Glucose Phosphotransferase System Research Articles

The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase.

The concentration of glucose in the medium influences the regulation of cAMP levels in Escherichia coli. Growth in minimal medium with micromolar glucose results in 8- to 10-fold higher intracellular cAMP concentrations than observed during growth with excess glucose. Current models would suggest that the difference in cAMP levels between glucose-rich and glucose-limited states is due to altered transport flux through the phosphoenolpyruvate: glucose phosphotransferase system (PTS), which in turn controls adenylate cyclase. A consequence of this model is that cAMP levels should be inversely related to the saturation of the PTS transporter. To test this hypothesis, the relationship between external glucose concentration and cAMP levels inside E. coli were investigated in detail, both through direct cAMP assay and indirectly through measurement of expression of cAMP-regulated genes. Responses were followed in batch, dialysis and glucose-limited continuous culture. A sharp rise in intracellular cAMP occurred when the nutrient concentration in minimal medium dropped to approximately 0.3 mM glucose. Likewise, addition of > 0.3 mM glucose, but not < 0.3 mM glucose, sharply reduced the intracellular cAMP level of starving bacteria. There was no striking shift in growth rate or [14C] glucose assimilation in bacteria passing through the 0.5 to 0.3 mM concentration threshold influencing cAMP levels, suggesting that neither metabolic flux nor transporter saturation influenced the sensing of nutrient levels. The (IIA/IIBC)Glc PTS is 96-97% saturated at 0.3 mM glucose so these results are not easily reconcilable with current models of cAMP regulation. Aside from the transition in cAMP levels initiated above 0.3 mM, a second shift occurred below 1 muM glucose. Approaching starvation, well below saturation of the PTS, cAMP levels either increased or decreased depending on unknown factors that differ between common E. coli K-12 strains.

Cystic fibrosis transmembrane conductance regulator: the first nucleotide binding fold targets the membrane with retention of its ATP binding function.

Most cases of cystic fibrosis are caused by a single deletion mutation (deltaF508) within the first nucleotide binding fold (NBF1) of the CFTR protein (cystic fibrosis transmembrane conductance regulator). NBF1 is classically defined as amino acid residues phenylalanine 433 through serine 589, encoded by exons 10-12, and only part of exon 9, of the CFTR gene. This assignment is based on sequence homology of this region of the CFTR protein with that of other nucleotide binding proteins. Here, we report that when the complete modular unit encoded precisely by exons 9-12 is expressed in Escherichia coli as glycine 404 through serine 589, i.e., as [G404-N432]NBF1 or as deltaF508[G404-N432]NBF1, the resultant proteins target the cytoplasmic membrane. Significantly, [G404-N432]NBF1 is readily labeled from the outside of intact E. coli spheroplasts with the water soluble, membrane impermeable probe Biotin-X-NHS, sulfosuccinimidyl-6-(biotinamido)-hexanoate. Similar findings were observed with the disease causing mutant deltaF508[G404-N432]NBF1. Three different control experiments which involved (1) assays for known cytosolic E. coli enzymes, (2) immuno-gold electron microscopy with antibody having an epitope for the biotin moiety, and (3) tests for biotinylation of the cytosolic component, Enzyme 1 of the glucose phosphotransferase system, demonstrated that the spheroplasts used in this study are neither leaky nor permeable to Biotin-X-NHS. In addition, membrane-associated [G404-N432]NBF1, upon solubilization with Triton X-100, was found to bind to an ATP-agarose column and be released therefrom by elution with ATP, emphasizing retention of a native-like structure. In sharp contrast, NBF1 localizes to the cytosol when the [G404-N432]-N-terminal region is replaced with the maltose binding protein. The novel findings reported here implicate a role of the N-terminal region of NBF1 in its subcellular localization and are directly relevant to our understanding of the membrane structure, function, and trafficking of CFTR.

Pathway engineering for the production of aromatic compounds in Escherichia coli.

