REGULATION OF THE GLUCOSE-H-ACTIVATED ATP-DEPENDENT PHOSPHORYLATION OF HPR IN LACTOBACILLUS-BREVIS( SYMPORTER BY METABOLITE)

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetaboliza ble lactose analoge thiomethyl beta-galactoside (TMG), via proton symp ort. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly effluxes from L. b revis cells or vesicles upon addition of glucose and that glucose inhi bits further accumulation of TMG. This regulation was shown to be medi ated by a metabolite-activated protein kinase that phosphorylates seri ne 46 in the HPr protein. We have now analyzed the regulation of 2DG u ptake and efflux and compared it with that of TMG. Uptake of 2DG was d ependent on an energy source, effectively provided by intravesicular A TP or by extravesicular arginine which provides ATP via an ATP-generat ing system involving the arginine deiminase pathway. 2DG uptake into t hese vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or glucona te 6-phosphate into the vesicles. Intravesicular but not extravesicula r wild-type or H15A mutant HPr of Bacillus subtilis promoted inhibitio n (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar t ransport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles wit h or without the phosphorylated metabolites, but the S46D mutant prote in promoted regulation, even in the absence of a metabolite. The V-max but not the K-m values for both TMG and 2DG uptake were affected. Upt ake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild type HPr in the pr esence of fructose 1,6-diphosphate or by S46D mutant HPr. These result s establish that HPr serine phosphorylation by the ATP-dependent, meta bolite-activated HPr kinase regulates glucose and lactose permease act ivities in L. brevis and suggest that other permeases may also be subj ect to this mode of regulation.