Phosphoenolpyruvate-dependent Phosphotransferase Research Articles

Engineering of Escherichia coli to facilitate efficient utilization of isomaltose and panose in industrial glucose feedstock

Industrial glucose feedstock prepared by enzymatic digestion of starch typically contains significant amounts of disaccharides such as maltose and isomaltose and trisaccharides such as maltotriose and panose. Maltose and maltosaccharides can be utilized in Escherichia coli fermentation using industrial glucose feedstock because there is an intrinsic assimilation pathway for these sugars. However, saccharides that contain α-1,6 bonds, such as isomaltose and panose, are still present after fermentation because there is no metabolic pathway for these sugars. To facilitate more efficient utilization of glucose feedstock, we introduced glvA, which encodes phospho-α-glucosidase, and glvC, which encodes a subunit of the phosphoenolpyruvate-dependent maltose phosphotransferase system (PTS) of Bacillus subtilis, into E. coli. The heterologous expression of glvA and glvC conferred upon the recombinant the ability to assimilate isomaltose and panose. The recombinant E. coli assimilated not only other disaccharides but also trisaccharides, including alcohol forms of these saccharides, such as isomaltitol. To the best of our knowledge, this is the first report to show the involvement of the microbial PTS in the assimilation of trisaccharides. Furthermore, we demonstrated that an l-lysine-producing E. coli harboring glvA and glvC converted isomaltose and panose to l-lysine efficiently. These findings are expected to be beneficial for industrial fermentation.

Molecular cloning and characterization of mannitol-1-phosphate dehydrogenase from Vibrio cholerae.

Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a 6×His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a K(m) of 1.54 +/- 0.1 mM and V(max) of 320.8 +/- 7.81 micronmol/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and 37°C, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.

Fine-Grained Parallel and Distributed Spatial Stochastic Simulation of Biological Reactions

Fine-Grained Parallel and Distributed Spatial Stochastic Simulation of Biological Reactions

Mechanism of Sugar Transport by a Phosphoenolpyruvate-Dependent Phosphotransferase

The phosphoenolpyruvate-dependent phosphotranferase system (PTS) is responsible for the uni-directional, phosphorylation-coupled uptake of sugar in bacteria. The multicomponent PTS differs from other carbohydrate uptake systems in that active transport is driven by coupling the translocation of the ligand across the membrane with its concomitant covalent modification to prevent efflux. Crystal structures have been solved previously for the numerous cytoplasmic proteins involved in the PTS pathway, but a complete understanding of the system has until now been hampered by the lack of any structures for the integral membrane component EIIC. The EIICs are a large, diverse family of carbohydrate transporters that selectively bind a cognate sugar, translocate it across the inner membrane, and assist in the phosphorylation reaction. In order to better understand how these functions are carried out, we have solved the 3.3 A structure of an ortholog of the transporter ChbC. This member of the glucose EIIC superfamily is specific for the uptake of N.N’-diacetylchitobiose, a component of chitin and important food source in a number of pathogens. The structure shows that ChbC possesses a novel fold, and has led us to propose a mechanism for how the transporter couples phosphorylation and transport of its substrate. We are now attempting a variety of functional assays to characterize the sugar selectivity of the transporter, as well as to elucidate the mechanism of phosphorylation and transport.

Cloning and molecular analysis of a mannitol operon of phosphoenolpyruvate-dependent phosphotransferase (PTS) type from Vibrio cholerae O395

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EII(Mtl)) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a mannitol operon repressor (MtlR). The transport of [(3)H]mannitol by the cloned mannitol operon in E. coli was 13.8 ± 1.4 nmol/min/mg protein. The insertional inactivation of EII(Mtl) abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram-positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon.

Spatial stochastic modelling of the phosphoenolpyruvate-dependent phosphotransferase (PTS) pathway in Escherichia coli

Many biochemical networks involve reactions localized on the cell membrane. This can give rise to spatial gradients of the concentration of cytosolic species. Moreover, the number of membrane molecules can be small and stochastic effects can become relevant. Pathways usually consist of a complex interaction network and are characterized by a large set of parameters. The inclusion of spatial and stochastic effects is a major challenge in developing quantitative and dynamic models of pathways. We have developed a particle-based spatial stochastic method (GMP) to simulate biochemical networks in space, including fluctuations from the diffusion of particles and reactions. Gradients emerging from membrane reactions can be resolved. As case studies for the GMP method we used a simple gene expression system and the phosphoenolpyruvate:glucose phosphotransferase system pathway. The source code for the GMP method is available at http://www.science.uva.nl/research/scs/CellMath/GMP.

Uncoupler-Resistant Glucose Uptake by the Thermophilic Glycolytic Anaerobe Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum)

The transport of glucose across the bacterial cell membrane of Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum) Rt8.B1 was governed by a permease which did not catalyze concomitant substrate transport and phosphorylation and thus was not a phosphoenolpyruvate-dependent phosphotransferase. Glucose uptake was carrier mediated, could not be driven by an artificial membrane potential (Deltapsi) in the presence or absence of sodium, and was not sensitive to inhibitors which dissipate the proton motive force (Deltap; tetrachlorosalicylanilide, N,N-dicyclohexylcarboiimide, and 2,4-dinitrophenol), and no uptake of the nonmetabolizable analog 2-deoxyglucose could be demonstrated. The glucokinase apparent K(m) for glucose (0.21 mM) was similar to the K(t) (affinity constant) for glucose uptake (0.15 mM), suggesting that glucokinase controls the rate of glucose uptake. Inhibitors of ATP synthesis (iodoacetate and sodium fluoride) also inhibited glucose uptake, and this effect was due to a reduction in the level of ATP available to glucokinase for glucose phosphorylation. These results indicated that T. thermosulfuricus Rt8.B1 lacks a concentrative uptake system for glucose and that uptake is via facilitated diffusion, followed by ATP-dependent phosphorylation by glucokinase. In T. thermosulfuricus Rt8.B1, glucose is metabolized by the Embden-Meyerhof-Parnas pathway, which yields 2 mol of ATP (G. M. Cook, unpublished data). Since only 1 mol of ATP is used to transport 1 mol of glucose, the energetics of this system are therefore similar to those found in bacteria which possess a phosphotransferase.