Glucose is the preferred substrate for certain fermentation processes. During its internalization and concomitant formation of glucose-6-phosphate through the glucose phosphotransferase system (PTS), one molecule of phosphoenolpyruvate (PEP) is consumed. Together with erythrose 4-phosphate (E4P), PEP is condensed to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP), the first intermediate of the common segment of the aromatic pathway. From this metabolic route, several commercially important aromatic compounds can be obtained. We have selected Escherichia coli mutants that can transport glucose efficiently by a non-PTS uptake system. In theory, this process should increase the availability of PEP for other biosynthetic reactions. Using these mutants, in a background where the DAHP synthase (the enzyme that catalyzes the condensation of PEP and E4P into DAHP) was amplified, we were able to show that at least some of the PEP saved during glucose transport, can be redirected into the aromatic pathway. This increased carbon commitment to the aromatic pathway was enhanced still further upon amplification of the E. coli tktA gene that encodes for a transketolase involved in the biosynthesis of E4P.

Regulation of Escherichia coli adenylate cyclase activity during hexose phosphate transport.

In Escherichia coli, cAMP levels vary with the carbon source used in the culture medium. These levels are dependent on the cellular concentration of phosphorylated EnzymeIIAglc, a component of the glucose-phosphotransferase system, which activates adenylate cyclase (AC). When cells are grown on glucose 6-phosphate (Glc6P), the cAMP level is particularly low. In this study, we investigated the mechanism leading to the low cAMP level when Glc6P is used as the carbon source, i.e. the mechanism preventing the activation of AC by phosphorylated EnzymeIIAglc. Glc6P is transported via the Uhp system which is inducible by extracellular Glc6P. The Uhp system comprises a permease UhpT and three proteins UhpA, UhpB and UhpC which are necessary for uhpT gene transcription. Controlled expression of UhpT in the absence of the regulatory proteins (UhpA, UhpB and UhpC) allowed us to demonstrate that (i) the Uhp regulatory proteins do not prevent the activation of AC by direct interaction with EnzymeIIAglc and (ii) an increase in the amount of UhpT synthesized (corresponding to an increase in the amount of Glc6P transported) correlates with a decrease in the cAMP level. We present data indicating that Glc6P per se or its degradation is unlikely to be responsible for the low cAMP level. It is concluded that the level of cAMP in the cell is determined by the flux of Glc6P through UhpT.

Glucose Transport in Stationary-Phase Cultures of an Asporogenous Strain of Bacillus licheniformis

The sporulation-deficient industrial organism Bacillus licheniformis HWL10 possesses two distinct glucose transport systems in log-phase cells, a glucose phosphotransferase system (PTS) and a non-PTS mechanism. The strain continues to take up glucose at a significant though reduced rate during prolonged stationary-phase incubation, but only the PTS is active.

A String of Enzymes, Purification and Characterization of a Fusion Protein Comprising the Four Subunits of the Glucose Phosphotransferase System of Escherichia coli

A multidomain protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli was constructed by fusion of the transmembrane subunit IICBGlc and the three cytoplasmic proteins, IIAGlc, HPr, and enzyme I. The subunits were linked in the above order with Ala-Pro-rich linkers; the fusion protein was overexpressed in E. coli and purified by Ni2+ chelate affinity chromatography. Approximately 3 mg of the fusion protein could be purified from 1 liter of culture. The phosphotransferase activity of the purified fusion protein was 3-4 times higher than that of an equimolar mixture of the isolated subunits. The mannose transporter, which also requires enzyme I and HPr, was not an effective competitor in the overall phosphoryltransfer reaction when the fusion protein was used, whereas it was a competitor when an equimolar mixture of the separate subunits was employed. Transphosphorylation activity of the fusion protein was almost indistinguishable from the wild-type IICBglc. Addition of extra IICBGlc subunit could significantly stimulate the phosphotransferase activity of the fusion protein, addition of extra IIAGlc subunit and enzyme I, in contrast, was slightly inhibitory, and HPr had almost no effect. An optimal detergent-lipid ratio is required for maximum activity of the fusion protein. Our results suggest that Ala-Pro-rich linker sequences may be of general use for the construction of catalytically active fusion proteins with novel properties.

