BglF, the Escherichia coli beta-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 act ivities, In the absence of beta-glucosides, it phosphorylates BglG, a posit ive regulator of bgl operon transcription, thus inactivating BglG, In the p resence of beta-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports beta-glucosides into the cell and phosphorylat es them. BglF is composed of two hydrophilic domains, IIA(bgl) and IIBbgl, and a membrane-bound domain, IICbgl, which are covalently linked in the ord er HBCA(bgl). Cys-24 in the IIBbgl domain is essential for all the phosphor ylation 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 IIB Cbgl and IICA(bgl), and tested which domains and which combinations are req uired for the catalysis of the different functions, both in vitro and in vi vo. We show here that the IIB and IIC domains, linked to each other (IIBCbg l), are required for the sugar-driven reactions, i.e., sugar phosphotransfe r and BglG activation by dephosphorylation. In contrast, phosphorylated IIB bgl alone can catalyze BglG inactivation by phosphorylation. Thus, the suga r-induced and noninduced functions have different structural requirements, Our results suggest that catalysis of the sugar-induced functions depends o n specific interactions between IIBbgl and IICbgl which occur upon the inte raction of BglF with the sugar.