Regulation of methyl-beta-d-thiogalactopyranoside-6-phosphate accumulation in Streptococcus lactis by exclusion and expulsion mechanisms

Abstract

Starved cells of Streptococcus lactis ML3 (grown previously on galactose, lactose, or maltose) accumulated methyl-beta-D-thiogalactopyranoside (TMG) by the lactose:phosphotransferase system. More than 98% of accumulated sugar was present as a phosphorylated derivative, TMG-6-phosphate (TMG-6P). When a phosphotransferase system sugar (glucose, mannose, 2-deoxyglucose, or lactose) was added to the medium simultaneously with TMG, the beta-galactoside was excluded from the cells. Galactose enhanced the accumulation of TMG-6P. Glucose, mannose, lactose, or maltose plus arginine, was added to a suspension of TMG-6P-loaded cells of S. lactis ML3, elicited rapid expulsion of intracellular solute. The material recovered in the medium was exclusively free TMG. Expulsion of galactoside required both entry and metabolism of an appropriate sugar, and intracellular dephosphorylation of TMG-6P preceded efflux of TMG. The rate of dephosphorylation of TMG-6P by permeabilized cells was increased two-to threefold by adenosine 5'-triphosphate but was strongly inhibited by fluoride. S. lactis ML3 (DGr) was derived from S. lactis ML3 by positive selection for resistance to 2-deoxy-D-glucose and was defective in the enzyme IIMan component of the glucose:phosphotransferase system. Neither glucose nor mannose excluded TMG from cells of S. lactic ML3 (DGr), and these two sugars failed to elicit TMG expulsion from preloaded cells of the mutant strain. Accumulation of TMG-6P by S. lactis ML3 can be regulation by two independent mechanisms whose activities promote exclusion or expulsion of galactoside from the cell.