A Fungal Membrane-Associated Phosphoenolpyruvate: glucose Phosphotransferase System, which Includes Adenylyl Cyclase, in the Lichen Evernia prunastri

Summary The lichen Evernia prunastri is capable of accumulating glucose from liquid media by passive diffusion as well as by active uptake and phosphorylation of the sugar. This latter mechanism of glucose uptake is inhibited by cyclic AMP whose levels, in turn, are inhibited by glucose in lichen cells. An enzyme complex that phosphorylates glucose using phosphoenolpyruvate as a phosphate donor has been purified 2770-fold from lichen membranes. This enzyme complex, which is composed of five distinct subunits, has a molecular mass of about 100 kDa. The enzyme uses glucose preferentially, but can also use fructose as a substrate. Phosphoenolpyruvate, but not ATP, serves as a phosphate donor. The enzyme contains adenylyl cyclase as a constitutive subunit and is completeley restricted to the fungal partner of the lichen thallus.

Control of glucose metabolism by the enzymes of the glucose phosphotransferase system in Salmonella typhimurium.

The quantitative role of the phosphoenolpyruvate:glucose phosphotransferase system (glucose phosphotransferase system) in glucose uptake and metabolism, and phosphotransferase-system-mediated regulation of glycerol uptake, was studied in vivo in Salmonella typhimurium. Expression plasmids were constructed which contained the genes encoding enzyme I (ptsI), HP (ptsH), IIAGlc (crr), and IICBGlc (ptsG) of the glucose phosphotransferase system behind inducible promoters. These plasmids allowed the controlled expression of each of the glucose phosphotransferase system proteins from about 30% to about 300% of its wild-type level. When enzyme I, HPr or IIAGlc were modulated between 30% and 300% of their wild-type value, hardly any effects on the growth rate on glucose, the glucose oxidation rate, the rate of methyl alpha-D-glucopyranoside (a glucose analog) uptake or the phosphotransferase-system-mediated inhibition of glycerol uptake by methyl alpha-D-glucopyranoside were observed. Employing the method of metabolic control analysis, it was shown that the enzyme flux control coefficients of these phosphotransferase system components on the different measured processes were close to zero. The enzyme flux control coefficient of IICBGlc on growth on glucose or glucose oxidation was also close to zero. In contrast, the enzyme flux control coefficient of IICBGlc on the flux through the glucose phosphotransferase system (transport and phosphorylation) was 0.72. The experimentally determined enzyme flux control coefficients allowed us to calculate the flux control coefficients of the phosphoenolpyruvate/pyruvate and methyl alpha-D-glucopyranoside/methyl alpha-D-glucopyranoside 6-phosphate couples and the process control coefficients of the phosphotransfer reactions of the glucose phosphotransferase system. We discuss the implications of these values and the possible control points in the glucose phosphotransferase system.

Control of Glucose Metabolism by the Enzymes of the Glucose Phosphotransferase System in Salmonella typhimurium

Control of Glucose Metabolism by the Enzymes of the Glucose Phosphotransferase System in Salmonella typhimurium

Effect of modified glucose uptake using genetic engineering techniques on high‐level recombinant protein production in escherichia coli dense cultures

Reduction of acetate excretion using a modified cellular glucose uptake rate was examined. An Escherichia coli strain bearing a mutationin ptsG, a gene encoding enzyme II in glucose phosphotransferase system (PTS), was constructed and characterized. The growth rate of the mutant strain was slower than its parent in glucose defined medium, butwas not affected in complex medium. Experimental results using this mutant strain showed a significant improvement in culture performance in simple batch cultivations due to reduced acetate excretion through the modified glucose uptake. Both biomass and recombinant protein productivity were increased by more than 50% with the ptsG mutant when compared to the parent strain. Recombinant protein productivity by the newly constructed strain at a level of more than 1.6 g/L was attained consistently in a simple batch bioreactor. (c) 1994 John Wiley & Sons, Inc.

