BglF, the Escherichia coli b-Glucoside Permease and Sensor of the bgl System: Domain Requirements of the Different Catalytic Activities

The Escherichia coli BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities. In the absence of b-glucosides, it phosphorylates BglG, a positive regulator of bgl operon transcription, thus inactivating BglG. In the presence of b-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports b-glucosides into the cell and phosphorylates them. BglF is composed of two hydrophilic domains, IIA and IIB, and a membrane-bound domain, IIC, which are covalently linked in the order IIBCA. Cys-24 in the IIB domain is essential for all the phosphorylation and dephosphorylation activities of BglF. We have investigated the domain requirement of the different functions carried out by BglF. To this end, we cloned the individual BglF domains, as well as the domain pairs IIBC and IICA, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo. We show here that the IIB and IIC domains, linked to each other (IIBC), are required for the sugar-driven reactions, i.e., sugar phosphotransfer and BglG activation by dephosphorylation. In contrast, phosphorylated IIB alone can catalyze BglG inactivation by phosphorylation. Thus, the sugar-induced and noninduced functions have different structural requirements. Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIB and IIC which occur upon the interaction of BglF with the sugar.