Promoter Mapping Analysis Using a GFP Reporter Plasmid Suggests that the lac Promoter May Drive brka Expression on Plasmid pDO6935 in Escherichia coli

Résumé

Autotransporters (AT) are a class of bacterial proteins that play a crucial role in bacterial virulence and pathogenesis. Understanding these proteins is essential to advance research in infectious disease and identify possible targets for therapeutic interventions. Bordetella pertussis autotransporter, BrkA belongs to the AT-1 subfamily of autotransporter proteins in Gram negative bacteria. Since its discovery, plasmids have been created to study brkA expression in various systems, pDO6935 being one of them. The mechanism by which brkA is expressed in pDO6935 is yet to be fully elucidated. We used a promoter trapping method to map the promoter regions that drive brkA expression. An analysis of pDO6935 gene sequence revealed the presence of a lac operon upstream of brkA. The lac operon is known to exhibit basal expression in the absence of lactose or the presence of a repressor. We studied whether the leaky expression caused by the lac operon may result in the expression of brkA in an Escherichia coli system. In our study, we utilized pSPPH21, a promoterless reporter plasmid that was designed to contain green fluorescent protein (GFP). Using this vector, we inserted regions of putative promoter sequences upstream of brkA in pDO6935. Fluorescence imaging and quantification on a plate reader suggested that the lac operon may be driving brkA expression in pDO6935. We further investigated this argument upon treatment with glucose, a known catabolite repressor of the lac operon. Glucose treatment resulted in repressed levels of GFP, providing additional evidence to our findings.