Quantification of the regulation of glycerol and maltose metabolism by IIAGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system in Salmonella typhimurium.

The amount of IIAGlc, one of the proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS), was modulated over a broad range with the help of inducible expression plasmids in Salmonella typhimurium. The in vivo effects of different levels of IIAGlc on glycerol and maltose metabolism were studied. The inhibition of glycerol uptake, by the addition of a PTS sugar, was sigmoidally related to the amount of IIAGlc. For complete inhibition of glycerol uptake, a minimal ratio of about 3.6 mol of IIAGlc to 1 mol of glycerol kinase (tetramer) was required. Varying the level of IIAGlc (from 0 to 1,000% of the wild-type level) did not affect the growth rate on glycerol, the rate of glycerol uptake, or the synthesis of glycerol kinase. In contrast, the growth rate on maltose, the rate of maltose uptake, and the synthesis of the maltose-binding protein increased two- to fivefold with increasing levels of IIAGlc. In the presence of cyclic AMP, the maximal levels were obtained at all IIAGlc concentrations. The synthesis of the MalK protein, the target of IIAGlc, was not affected by varying the levels of IIAGlc. The inhibition of maltose uptake was sigmoidally related to the amount of IIAGlc. For complete inhibition of maltose uptake by a PTS sugar, a ratio of about 18 mol of IIAGlc to 1 mol of MalK protein (taken as a dimer) was required.

Pentose utilization by the ruminal bacterium Ruminococcus albus.

Ruminococcus albus is an important fibrolytic ruminal bacteria which degrades hemicellulose and ferments the resulting pentose sugars. However, little information is available on the utilization of pentoses by this organism or the effect of hexose sugars on pentose metabolism. Enzymatic studies indicated that R. albus metabolized pentoses via the pentose phosphate pathway and possessed constitutive transketolase activity. Cellobiose was preferred over xylose and arabinose, and it appeared that the disaccharide decreased pentose metabolism by repression of transport activity and catabolic enzymes (isomerases and kinases). Glucose and xylose were co-utilized, and transport studies suggested that there was a common transport system for both sugars. In contrast, glucose was preferred over arabinose and the hexose noncompetitively inhibited the transport of arabinose. Since R. albus lacks a glucose phosphotransferase system, the inhibition of arabinose uptake could not be explained by previously described models of inducer exclusion involving such a system. Because accumulation of radiolabeled xylose, arabinose, and glucose proceeded in the absence of a proton motive force and since transport was correlated with the intracellular ATP concentration, it appeared that monosaccharide uptake was driven by ATP hydrolysis.

Control and regulation of metabolic fluxes in microbes by substrates and enzymes.

The control of enzymes and substrates on the flux through microbial metabolic pathways can be quantified in terms of flux control coefficients. In pathways involving group transfer, the summation theorem for flux control by the enzymes has to be modified: the sum of control by all enzymes is between 1 and 2. The phosphoenolpyruvate:glucose phosphotransferase system is such a pathway. Experimental determination of the control by the enzymes in this pathway is under way. The control of the enzymes on the glycolytic flux in yeast is low, with the possible exception of the uptake step. In Klebsiella pneumoniae potassium and ammonium ions can simultaneously be 'limiting', (i.e. have significant control on growth) at pH6, but not at pH8. This may be due to the fact that at pH8 the high-affinity potassium uptake system is absent.

