Application of Pepper aptamer-based reporter system for bacterial transcription studies.

This study introduces a novel bacterial reporter system using the fluorescent RNA aptamer Pepper to directly monitor mRNA-level transcriptional changes, overcoming limitations of traditional protein-based reporters. First, the Pepper reporter system was used to characterize the strength differences of promoters used in Escherichia coli, and the result were compared with enhanced green fluorescent protein (eGFP) reporter and quantitative real-time PCR (qRT-PCR) analysis. Then the Pepper reporter system was used to examine six pairs of known transcriptional regulatory relationships: RacR activating PlysN and SoxS activating PydbK in E. coli, EsrB activating PevpP and repressing PglnA in Edwardsiella piscicida, as well as Spo0A activating PyloA and AbrB repressing PyloA in Bacillus subtilis. Additionally, we tested the capacity of Pepper reporter to verify the binding of LmbU, a transcriptional regulator from Streptomyces lincolnensis, to known or putative binding sequences in E. coli. These results demonstrate that the Pepper reporter system is a reliable and versatile tool for probing promoter activity, transcriptional regulation, and regulator-DNA interactions in diverse bacteria.