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

Abstract

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 P_lysN and SoxS activating P_ydbK in E. coli, EsrB activating P_evpP and repressing P_glnA in Edwardsiella piscicida, as well as Spo0A activating P_yloA and AbrB repressing P_yloA 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.