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At least 19 recordsLinked to original sources
PubMed1993

Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate and loss of trehalose-6-phosphate phosphatase activity.

Preparations of the trehalose-6-phosphate synthase/phosphatase complex from Saccharomyces cerevisiae contain three polypeptides with molecular masses 56, 100 and 130 kDa, respectively. Recently, we have cloned the gene for the 56-kDa subunit of this complex (TPS1) and found it to be identical with CIF1, a gene essential for growth on glucose and for the activity of trehalose-6-phosphate synthase. Peptide sequencing of the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex (TPS2) revealed one sequence to be 100% identical with the deduced amino acid sequence of the upstream region of PPH3 on the right arm of chromosome IV. This sequence was used to clone an upstream region of PPH3 containing an open reading frame of 2685 nucleotides, predicted to encode a polypeptide of 102.8 kDa. The N-terminal sequence, as well as three internal amino acid sequences, obtained from peptide sequencing of the 100-kDa subunit, were identical with specific regions of the deduced amino acid sequence. Thus, the sequence cloned represents TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex. Interestingly, a stretch of about 500 amino acids from the first part of TPS2 was 33% identical with the entire TPS1 sequence. Disruption of TPS2 had no effect on trehalose-6-phosphate synthase activity but caused complete loss of trehalose-6-phosphate phosphatase activity, measured in vitro, and accumulation of excessive amounts of trehalose-6-phosphate instead of trehalose upon heat shock or entrance into stationary phase in vivo. These results suggest that TPS2 codes for the structural gene of the trehalose-6-phosphate phosphatase. Heat shock induced an increase in trehalose-6-phosphate phosphatase activity and this was preceded by an accumulation in TPS2 mRNA, suggesting that the trehalose-6-phosphate phosphatase is subjected to transcriptional control under heat-shock conditions.

Amino Acid Sequence↗
PubMed1998

Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.

Bacterial Proteins↗
PubMed2004

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.

Corynebacterium↗
PubMed2001

Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues.

Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.

Amino Acid Sequence↗
PubMed2003

Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth.

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1). This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.

Adaptation, Physiological↗
PubMed2004

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98 degrees C and pH near 6.0; TPP had a maximal activity near 70 degrees C and at pH 7.0. The enzymes were extremely thermostable: at 100 degrees C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co2+ and Mg2+ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.

Adenosine Diphosphate↗
PubMedDate not supplied

The control of trehalose biosynthesis in Saccharomyces cerevisiae: evidence for a catabolite inactivation and repression of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.

During diauxic growth of yeast in glucose-rich medium, the accumulation of trehalose started well after complete exhaustion of glucose from the medium. The accumulation of the disaccharide was concomitant with a resumption of cell growth on the ethanol accumulated in the medium, but not with a degradation of glycogen which occurred as soon as glucose had been consumed. In contrast, in a mutant deficient in phosphoenolpyruvate carboxykinase, the synthesis of trehalose coincided exactly with the degradation of glycogen. Upon inoculation of stationary phase wild-type cells into a glucose medium, the activities of trehalose-6-phosphate (Tre6P) synthase and Tre6P phosphatase dropped in parallel to reach only 15% of their initial values after 3 h, and only recovered their original values as cells re-entered stationary phase. In the presence of cycloheximide, the decrease in Tre6P synthase and Tre6P phosphatase activities was restricted to 50-60%, the remaining decrease being inhibited by the drug. Furthermore, the reappearance of the enzyme activities following transfer of cells to an acetate medium was blocked by cycloheximide. It was also shown that loss of activity of these two enzymes required a combination of metabolizable sugars together with a nitrogen source. Low activities of Tre6P synthase and Tre6P phosphatase were measured in mutants with increased adenylate cyclase activity (RAS2ala18val19 mutants). Moreover, derepression of these enzymes at the approach of stationary phase was prevented in a pde2 mutant when it was cultivated in the presence of exogenous cyclic nucleotide. The mechanism of this effect is not clear, but may involve a transcriptional regulation by cAMP of the genes encoding these proteins.

Culture Media↗
PubMed1997

Trehalose accumulation in mutants of Saccharomyces cerevisiae deleted in the UDPG-dependent trehalose synthase-phosphatase complex.

