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An analysis of the biochemical basis for the lack of phosphoenolpyruvate:glycose phosphotransferase activity in heterofermentative lactobacilli was carried out. Extracts of Lactobaciflus brevis and Lactobacilus buchneri failed to reconstitute phosphotransferase activity of extracts of Staphylococcus aureus mutants impaired in the phosphotransferase system due to the absence of enzyme I, enzyme IILc, or enzyme IIILac activity, suggesting that these lactobacilli lack those phosphotransferase system components. In contrast, complementation tests with an extract of a S. aureus mutant deficient in heat-stable protein (HPr) indicated the presence of HPr activity in heterofermentative lactobacilli. The HPr of L. brevis was purified and shown to have properties similar to those of a typical HPr. In addition, L. brevis possesses an ATP-dependent protein kinase that phosphorylates a serine residue of the endogenous HPr as well as other HPrs of Gram-positive origin. The kinase activity is markedly stimulated by phosphorylated compounds related to sugar metabolism and is negatively modulated by orthophosphate, pyrophosphate, or arsenate and by a low molecular weight endogenous factor. In keeping with the idea of a regulatory role for the phosphorylation of HPr in lactobacilli, a HPr[Ser(P)] phosphatase activity in L. brevis was also demonstrated. On the basis of the rinding of HPr and a system for its reversible covalent modification in an organism devoid of a functional phosphotransferase system we propose that, in lactobacilli, HPr has a role in the regulation of pathways other than the phosphotransferase system.One of the general energy coupling components of the phosphoenolpyruvate (P-ePrv):glycose phosphotransferase system (PTS) is a low molecular weight protein known as HPr (heat-stable protein) that is phosphorylated by P-ePrv and enzyme I on a single histidine residue (1). Recent studies have established that, in Gram-positive bacteria, HPr can also be reversibly phosphorylated on a serine residue by a unique, metabolite-activated, ATP-dependent protein kinase-the phosphorylated HPr is designated HPr[Ser(P)]-and a Pi-dependent HPr[Ser(P)] phosphatase (for reviews see refs. 2 and 3). This reversible modification appears to play a regulatory role in energy and carbon metabolism by modulating the flow of phosphate groups at the level of HPr (3, 4). HPr[Ser(P)] has also been implicated in a vectorial regulatory process, termed inducer expulsion, that effectively displaces intracellularly accumulated sugars and functions in response to variations in energy availability and metabolite concentrations (3,5,6).Heterofermentative lactobacilli, such as Lactobacillus brevis or Lactobacillus buchneri, appear to lack a functional PTS (refs. 7 and 8 and this paper). However, these organisms possess a metabolite-activated vectorial process that displaces intracellularly accumulated galactosides (8). This regulatory mechanism resembles the expulsion process of homofermentative lactic acid bacteria, which possess a PTS and...

In this study, high-affinity maltose-and glucose-binding activities in cell-free extracts of Thermotoga maritima were detected ; these activities were distinct and specific. At the gross level, the expression of binding-protein activities was repressed by growth of T. maritima in the presence of the cognate sugar. Growth of the organism in the presence of maltose reduced maltose-binding activity but not glucose-binding activity, while growth in the presence of glucose reduced glucose-binding activity but not maltose-binding activity. In competition assays, these binding activities showed distinct patterns of substrate specificity : whereas the maltose-binding activity showed specificity for α-linked glucosides, the glucose-binding activity showed a broader specificity. All maltose-and glucose-binding activity was found in the supernatant retrieved following centrifugation (100 000 g) of the cell-free extracts prepared by French-pressure-cell treatment ; no activity was found in an octyl-glucoside-treated extract of the membrane fraction. The maltosebinding-protein activity was recovered from the periplasmic fraction by selective release of the periplasmic contents of T. maritima cells using a newly developed freeze-thaw procedure. Annotation of the complete genome sequence of T. maritima suggests that there may be at least two maltosebinding proteins, MalE1 and MalE2, encoded in the genome. The maltosebinding activity corresponded to a protein of 43 kDa, which was consistent in size with either of the putative proteins. These data demonstrate that the hyperthermophilic bacterium T. maritima possesses separate maltose-and glucose-binding-protein activities that are freely soluble in its periplasm, in contrast to the membrane-bound sugar-binding proteins found in archaeal hyperthermophiles.

Galactose-grown cells of the heterofermentative lactic acid bacteria Lactobacilus brevis and Lactobacillus buchneri transported methyl-4-D-thiogalactopyranoside (TMG) by an active transport mechanism and accumulated intracellular free TMG when provided with an exogenous source of energy, such as arginine. The intracellular concentration of TMG resultant under these conditions was approximately 20-fold higher than that in the medium. In contrgst, the provision of energy by metabolism of glucose, gluconate, or glucosamine promoted a rapid but transient uptake of TMG followed by efflux that established a low celular concentration of the galactoside, i.e., only two-to fourfold higher than that in the medium. Furthermore, the addition of glucose to cells preloaded with TMG in the presence of arginine elicited a rapid efflux of the intracellular galactoside. The extent -of cellular TMG displacement and the duration of the transient effect of glucose on TMG transport were related to the initial concentration of glucose in the medium. Exhaustion of glucose from the medium restored uptake and accumulation of TMG, providing arginine was available for ATP generation. The nonmetabolizable sugar 2-deoxyglucose elicited efflux of TMG from preloaded cells of L. buchneri but not from those of L. brevis. Phosphorylation of this glucose analog was catalyzed by cell extracts of L. buchneri but not by those of L. brevis. Iodoacetate, at a concentration that inhibits growth and ATP production from glucose, did not prevent efflux of cellular TMG elicited by glucose. The results suggested that a phosphorylated metabolite(s) at or above the level of glyceraldehyde-3-phosphate was required to evoke displacement of intracellular TMG from the cells. Counterflow experiments suggested that glucose converted the active uptake of TMG in L. brevis to a facilitated diffusion mechanism that allowed equilibration of TMG between the extraand intracellular milieux. The means by which glucose metabolites elicited this vectorial regulation is not known, but similarities to the inducer expulsion that has been described for homofermentative Streptococcus and Lactobacillus species suggested the involvement of HPr, a protein that functions as a phosphocarrier protein in the phosphotransferase system, as well as a presumptive regulator of sugar transport. Indeed, complementation assays with extracts of a Staphylococcus aureus ptsH mutant revealed the presence of HPr in L. brevis, although this lactobacillus lacked a functional phosphoenolpyruvate-dependent phosphotransferase system for glucose, 2-deoxyglucose, or TMG.A number of bacterial mechanisms for the regulation of sugar transport and accumulation have been described previously (for reviews, see references 6, 13, 19, and 22). These mechanisms include those that are operative at the level of transcription of genetic determinants encoding specific transport systems (induction and repression), those that are operative at the level of activity of specific permeases (inducer exclusion), and those that op...

A survey of the occurrence of the phosphoenolpyruvate-dependent glucose phosphotransferase system was carried out in a number of bacteria, representing both gram-positive and gram-negative facultative anaerobic and strictly aerobic types. The system was found to be present in representatives of genera that are characteristically facultative anaerobes, but the system was absent in members of those genera that are strictly aerobic. Thus, although the phosphoenolpyruvate phosphotransferase system is an important system for the transport of sugars in bacteria carrying out anaerobic glycolysis, it plays no role in sugar transport by those organisms having a strictly oxidative physiology. A fundamentally different system, probably not involving phosphorylation during transport, is indicated in this latter group.