Trehalose-6-phosphate-mediated phenotypic change in Acinetobacter baumannii

The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD-glucose and glucose-6-phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose-6-phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose-6-phosphate phosphatase activity accumulated trehalose-6-phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose-6-phosphate. Our results demonstrate that trehalose-6-phosphate affects multiple physiological activities in A. baumannii ATCC 19606.     

Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation

Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1-1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.     

Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation

A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation.     

A unique combination of genetic systems for the synthesis of trehalose in Rubrobacter xylanophilus: properties of a rare actinobacterial TreT

Trehalose is the primary organic solute in Rubrobacter xylanophilus under all conditions tested, including those for optimal growth. We detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (Tps)/trehalose-6-phosphate phosphatase (Tpp), TreS, TreY/TreZ, and TreT pathways. Moreover, R. xylanophilus is the only known member of the phylum Actinobacteria to harbor TreT. The Tps sequence is typically bacterial, but the Tpp sequence is closely related to eukaryotic counterparts. Both the Tps/Tpp and the TreT pathways were active in vivo, while the TreS and the TreY/TreZ pathways were not active under the growth conditions tested and appear not to contribute to the levels of trehalose observed. The genes from the active pathways were functionally expressed in Escherichia coli, and Tps was found to be highly specific for GDP-glucose, a rare feature among these enzymes. The trehalose-6-phosphate formed was specifically dephosphorylated to trehalose by Tpp. The recombinant TreT synthesized trehalose from different nucleoside diphosphate-glucose donors and glucose, but the activity in R. xylanophilus cell extracts was specific for ADP-glucose. The TreT could also catalyze trehalose hydrolysis in the presence of ADP, but with a very high K_m. Here, we functionally characterize two systems for the synthesis of trehalose in R. xylanophilus, a representative of an ancient lineage of the actinobacteria, and discuss a possible scenario for the exceptional occurrence of treT in this extremophilic bacterium.     

Uncoupling of the glucose growth defect and the deregulation of glycolysis in Saccharomyces cerevisiae Tps1 mutants expressing trehalose-6-phosphate-insensitive hexokinase from Schizosaccharomyces pombe

In the yeast Saccharomyces cerevisiae inactivation of trehalose-6-phosphate (Tre6P) synthase (Tps1) encoded by the TPS1 gene causes a specific growth defect in the presence of glucose in the medium. The growth inhibition is associated with deregulation of the initial part of glycolysis. Sugar phosphates, especially fructose-1,6-bisphosphate (Fru1,6bisP), hyperaccumulate while the levels of ATP, Pi and downstream metabolites are rapidly depleted. This was suggested to be due to the absence of Tre6P inhibition on hexokinase. Here we show that overexpression of Tre6P (as well as glucose-6-phosphate (Glu6P))-insensitive hexokinase from Schizosaccharomyces pombe in a wild-type strain does not affect growth on glucose but still transiently enhances initial sugar phosphate accumulation. We have in addition replaced the three endogenous glucose kinases of S. cerevisiae by the Tre6P-insensitive hexokinase from S. pombe. High hexokinase activity was measured in cell extracts and growth on glucose was somewhat reduced compared to an S. cerevisiae wild-type strain but expression of the Tre6P-insensitive S. pombe hexokinase never caused the typical tps1Delta phenotype. Moreover, deletion of TPS1 in this strain expressing only the Tre6P-insensitive S. pombe hexokinase still resulted in a severe drop in growth capacity on glucose as well as sensitivity to millimolar glucose levels in the presence of excess galactose. In this case, poor growth on glucose was associated with reduced rather than enhanced glucose influx into glycolysis. Initial glucose transport was not affected. Apparently, deletion of TPS1 causes reduced activity of the S. pombe hexokinase in vivo. Our results show that Tre6P inhibition of hexokinase is not the major mechanism by which Tps1 controls the influx of glucose into glycolysis or the capacity to grow on glucose. In addition, they show that a Tre6P-insensitive hexokinase can still be controlled by Tps1 in vivo.     

