promoters controlled by the Spx paralogue MgsR. We found evidence for (i) an intrinsic promoter silencer element located upstream of the SigB-type -35 core region, preventing target gene expression in the absence of MgsR and (ii) an MgsR-dependent antagonist enhancer element necessary to overcome the negative element and to achieve full stress-dependent promoter activation. Our data demonstrate the universal applicability of our novel reporter gene system pHIS and support an RNA polymerase repositioning model for transcription initiation at MgsR-regulated SigB-type promoters in B. subtilis.