Mapping Potential Transcription Start Sites of the brka Gene on pDO6935 in Escherichia coli Using the ARF-TSS Method

Autotransporters are membrane proteins expressed on the outer membrane in gram-negative bacteria. BrkA is an autotransporter and virulence factor of the whooping cough-causing bacterium Bordetella pertussis. Previous studies aimed at characterizing the autotransporter BrkA have used pDO6935-expressing Escherichia coli cells; however, the promoter driving brkA gene expression in pDO6935-expressing E. coli cells remains unknown. Our study aims to determine the transcription start site (TSS) of the brkA gene in pDO6935 in E. coli (DH5α). In order to map the TSS, we implemented the adaptor- and radioactivity-free identification of the transcription start site method (ARF-TSS) which involves reverse transcribing brkA mRNA into cDNA using a 5'-phosphorylated primer, ligating the cDNA, and PCR-amplifying it before sending it for sequencing to determine the TSS. We were able to more precisely map the region of the plasmid that the transcription start site driving the expression of the brkA gene in pDO6935 in E. coli which is located in a region more than 270 bp upstream of the brkA translation start site. Finding the transcription start site aids in locating the promoter and contributes to our understanding of gene expression and regulation. This knowledge can help improve future studies aimed at characterizing BrkA autotransporter using pDO6935-expressing E. coli cells.