Regulation of the glucose:H+ symporter by metabolite-activated ATP-dependent phosphorylation of HPr in Lactobacillus brevis

Abstract

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl I-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly efiluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylates serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of BaciUlus subtilis promoted inhibition (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 transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.Many but not all low-GC gram-positive bacteria possess the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that catalyzes the concomitant uptake and phosphorylation of its sugar substrates. The PTS-catalyzed process requires the sequential phosphorylation of four proteins or protein domains, enzyme I, HPr, IlAsugar and IIBsusgar, before sugar phosphorylation and concomitant transport can occur (8,20). The PTS proteins function in numerous biochemical and genetic regulatory capacities (17)(18)(19)21). In early studies, it was shown that addition of a rapidly metabolizable sugar such as glucose to streptococci, lactococci, or lactobacilli resulted in inhibition of the uptake of other sugars (inducer exclusion) as well as rapid efflux of preaccumulated sugars or sugar metabolites (inducer expulsion). For example, lactose and its nonmetabolizable analog...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…Moreover, Ser46Asp mutant HPr slowed TMG uptake even in the absence of extravesicular glucose or intravesicular metabolites. Studies of 2DG uptake by L. brevis vesicles electroporated with wild-type or mutant HPrs and with glycolytic intermediates provided results that were almost identical to those obtained for TMG uptake (968). In addition, by expressing the L. brevis galP gene (encodes the galactose/H ϩ symporter) in a B. subtilis wild-type strain and an hprK mutant, both B. subtilis strains gained the capacity to efficiently take up TMG.…”

Identification of an anaerobically induced phosphoenolpyruvate-dependent fructose-specific phosphotransferase system and evidence for the Embden-Meyerhof glycolytic pathway in the heterofermentative bacterium Lactobacillus brevis

“…These enzymes had not previously been demonstrated in a heterofermentative lactic acid gram-positive bacterium. These results show that with the exception of HPr, which is used for the regulation of non-PTS transport (10,20,21), all of these enzymes are fructose inducible and oxygen repressible. It will be interesting to see if other heterofermentative lactic acid bacteria similarly possess these enzymes.…”

“…Lactobacillus brevis is one heterofermentative lactic acid bacterium which has been reported to lack PTSs specific for glucose, fructose, and lactose (10,12). Glucose and lactose are known to be transported into L. brevis by sugar:H ϩ symport (10,20,21). Phosphofructokinase and fructose-1,6-bisphosphate aldolase have not been identified in this organism.…”

“…It was therefore postulated that of the PTS proteins, this heterofermentative organism possesses only HPr and uses it exclusively for regulatory purposes. The use of HPr, the target of an ATP-dependent, metabolite-activated HPr(ser) kinase, to regulate both lactose:H ϩ symport and glucose:H ϩ symport in L. brevis has been extensively documented (9,10,13,(20)(21)(22)(23).We have previously reported that Listeria and Streptomyces species possess fructose-specific PTSs (8,17,18). While examining the properties of L. brevis, as described in Bergey's Manual of Systematic Bacteriology (5), we noted that this organism has been reported to utilize fructose anaerobically but not aerobically.…”

“…It was therefore postulated that of the PTS proteins, this heterofermentative organism possesses only HPr and uses it exclusively for regulatory purposes. The use of HPr, the target of an ATP-dependent, metabolite-activated HPr(ser) kinase, to regulate both lactose:H ϩ symport and glucose:H ϩ symport in L. brevis has been extensively documented (9,10,13,(20)(21)(22)(23).…”