A temperature-sensitive mutant of Sacchromyces cerevisiae defective in the specific phosphatase of trehalose biosynthesis

Abstract A temperature-sensitive mutant of Saccharomyces cerevisiae has been isolated which accumulates a large pool of trehalose-6-phosphate when shifted to temperatures above 34°C nonpermissive for growth. This indicates that its defect is in the second enzyme of trehalose biosynthesis, the hydrolase that converts trehalose-6-phosphate to trehalose. Trehalose is made continouosly when yeast is growing on high glucose or when it is starved for a nitrogen source, and accumulates as cells enter the stationary phase. Revertants of the mutant able to grow at 37°C arise spontaneously and no longer accumulate trehalose-6-phosphate at this temperature. Also the kinetics of trehalose-6-phosphate accumulation in the mutant following a 25–37°C shift resemble the kinetics of inhibition of RNA and protein synthesis. It is probable therefore that accumulation of high levels of this metabolic intermediate is inhibitory to growth.     

Accumulation of Trehalose by Overexpression of tps1, Coding for Trehalose-6-Phosphate Synthase, Causes Increased Resistance to Multiple Stresses in the Fission Yeast Schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.     

Overexpression of a trehalose-6-phosphate synthase/phosphatase fusion gene enhances tolerance and photosynthesis during drought and salt stress without growth aberrations in tomato

Trehalose is a non-reducing disaccharide of glucose that confers tolerance against abiotic stresses in many diverse organisms, including higher plants. It was previously reported that overexpression of the yeast trehalose-6-phosphate synthase gene in tomato results in improved tolerance against abiotic stresses. However, these transgenic tomato plants had stunted growth and pleiotropic changes in appearance. In this study, transgenic tomato plants were generated by the introduction of a gene encoding a bifunctional fusion of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase genes from Escherichia coli under the control of the CaMV35S promoter. Transgenic plants accumulated higher levels of trehalose in their leaves and exhibited enhanced drought and salt tolerance and photosynthetic rates under salt stress conditions than wild-type plants. All of the transgenic plants had normal growth patterns and appearances. Therefore, the system described in this study can be used for practical application of the gene in crop improvement.     

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.     

Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H⁺-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.     

Trehalose Metabolism-Related Genes in Maize

Maize is a cereal crop that is grown widely throughout the world in a range of agro-ecological environments. Trehalose is a nonreducing disaccharide of glucose that has been associated with tolerance to different stress conditions, including salt and drought. Bioinformatic analysis of genes involved in trehalose biosynthesis and degradation in maize has not been reported to date. Through systematic analysis, 1 degradation-related and 36 trehalose biosynthesis-related genes were identified. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs, isolated from other plant species such as Arabidopsis and rice, were revealed. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to salt stress, jasmonic acid, and abscisic acid treatment were analyzed. Microarray data demonstrated that some of the genes show differential, organ-specific expression patterns in the 60 different developmental stages of maize. It was discovered that some of the key enzymes such as hexokinase, trehalose-6-phosphate synthase, and trehalose-6-phosphate phosphatase are encoded by multiple gene members with different expression patterns. The results highlight the complexity of trehalose metabolism and provide useful information for improving maize stress tolerance through genetic engineering.     

Enhanced freeze tolerance of baker’s yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker’s yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301_TPS1 overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301_TPS1 were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301_TPS1 was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker’s yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker’s yeast.     

Kinetic evidence that the high-affinity glucose 6-phosphate site on hexokinase I Is the active site

Two mechanisms have been suggested to account for the regulation of brain hexokinase by glucose 6-phosphate. One mechanism places glucose-6-P at an allosteric site, remote from the active site, while the second describes glucose-6-P as a simple product inhibitor of the enzyme, binding at the γ phosphate subsite within the ATP locus of the active site. To distinguish between these possibilities, we have undertaken a study of the back reaction of hexokinase I. Our data indicate that glucose-6-P displays classical Michaelis-Menten kinetics with brain hexokinase. This finding is consistent only with the high-affinity glucose-6-P site on the enzyme being the catalytic site. The dissociation constant, estimated from the initial-rate experiments is approximately 25 μm, a value that agrees well with the inhibition constant for glucose-6-P in the forward direction. These findings are consistent with an earlier model (W. R. Ellison, J. D. Lueck and H. J. Fromm, (1975) J. Biol. Chem.250, 1864–1871), which maintains that glucose-6-P inhibition of brain hexokinase is a manifestation of product inhibition. In a recent paper, Lazo et al. (P. A. Lazo, A. Sols, and J. E. Wilson, (1980) J. Biol. Chem.255, 7548–7551) reported data obtained from binding studies with rat brain hexokinase at an elevated (250 μm) level of glucose-6-P. These authors believe that their results indicate multiple binding of glucose-6-P to the enzyme and interpret the data in terms of a high-affinity allosteric site and a low-affinity catalytic site. Our results are at variance with this interpretation and are consistent only with the high-affinity site for glucose-6-P on brain hexokinase being the active site.     

Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose‐6‐phosphate synthase

An Arabidopsis thaliana cDNA clone, AtTPS1, that encodes a trehalose-6-phosphate synthase was isolated. The identity of this protein is supported by both structural and functional evidence. On one hand, the predicted sequence of the protein encoded by AtTPS1 showed a high degree of similarity with trehalose-6-phosphate synthases of different organisms. On the other hand, expression of the AtTPS1 cDNA in the yeast tps1 mutant restored its ability to synthesize trehalose and suppressed its growth defect related to the lack of trehalose-6-phosphate. Genomic organization and expression analyses suggest that AtTPS1 is a single-copy gene and is expressed constitutively at very low levels.     

Influence of the StubSNF1 kinase complex and the expression of the yeast TPS1 gene on growth and tuber yield in potato

Expression of the yeast trehalose-6-phosphate synthase-1 (TPS1) gene in potato results in growth aberrations and arrest of development. Recent studies have shown that this phenomenon could be related to the inhibitory effect of trehalose-6-phosphate on SnRK1s, a family of sucrose non-fermenting-1 (SNF1)-related protein kinases that link metabolic and stress signalling in plants. SnRK1s are heterotrimeric enzymes similar to yeast SNF1 and mammalian AMP-activated protein kinases (AMPKs). Previously, we showed that antisense repression of StubGAL83, one of the three subunits of the potato SnRK1 complex, results in a delay in rooting and increases sensitivity to salt stress. Here we report that StubGAL83 is a positive regulator of SNF1 kinase activity in potato and that repression of the kinase subunit of the SnRK1 complex, StubSNF1, reduces growth and tuber yield in potato plants. Co-repression of StubGAL83 and StubSNF1 at a certain level, however, can result in larger plants and increased tuber yield. We found that repression of StubGAL83, but not repression of StubSNF1 attenuated growth aberrations caused by TPS1 expression. We provide evidence that the increased plant size and yield in StubGAL83-StubSNF1 co-repressed plants as well as the attenuation of aberrations caused by TPS1 expression are related to increased nitrate reductase activity.     

Regulation of mammalian hexokinase: regulatory differences between isoenzyme I and II

Mammalian hexokinase isoenzymes I and II have been shown to differ qualitatively in response to various modifiers. Although both enzymes are inhibited by glucose 6-phosphate, only isoenzyme II exhibits a slow response to the presence of this inhibitor. P_i decreases the affinity of glucose 6-phosphate for Sarcoma 37 hexokinase I, but has no effect on hexokinase II from the same cell. P_i overcomes all of the inhibition of red cell hexokinase by glucose-6-P and hence the two effectors act competitively. At pH 6.5, catecholamines increase the V of isoenzyme I of Sarcoma 37 and brain in the soluble and mitochondrial forms but do not activate these forms of tumor isoenzyme II. Citrate activates brain and tumor isoenzyme I when they are inhibited by tris(hydroxy-methyl)aminomethylethane sulfonate (TES) and ADP; however, tumor isoenzyme II is not activated.     

Effects of Cadmium and Mercury on the Upper Part of Skeletal Muscle Glycolysis in Mice

The effects of pre-incubation with mercury (Hg²⁺) and cadmium (Cd²⁺) on the activities of individual glycolytic enzymes, on the flux and on internal metabolite concentrations of the upper part of glycolysis were investigated in mouse muscle extracts. In the range of metal concentrations analysed we found that only hexokinase and phosphofructokinase, the enzymes that shared the control of the flux, were inhibited by Hg²⁺ and Cd²⁺. The concentrations of the internal metabolites glucose-6-phosphate and fructose-6-phosphate did not change significantly when Hg²⁺ and Cd²⁺ were added. A mathematical model was constructed to explore the mechanisms of inhibition of Hg²⁺ and Cd²⁺ on hexokinase and phosphofructokinase. Equations derived from detailed mechanistic models for each inhibition were fitted to the experimental data. In a concentration-dependent manner these equations describe the observed inhibition of enzyme activity. Under the conditions analysed, the integral model showed that the simultaneous inhibition of hexokinase and phosphofructokinase explains the observation that the concentrations of glucose-6-phosphate and fructose-6-phosphate did not change as the heavy metals decreased the glycolytic flux.     

Trehalose‐6‐phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6°C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.