Genes Involved in Control of Galactose Uptake in Lactobacillus brevis and Reconstitution of the Regulatory System in Bacillus subtilis

The phosphoenolpyruvate: sugar phosphotransferase system (PTS) plays a major role in the regulation of carbon and energy metabolism in many diverse prokaryotes (26). However, different PTS proteins are involved, and they function by entirely different mechanisms in the various organisms (34). In gram-positive bacteria, glucose represses synthesis of PTS and non-PTS carbohydrate catabolic enzymes (catabolite repression), inhibits the uptake of PTS and non-PTS sugars (inducer exclusion), and stimulates dephosphorylation of intracellular sugar phosphates and/or elicits efflux of free sugars (inducer expulsion) (35, 40). A principal role of a metabolite-activated ATP-dependent protein kinase which phosphorylates seryl residue 46 (Ser-46) in HPr in the regulation of enzyme synthesis, inducer exclusion, and inducer expulsion via an allosteric mechanism that acts on several different target proteins has been suggested (4, 5, 30, 34, 36, 44). Homofermentative lactic acid bacteria transport most sugars via the PTS. In contrast, heterofermentative lactobacilli such as Lactobacillus brevis transport glucose and galactose as well as their nonmetabolizable analogues 2-deoxyglucose and thiomethyl-β-galactoside (TMG), respectively, by active transport energized by the proton motive force (33). Previous biochemical studies have shown that L. brevis possesses a galactose:H+ symport permease, here designated GalP, which transports and accumulates cytoplasmic TMG. An ATP-dependent HPr(Ser) kinase was also identified, and evidence was presented suggesting that it participates in GalP regulation. A complex mechanism was proposed that led to metabolite-activated vectorial sugar exclusion and expulsion (30, 44). When provided with an exogenous energy source such as arginine, galactose-grown cells of L. brevis were shown to transport [14C]TMG and accumulate the free sugar analogue in the cytoplasm to a concentration at least 20-fold higher than that in the medium. However, addition of exogenous glucose, gluconate, or glucosamine to cells loaded with [14C]TMG resulted in efflux of the intracellular galactoside, giving rise to a reduced cytoplasmic concentration of the sugar. Counterflow experiments suggested that metabolism of glucose by intact L. brevis cells results in conversion of the active sugar:H+ transporter (symporter) into a facilitated diffusion system (uniporter [33]). When right-side-out vesicles of L. brevis were loaded with free [14C]TMG and electroporated with purified HPr of Bacillus subtilis, rapid efflux of the galactoside was observed upon addition of glucose (44). Glucose could be replaced by any one of several glycolytic phosphorylated metabolites, such as fructose 1,6-bisphosphate, gluconate-6-phosphate, and 2-phosphoglycerate, when the compounds were electroporated into the vesicles. Allosteric activating effects of these compounds on the HPr(Ser) kinase in vitro were also observed (30). In the absence of glucose or phosphorylated metabolites, intravesicular HPr(S46D), a structural analogue of the seryl-phosphorylated form of HPr, promoted expulsion of accumulated free [14C]TMG. The noncharged HPr analogue HPr(S46A) did not promote expulsion of the accumulated galactoside, clearly implying a critical role for phosphorylation of HPr at Ser-46 by the HPr(Ser) kinase in inducer expulsion. In addition, uptake of lactose, glucose, mannose, and ribose was inhibited by wild-type HPr in the presence of fructose 1,6-bisphosphate or by HPr(S46D) alone, indicating that the metabolite-activated, ATP-dependent HPr(Ser) kinase regulates sugar permeases in L. brevis by a feedback-controlled process. Direct binding of 125I-labeled HPr(S46D) to L. brevis membranes containing high levels of the galactose permease and inhibition of the binding by nonradioactive HPr(S46D) were demonstrated (45). Based on the extensive biochemical evidence summarized above, an allosteric model was proposed for the observed vectorial sugar expulsion in L. brevis (Fig. ​(Fig.1).1). To elucidate the details of the molecular mechanism of this process in vivo and to eventually determine the physiological significance of this process to growth and enzyme induction via the control of inducer levels, we have cloned, sequenced, and expressed the relevant L. brevis genes in B. subtilis. In this organism, which does not normally exhibit the phenomenon of PTS-mediated inducer control, we could observe regulation of the L. brevis GalP activity by mutant derivatives of the L. brevis HPr. We have therefore reconstituted the regulatory system in a bacterium that is readily amenable to genetic manipulation. This system should allow detailed studies of the type necessary to establish the mechanistic details of inducer control. FIG. 1 Proposed model for PTS-mediated regulation of vectorial sugar expulsion in L. brevis. Inducer expulsion in L. brevis is envisioned as a multicomponent signal transduction process. The galactose:H+ symporter GalP cotransports sugar and H+ ...