A reporter gene assay for inhibitors of the bacterial phosphoenolpyruvate: sugar phosphotransferase system

Abstract

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) plays a key role in sugar uptake and metabolic regulation in bacteria. PTS proteins form a divergent phosphorylation cascade. Enzyme I (EI) is at the top of the cascade and mediates phosphoryl-transfer from phosphoenolpyruvate to the phosphoryl-carrier protein HPr, which then distributes the phosphoryl-groups to the different carbohydrate transporters. In addition, some PTS proteins have a regulatory function in catabolite repression, inducer exclusion and chemotaxis which is modulated by their degree of phosphorylation in response to the availability of substrates. Using as a reporter the IacZ gene under control of the bgl t2 transcriptional terminator and as an effector the transcriptional antiterminator LicT from B. subtilis, a two-plasmid reporter gene system was constructed in order to monitor PTS activity. LicT, when present at low concentration in E. coli, is inactivated by EI/HPr-dependent phosphorylation and conversely is active in a ptsl- mutant lacking El. Active LicT allows for transcriptional readthrough at bgl t2, resulting in a full-length lacZ transcript. Beta-galactosidase activities are increased 4-8-fold in a ptsl+ strain growing on PTS substrates relative to growth on non-PTS substrates and approximately 30-fold in a ptsl- mutant. This gain-of-function in response to dephosphorylation of El or lack of active El can be used to monitor changes of El activity caused by mutations and environmental factors and for screening and validation of inhibitors of the PTS as potentially novel antibacterial compounds.