Effects of N-acetylglucosamine on carbohydrate fermentation by Streptococcus mutans NCTC 10449 and Streptococcus sobrinus SL-1

We have investigated the ability of two species of streptococci isolated from the human oral cavity (Streptococcus mutans NCTC 10449 and Streptococcus sobrinus SL-1) to metabolize N-acetylglucosamine (GlcNAc), a naturally occurring amino sugar present in saliva and human glycoproteins, when provided as the sole fermentable carbohydrate and determined the effects of the presence of GlcNAc on the fermentation of other carbohydrates. S. mutans used GlcNAc at concentrations of up to 10 mM to increase cell numbers, but S. sobrinus was unable to ferment the amino sugar alone and its uptake only occurred in the presence of a fermentable carbohydrate. GlcNAc had a marked inhibitory effect on the ability of S. sobrinus to produce lactic acid from glucose, sucrose, and fructose, at the same time increasing the lag period and doubling time of batch-grown cells. Such patterns of inhibition were found with S. mutans, but the effects were less than those seen in S. sobrinus. In mixed culture studies of the two species, S. sobrinus became the predominant organism when 10 mM glucose was supplied as the sole fermentable carbohydrate, with a concomitant decrease in the numbers of S. mutans cells, but supplementation of the broth with 10 mM glucose and 10 mM GlcNAc resulted in the emergence of S. mutans as the predominant organism. S. mutans and S. sobrinus grown in media containing glucose possessed the ability to transport glucose and GlcNAc, probably via the same glucose-phosphotransferase system at similar rates. However, intracellular levels of N-acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase were markedly higher in S. mutans grown on glucose and GlcNAc than in S. sobrinus: 34 and 398 and 8 and 17 nmol of NADPH formed per mi per mg of protein for S. mutans and S. sobrinus, respectively. We propose that GlcNAc inhibited growth of S. sobrinus in media containing glucose and GlcNAc by competing with glucose for the glucose phosphotransferase, depleting intracellular levels of phosphoenolpyruvate, and possessing, in contrast to S. mutans, low levels of N-acetyl-glucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase activity. Together, these data suggest that in dental plaque, S. sobrinus when exposed to GlcNAc will have a reduced ability to compete with S. mutans for dietary carbohydrates, contributing to the greater frequency of isolation of S. mutans from human populations.

Maltose uptake and its regulation inBacillus subtilis

Extracts prepared from cultures of Bacillus subtilis, grown on maltose as the sole carbon source, lacked maltose phosphotransferase system activity. There was, however, evidence for a maltose phosphorylase activity, and such extracts also possessed both glucokinase and glucose phosphotransferase system activities. Maltose was accumulated by whole cells of B. subtilis by an energy-dependent mechanism. This uptake was sensitive to the effects of uncouplers, suggesting a role for the proton-motive force in maltose transport. Accumulation of maltose was inhibited in the presence of glucose, and there was no accumulation of maltose by a strain carrying the ptsl6 null-mutation. A strain carrying the temperature-sensitive ptsl1 mutation accumulated maltose normally at 37°C but, in contrast to the wild-type, was devoid of maltose transport activity at 47°C. The results indicate a role for the phosphotransferase system in the regulation of maltose transport activity in this organism.

Maltose uptake and its regulation in Bacillus subtilis

Extracts prepared from cultures of Bacillus subtilis, grown on maltose as the sole carbon source, lacked maltose phosphotransferase system activity. There was, however, evidence for a maltose phosphorylase activity, and such extracts also possessed both glucokinase and glucose phosphotransferase system activities. Maltose was accumulated by whole cells of B. subtilis by an energy-dependent mechanism. This uptake was sensitive to the effects of uncouplers, suggesting a role for the proton-motive force in maltose transport. Accumulation of maltose was inhibited in the presence of glucose, and there was no accumulation of maltose by a strain carrying the ptsl6 null-mutation. A strain carrying the temperature-sensitive ptsl1 mutation accumulated maltose normally at 37°C but, in contrast to the wild-type, was devoid of maltose transport activity at 47°C. The results indicate a role for the phosphotransferase system in the regulation of maltose transport activity in this organism.

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis.

