Glucose Phosphotransferase System Research Articles

The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for IIGlc and IIIGlc domains

Glucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose-specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane-bound, polypeptide with an apparent molecular weight of 80,000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40-50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl alpha-glucoside. Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl alpha-glucoside transport but retained part of the glucose-transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS. The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non-PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non-phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat- and batch cultures were compared. Strain PP799 transported and phosphorylated glucose via an intact PTS, while strain PP952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP799 and PP952 exhibited similar YGlc values. Assuming equal YATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc.

Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli

Osmotic upshock of E. coli cells in NaCl or sucrose medium resulted in a large decrease in the cytoplasmic volume and the inhibition of growth, of the electron transfer chain and of four different types of sugar transport system: the lactose proton symport, the glucose phosphotransferase system, the binding-protein dependent maltose transport system and the glycerol facilitator. In contrast to NaCl and sucrose, the permeant solute glycerol had no marked effect. These inhibitions could be partially relieved by glycine betaine. Despite these inhibitions, the internal pH, the protonmotive force and the ATP pool were maintained. It is concluded that inhibition of electron transfer and of sugar transport is the consequence of conformational changes caused by the deformation of the membrane. It is also concluded that the arrest of growth observed upon osmotic upshock is not due to energy limitations and that it cannot be explained by the inhibition of carbohydrate transport.

Low-affinity, high-capacity system of glucose transport in the ruminal bacterium Streptococcus bovis: evidence for a mechanism of facilitated diffusion

The glucose phosphotransferase system (PTS) of Streptococcus bovis could not account for the glucose consumption of exponential cultures, and the kinetics of glucose transport were biphasic. A PTS-deficient mutant lost the high-affinity, low-capacity system but retained its ability to take up glucose at high substrate concentrations. The low-affinity, high-capacity system did not require a proton motive force or ATP and could not be driven by an artificial membrane potential in the presence or absence of sodium. Since low-affinity transport was directly proportional to the external substrate concentration and exhibited counterflow kinetics, it appeared that a facilitated-diffusion mechanism was responsible for glucose transport at high substrate concentrations.

Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP):glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations.

ATPase-dependent energy spilling by the ruminal bacterium, Streptococcus bovis

When the ruminal bacterium Streptococcus bovis was grown in batch culture with glucose as the energy source, the doubling time was approximately 21 min and the rate of bacterial heat production was proportional to the optical density (1.72 microW/micrograms protein). If exponentially growing cultures were treated with chloramphenicol, there was a decline in heat production, but the rate was greater than 0.30 microW/micrograms protein even after growth ceased. Since there was no heat production after glucose depletion, this growth-independent energy dissipation (spilling) was not simply due to endogenous metabolism. Stationary cells which were washed and incubated in nitrogen-free medium containing an excess of glucose produced heat at a rate of 0.17 microW/micrograms protein. Monensin and tetrachlorosalicylanilide (TCS), compounds which facilitate an influx of protons, caused a more than 2-fold increase in heat production. Dicyclohexylcarbodiimide (DCCD) virtually eliminated growth-independent heat production regardless of the mode of growth inhibition. Because DCCD had little effect on the glucose phosphotransferase system, it appeared that the combined action of proton influx and the membrane bound F1F0 proton ATPase was responsible for energy spilling.

Thiolutin inhibits utilization of glucose and other carbon sources in cells of Escherichia coli.

Thiolutin was found to inhibit the utilization of glucose and other growth substrates in Escherichia coli. The inhibition was detected by a sharp drop of the respiration rate after addition of the antibiotic. The actual function affected was allocated to the cytoplasmic membrane of the bacterial cells by the following evidence: --spheroplasts were affected like intact cells, --individual reactions of either the electron transport chain or the glycolytic pathway were not inhibited, --glucose consumption in the culture stopped and the cells accumulated guanosine tetraphosphate as under starvation conditions, --activation of the cell's apo-glucose dehydrogenase restored respiration via bypassing the glucose phosphotransferase system. It was concluded that the transport of certain substrates across the membrane was inhibited.

Evolutionary divergence of enterobacterial genes coding for the glucose phosphotransferase system components

The DNA relatedness of 64 enterobacterial species to Escherichia coli genes ptsI, ptsH, crr, ptsG, and ptsLPM, was determined by quantitative filter hybridization. DNA relatedness was expressed relative to E. coli K-12 DNA. Enterobacterial DNAs were 0 to 100% related to E. coli genes and the level of relatedness (except for crr data) reflected the known taxonomic (phylogenetic) position of species with respect to E. coli. When ptsI relatedness data were plotted against ptsH data, correlation was excellent. In ptsG versus ptsLPM plots, the data points (species) were scattered along the diagonal with a large gap separating E. coli strains (80–100% relatedness to both probes) from the 63 other species (1 to 40% relatedness to E. coli genes). Serratia (9 species), Buttiauxella agrestis, and Klebsiella planticola gave higher relatedness values with crr probe than with the other probes tested.