Spiroplasma cells utilize carbohydrates via the phosphoenolpyruvate-dependent sugar phosphotransferase system

Ten Spiroplasma species tested were found capable of fermenting glucose, mannose, fructose, and sucrose, but not ribose, maltose, 2-deoxyglucose, xylose, sorbitol, glactose, lactose, and arabinose. Sugar utilization was measured by a direct measurement of the changes in pH of a washed cell suspension upon the addition of the various sugars. Sulfhydryl reagents, uncouplers, and glycolysis inhibitors prevented the sugar-induced pH shifts. The spiroplasmas were capable of phosporylating α-methylgucoside in a reaction that required phosphoenolypyruvate, but not ATP, as a phosphate donor, suggesting that Spiroplasma species possess a phosphoenolpyruvate-dependent sugar phosphotransferase system. Key words: Spiroplasma, carbohydrate utilization, pH changes, phosphenolpyruvate-dependent sugar phosphotransferase system.

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.

Phosphotransferase Activity in Clostridium acetobutylicum from Acidogenic and Solventogenic Phases of Growth

Clostridium acetobutylicum cells, when energized with fructose, transported and phosphorylated the glucose analog 2-deoxyglucose by a phosphoenolpyruvate-dependent phosphotransferase (PT) system. Butanol up to 2% did not inhibit PT activity, although its chaotropic effect on the cell membrane caused cellular phosphoenolpyruvate and the 2-deoxyglucose-6-phosphate to leak out. Cells harvested from the solventogenic phase of batch growth had a significantly lower PT activity than did cells from the acidogenic phase.

Effect of growth conditions on sucrose phosphotransferase activity of Streptococcus mutans.

Sucrose and glucose phosphoenolpyruvate-dependent phosphotransferase (PTS) activities were studied in growing cultures of Streptococcus mutans serotype c and d/g cells adapted to either glucose or sucrose. Both acid production and optical absorbance were used to monitor growth in pH-controlled defined growth medium. The sucrose PTS activity appeared to be significant only under conditions of substrate limitation or slow growth as a result of low environmental pH. However, under environmental conditions which permitted rapid growth sucrose PTS activity appeared to be repressed, and only when the cells approached substrate-limited stationary phase after growth on high sucrose-supplemented medium was significant sucrose PTS activity again observed. A mutant apparently defective in sucrose PTS activity grew rapidly and produced acid under conditions of high environmental sucrose level but showed no sucrose PTS activity when the culture approached stationary phase. The mutant, however, after adaptation to glucose, demonstrated significant glucose PTS once the culture had attained the stationary growth phase. During diauxie growth in the presence of glucose and sucrose, there were sequential apparent inductions and repressions of glucose and sucrose PTS activities corresponding to decreases and increases of growth rate on the two substrates. Thus, S. mutans possesses at least two transport mechanisms for each substrate studied. One system (PTS) functions under conditions permitting slow growth and another functions under conditions permitting rapid growth.

The uptake of glucose by a thermophilic Bacillus sp.

A phospho enol pyruvate-dependent phosphotransferase that effects the uptake and phosphorylation of glucose and of methyl α -glucoside is inducibly formed when glucose is added to cultures of Bacillus stearothermophilus , var. non-diastaticus , that are growing at 55 °C on media containing acetate or succinate, but not when growing on fructose, as carbon source. This phosphotransferase is no less stable in intact cells than are other proteins: it is diluted out, but not preferentially destroyed, when glucose-grown inocula grow on fructose. Suspensions of non-growing cells kept at 55 °C lose half their glucose phosphotransferase activity in 2 to 3 h; this is similar to the rate at which this activity is lost from suspensions of mesophilic bacilli kept at 35 °C, which is close to their optimal growth temperature. However, in absolute terms, the glucose phosphotransferase activity of mesophiles is much more sensitive to heat denaturation than that of thermophiles.

Role of phosphoenolpyruvate-phosphotransferase in glucose utilization by bacilli.

The growth of mutantZ4ofBacillus licheniformison glucose and on a number of other carbohydrates is impaired, but growth on fructose, glycerol and on glucuronate is not. There are no significant differences between the mutant and its parent in the levels of glycolytic enzymes and in the ability of the organisms to take up labelled fructose; in contrast, the mutant takes up and phosphorylates labelled glucose, and its analogues methylα-glucoside and 2-deoxyglucose, to a much smaller extent than does the wild-type. Extracts of the mutant are virtually devoid of the inducible phosphoenolpyruvate-dependent glucose phosphotransferase present in the parent, though fructose phosphotransferase activity is present in both organisms. Revertants ofZ4, selected for growth on glucose, fully regain the wild-type phenotype. These results show that the phosphotransferase system plays a necessary role in the utilization of glucose by bacilli.