Regulation of 2 - deoxyg I ucose phosphate accumulation in Lactococcus lactis vesicles by metabolite - activated , ATP - dependent phosphorylation of serine - 46 in HPr of the phosphotransferase system

Department o f Biology, University of California at San Diego, La Jolla, CA 92093-01 16, USA Lactococcus lactis takes up glucose and the nonmetabolizable glucose analogue 2-deoxyglucose (2DG) via the phosphotransferase system and extrudes the accumulated sugar phosphates in a process apparently dependent on a cytoplasmic sugar-phosphate phosphatase. Uptake of 2DG into L. lactis vesicles was shown to be dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate (PEP). 2DG phosphate (2DG-P) accumulation in these vesicles was not inhibited, and preaccumulated 2DG-P was not released from them, upon electroporation of fructose 1,6-diphosphate (FDP), gluconate 6-phosphate or 2-phosphoglycerate into the vesicles. lntravesicular but not extravesicular wild-type HPr of Bacillus subtiljs alone stimulated uptake, but in the presence of any one of these metabolites, it prevented accumulation of 2DC-P. lntravesicular H I 5A mutant HPr inhibited uptake and allowed further inhibition of 2DG-P accumulation in the presence of the intravesicular metabolites. lntravesicular S46A mutant HPr stimulated uptake but could not promote inhibition in the presence of the phosphorylated metabolites. The 5460 mutant HPr protein promoted regulation, even in the absence of a metabolite. The V,,, but not the Km value for 2DG uptake was affected. Accumulation of the natural, metabolizable substrates of the lactose, glucose, mannose and ribose permeases was inhibited by wild-type HPr in the presence of FDP or by S46D mutant HPr. The results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase selectively determines the levels of sugar accumulation via the glucose and lactose permeases in L. lactis. They suggest that two primary functions of HPr(Ser) phosphorylation are: (1) to feedback-inhibit the activities of carbohydrate permeases and not merely to create a hierarchy of preferred carbon sources, and (2) to regulate the cytoplasmic concentrations of carbohydrate inducers by exclusion and expulsion mechanisms.