The ptsG gene of Bacillus subtilis encodes Enzyme IIGlc of the phosphoenolpyruvate: glucose phosphotransferase system. The 3' end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes IIIGlc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5' end. These results, combined with the sequence of the 3' end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes IIGlc and IINag of E. coli but which are present in the IIGlc-like protein encoded by the E. coli malX gene.

Effect of growth conditions on the Streptococcus bovis phosphoenolpyruvate glucose phosphotransferase system1

Four strains of the ruminal bacterium Streptococcus bovis were surveyed for phosphoenolpyruvate (PEP) and ATP-dependent phosphorylation of glucose and the nonmetabolizable glucose analog 2-deoxyglucose. All four strains had high rates of glucose phosphorylation with either phosphoryl donor, but 2-deoxyglucose activity was much higher in the presence of PEP. These results provide evidence for a PEP-dependent glucose phosphotransferase system in these bacteria. Mannose and 2-deoxyglucose inhibited PEP-dependent phosphorylation of glucose by S. bovis JB1 by 50 and 38%, respectively, whereas alpha-methylglucoside had little effect. Mannose was a competitive inhibitor of PEP-dependent phosphorylation of glucose with an inhibition constant of 2.8 mM, and PEP-dependent activity in cells grown in batch culture was optimal at pH 7.2. When S. bovis JB1 was grown in continuous culture, PEP-dependent phosphorylation of glucose and 2-deoxyglucose was highest in cells grown at a dilution rate of .10/h and at low glucose concentrations. Phosphoenolpyruvate-dependent activity was optimum at a growth pH of 5.0 for cells grown in medium that contained less than 6.0 g/liter of glucose. These data indicate that PEP-dependent glucose phosphotransferase system activity can be influenced depending on the growth conditions used to culture S. bovis. Furthermore, these results suggest that environmental conditions within the rumen will affect how glucose is transported by S. bovis.

Control of glucose metabolism by enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli

The quantitative effects of variations in the amount of enzyme IIGlc of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) on glucose metabolism in Escherichia coli were studied. The level of enzyme IIGlc could be adjusted in vivo to between 20 and 600% of the wild-type chromosomal level by using the expression vector pTSG11. On this plasmid, expression of the structural gene for enzyme IIGlc, ptsG, is controlled by the tac promoter. As expected, the control coefficient (i.e., the relative increase in pathway flux, divided by the relative increase in amount of enzyme) of enzyme IIGlc decreased in magnitude if a more extensive pathway was considered. Thus, at the wild-type level of enzyme IIGlc activity, the control coefficient of this enzyme on the growth rate on glucose and on the rate of glucose oxidation was low, while the control coefficient on uptake and phosphorylation of methyl alpha-glucopyranoside (an enzyme IIGlc-specific, nonmetabolizable glucose analog) was relatively high (0.55 to 0.65). The implications of our findings for PTS-mediated regulation, i.e., inhibition of growth on non-PTS compounds by glucose, are discussed.

Properties of the glucose phosphotransferase system of Clostridium acetobutylicum NCIB 8052

The glucose phosphotransferase system (PTS) of Clostridium acetobutylicum was studied by using cell extracts. The system exhibited a Km for glucose of 34 microM, and glucose phosphorylation was inhibited competitively by mannose and 2-deoxyglucose. The analogs 3-O-methylglucoside and methyl alpha-glucoside did not inhibit glucose phosphorylation significantly. Activity showed no dependence on Mg2+ ions or on pH in the range 6.0 to 8.0. The PTS comprised both soluble and membrane-bound proteins, which interacted functionally with the PTSs of Clostridium pasteurianum, Bacillus subtilis, and Escherichia coli. In addition to a membrane-bound enzyme IIGlc, sugar phosphorylation assays in heterologous systems incorporating extracts of pts mutants of other organisms provided evidence for enzyme I, HPr, and IIIGlc components. The HPr was found in the soluble fraction of C. acetobutylicum extracts, whereas enzyme I, and probably also IIIGlc, was present in both the soluble and membrane fractions, suggesting a membrane location in the intact cell.