Evolutionary divergence of enterobacterial genes coding for the glucose phosphotransferase system components

Evolutionary divergence of enterobacterial genes coding for the glucose phosphotransferase system components

The roles of osmotic stress and water activity in the inhibition of the growth, glycolysis and glucose phosphotransferase system of Clostridium pasteurianum.

Growth of Clostridium pasteurianum was inhibited in media of high solute content. At equal osmolarities, 'permeant' solutes (glycerol and acetamide) were much less growth-inhibitory than 'non-permeant' solutes (KC1 and xylitol). Glycolysis by washed cell suspensions was inhibited by these solutes in parallel with growth. However, in their inhibition of glucose 6-phosphate dissimilation by permeabilized cells the distinction between 'permeant' and 'impermeant' solutes was significantly less marked. The glucose phosphotransferase system (PTS) of intact cells was much more strongly inhibited by 'non-permeant' than by 'permeant' solutes. It was concluded that the predominant inhibitory effects on this organism of media of high solute content are due not to the low water activity of such media per se, but to the creation of an osmotic pressure across the bacterial cytoplasmic membrane, which acts to inhibit the glucose PTS by which the organism effects glucose uptake. Parallel measurements of the effects of xylitol on both glycolysis and the activity of the glucose PTS suggested that despite this correlation between the osmotic inhibition of growth, glycolysis and the PTS, the flux-control coefficient of the PTS on glycolysis did not exceed 0.2 under the conditions used.

Phosphoenolpyruvate: Sugar phosphotransferase systems and sugar metabolism in Brevibacterium flavum.

Brevibacterium flavum mutants defective in the phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system (PTS) were selected with high frequency by 2-deoxyglucose-resistance. Most of them (DOGr) still had the fructose-PTS and grew not only on fructose but also on glucose like the wild-type strain. A mutant having 1/8th the fructose-PTS activity of the wild strain but normal glucose-PTS activity was isolated as a xylitol-resistant mutant. It grew on glucose but not on fructose. The glucose-PTS was active on glucose, glucosamine, 2-deoxyglucose and mannose, and slightly on methyl-α-glucoside and N-acetylglucosamine, but not on fructose or xylitol. The fructose-PTS acted on fructose and xylitol, and to some extent on glucose but not on glucosamine or 2-deoxyglucose. Mutants unable to grow on glucose (DOGGlc-) derived from a DOGr mutant were all defective in the fructose-PTS. All revertants able to grow on glucose derived from a DOGrGlc- mutant had the fructose-PTS. The glucokinase activity was about 2/3rds the glucose activity of the fructose-PTS. All the DOGsprGlc- mutants had normal levels of glucokinase. One of these mutants grew on maltose and sucrose, which were hydrolyzed to glucose. Thus, glucokinase seems to contribute to the phosphorylation of glucose liberated inside the cell. The fructose-PTS was induced by fructose and repressed by glucose. The glucose repression was not observed in a mutant defective in the glucose-PTS.

Pyruvate formation and sugar metabolism in an amino acid-producing bacterium, Brevibacterium flavum.

A Brevibacterium flavum mutant lacking pyruvate kinase, No. 70, grew on glucose, fructose and sucrose as well as the original wild strain did, but was unable to grow on ribose or gluconate unless pyruvate was added. Mutants that required pyruvate for growth on ribose were derived directly from the wild strain. Many of them were completely or partially defective in pyruvate kinase activity. These pyruvate kinase mutants were also unable to grow on gluconate. A phosphoenolpy- ruvate (PEP): sugar phosphotransferase system (PTS) was found in B. flavum, which catalyzed the formation of pyruvate and sugar phosphate from PEP and sugar. The system required Mg2 +, acted on glucose, fructose, mannose, glucosamine and 2-deoxyglucose, and existed in the cells grown on any of the carbon sources tested. Cells grown on fructose, mannitol and sucrose, however, exhibited higher PTS activities on fructose than those grown on others. Glucose PTS activity was about 20-fold stronger than that of glucokinase. Other sugar metab...

Transport of trehalose in Salmonella typhimurium.

We have studied trehalose uptake in Salmonella typhimurium and the possible involvement of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in this process. Two transport systems could recognize and transport trehalose, the mannose PTS and the galactose permease. Uptake of trehalose via the latter system required that it be expressed constitutively (due to a galR or galC mutation). Introduction of a ptsM mutation, resulting in a defective IIMan/IIIMan system, in S. typhimurium strains that grew on trehalose abolished growth on trehalose. A ptsG mutation, eliminating IIGlc of the glucose PTS, had no effect. In contrast, a crr mutation that resulted in the absence of IIIGlc of the glucose PTS prevented growth on trehalose. The inability of crr and also cya mutants to grow on trehalose was due to lowered intracellular cyclic AMP synthesis, since addition of extracellular cyclic AMP restored growth. Subsequent trehalose metabolism could be via a trehalose phosphate hydrolase, if trehalose phosphate was formed via the PTS, or trehalase. Trehalose-grown cells contained trehalase activity, but we could not detect phosphoenolpyruvate-dependent phosphorylation of trehalose in toluenized cells.

