Mapping the brkA Promoter Region on pDO6935 in Escherichia coli Using pLISA, A Novel Promoterless GFP Reporter Plasmid

Résumé

Bordetella resistance to killing A (brkA) is a serum resistance gene encoding an autotransporter native to Bordetella pertussis, the causative agent of whooping cough (1). Using the expression construct pDO6935, previous studies have demonstrated brkA expression in Escherichia coli. However, the promoter driving brkA expression from this construct is unknown. pDO6935 harbors a lac regulatory region inclusive of a promoter, operator, and catabolite activator protein binding site (CAP BS) upstream of brkA which may be implicated in its expression. The lactose, or lac, operon regulatory elements are known for their ability to control gene expression via transcriptional regulation in E. coli. To characterize the brkA promoter on pDO6935 in E. coli, we created pLISA, a promoterless green fluorescent protein (GFP) vector to be used in a promoter trap experiment. In our work, regions upstream of the brkA locus on pDO6935, either inclusive or exclusive of the lac regulatory region, were cloned into pLISA. GFP expression was observed as an indicator of promoter activity. We showed that GFP expression was induced when amplicons contained the full lac regulatory region. This observation suggests that the lac regulatory region is likely both necessary and sufficient to drive brkA expression from pDO6935 in E. coli - informing future efforts to study virulence gene brkA using E. coli as a model system.