Phosphate transfer between acetate kinase and enzyme I of the bacterial phosphotransferase system.

Interactions between homogeneous acetate kinase and proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) were studied. The phosphorylation of D-glucose was followed spectrophotometrically using a coupled assay system, and acetate kinase and GTP were found to substitute for phosphoenolpyruvate provided that each of the PTS proteins was present in the mixture. To further define the phosphoryl transfer reaction pathway, the system was simplified to include only the homogeneous, soluble PTS proteins. 32P was transferred from [gamma-32P]ATP to the protein IIIGlc, but this transfer reaction required acetate kinase, and the PTS proteins Enzyme I and HPr. These results suggested that acetate kinase interacts with the first protein in the PTS sequence, Enzyme I. Acetate kinase was therefore incubated with [32P] phospho-Enzyme I, and a direct transfer of the phosphoryl group was observed without the addition of any other protein. These results show that there is a reversible transfer of the phosphoryl group between Enzyme I and acetate kinase. The possible role of this interaction in regulating sugar uptake by the Krebs cycle is discussed.

The glucose phosphotransferase system as the major site of growth inhibition of Clostridium pasteurianum in media of high solute content

The glucose phosphotransferase system as the major site of growth inhibition of <i>Clostridium pasteurianum</i> in media of high solute content

Glucose uptake and phosphorylating activities in two species of slow-growingRhizobium

Glucose uptake and phosphorylating activities were studied in two strains of slow-growing Rhizobium: Rhizobium japonicum (USDAI-110) and cowpea Rhizobium (USDA3278). Cultured cells of both species actively took up glucose, and at least two systems appeared to be involved, whereas purified bacteroids of both species failed to accumulate glucose actively. In both cell types, glucose phosphorylation was ATP-dependent, and no evidence was obtained for a phosphoenolpyruvate-dependent glucose phosphotransferase system.

Transport and Metabolism of Lactose, Glucose, and Galactose in Homofermentative Lactobacilli

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained beta-galactosidase activity, and no significant phospho-beta-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.

The phosphoenolpyruvate:glucose phosphotransferase system of Salmonella typhimurium. The phosphorylated form of IIIGlc.

Enzyme IIIGlc of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Salmonella typhimurium can occur in two forms: phosphorylated and nonphosphorylated. Phosphorylated IIIGlc (P-IIIGlc) has a slightly lower mobility during sodium dodecyl sulphate/polyacrylamide gel electrophoresis than IIIGlc. In bacterial extracts both phosphoenolpyruvate (the physiological phosphoryl donor of the PTS) as well as ATP can phosphorylate IIIGlc. The ATP-catalyzed reaction is dependent on phosphoenolpyruvate synthase, however, and is due to prior conversion of ATP to phosphoenolpyruvate. The phosphoryl group of phosphorylated IIIGlc is hydrolysed after boiling in sodium dodecyl sulfate but phosphorylated IIIGlc can be discriminated from IIIGlc if treated with this detergent at room temperature. We have used the different mobilities of IIIGlc and P-IIIGlc to estimate the proportion of these two forms in intact cells. Wild-type cells contain predominantly P-IIIGlc in the absence of PTS sugars. In an S. typhimurium mutant containing a leaky ptsI17 mutation (0.1% enzyme I activity remaining) both forms of IIIGlc occur in approximately equal amounts. Addition of PTS sugars such as glucose results, both in wild-type and mutant, in a dephosphorylation of P-IIIGlc. This correlates well with the observed inhibition of non-PTS uptake systems by PTS sugars via nonphosphorylated IIIGlc.

Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat.

Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.

Regulation of the glucose phosphotransferase system in Brochothrix thermosphacta by membrane energization.

Uptake of 2-deoxyglucose, alpha-methylglucopyranoside, and glucose into intact cells of Brochothrix thermosphacta (formerly Microbacterium thermosphactum, ATCC 11509) was stimulated by KCN or CCCP. The glucose analogs were recovered almost totally as the sugar phosphates. Membrane vesicles were isolated from protoplasts and shown to be right side out by freeze fracturing and by using ATPase as a marker for the cytoplasmic membrane surface. Uptake of glucose into vesicles was dependent on the presence of phosphoenolpyruvate. NADH oxidation, K+ -diffusion gradients, and externally directed lactate gradients (pH greater than 7 initially) were used to generate transmembrane potentials across membrane vesicles. Above a threshold value of about -50 mV, uptake of glucose into membrane vesicles was reduced. Likewise, the maximum uptake of glucose and its two analogs into cells occurred when the protonmotive force was less than about -50 mV.