In Saccharomyces cerevisiae, trehalose-6-phosphate synthase converts uridine-5'-diphosphoglucose and glucose 6-phosphate to trehalose 6-phosphate which is dephosphorylated by trehalose 6-phosphatase to trehalose. These two steps take place within a complex consisting of three proteins: trehalose-6-phosphate synthase encoded by the GGS1/TPS1 (= FDP1, = BYP1, = CIF1) gene, trehalose 6-phosphatase encoded by the TPS2 gene and by a third protein encoded by both the TSL1 and TPS3 genes. Using three different methods for trehalose determination, we observed trehalose accumulation in ggs1/tps1delta, tps2delta and tsl1delta mutants, and in the double mutants ggs1/tps1delta/tps2delta and also in ggs1/tps1delta deleted mutants suppressed for growth on glucose. All these mutants harbor MAL genes. Trehalose synthesis in these mutants is probably performed by the adenosine-5'-diphosphoglucose-dependent trehalose synthase, (ADPG-dependent trehalose synthase) which was detected in all strains tested. It is noteworthy that trehalose accumulation in these mutants was detected only in cells grown on weakly repressive carbon sources such as maltose and galactose or during the transition phase from fermentable to non-fermentable growth on glucose. alpha-Glucosidase activity was always present in high amounts. We also describe an adenosine-diphosphoglucosepyrophosphorylase (ADPG-pyrophosphorylase) activity in Saccharomyces cerevisiae which increased concomitantly with the accumulation of trehalose during the transition phase from fermentable to non-fermentable growth in MAL-constitutive (MAL2-8c) strains. The same was observed when MAL-induced (MAL1) strains were compared during growth on glucose and maltose. These results led us to conclude that maltose-induced trehalose accumulation is independent of the UDPG-dependent trehalose-6-phosphate synthase/phosphatase complex; that the ADPG-dependent trehalose synthase is responsible for maltose-induced trehalose accumulation probably by forming a complex with a specific trehalose-6-phosphatase activity and that ADPG synthesis is activated during trehalose accumulation under these conditions.

Cell-Free System↗
PubMed1991

The repression of trehalose transport and metabolism in Escherichia coli by high osmolarity is mediated by trehalose-6-phosphate phosphatase.

Trehalose transport and metabolism in Escherichia coli are induced by trehalose in the growth medium but only at low osmolarity. In contrast, synthesis of internal trehalose as an osmoprotectant occurs only at high osmolarity, independent of the carbon source. The synthesis of internal trehalose proceeds via the UDP-glucose-mediated transfer of glucose to glucose-6-phosphate, forming trehalose-6-phosphate, which is then hydrolysed to trehalose. We demonstrate that the inducer for the synthesis of the trehalose transport system as well as of amylotrehalase, the key enzyme in trehalose metabolism at low osmolarity, is trehalose-6-phosphate. We found that the inability to induce these proteins at high osmolarity is primarily due to activity of trehalose-6-phosphate phosphatase, the enzyme responsible for the final step in the synthesis of internal trehalose under these conditions. A gene, otsP, necessary for the synthesis of the biosynthetic trehalose-6-phosphate phosphatase, is located at min 42 closely linked to otsA/B the structural genes for the trehalose-6-phosphate synthase. There is another gene locus near 84 min on the chromosome, that we termed otsR, which is involved in the regulation of otsA/B and possibly otsP. The nature of this regulatory gene is unclear at present.

Biological Transport↗
PubMed2003

NMR and quantum chemical study on the OH...pi and CH...O interactions between trehalose and unsaturated fatty acids: implication for the mechanism of antioxidant function of trehalose.

Trehalose is a disaccharide that attracts much attention as a stress protectant. In this study, we investigated the mechanism of the antioxidant function of trehalose. The spin-lattice relaxation times (T(1)) of (1)H and (13)C NMR spectra were measured to investigate the interaction between trehalose and unsaturated fatty acid (UFA). We selected several kinds of UFA that differ in the number of double bonds and in their configurations (cis or trans). Several other disaccharides (sucrose, maltose, neotrehalose, maltitol, and sorbitol) were also analyzed by NMR. The T(1) values for the (1)H and (13)C signals assigned to the olefin double bonds in UFA decrease with increasing concentration of trehalose and the changes reaches plateaus at integer ratios of trehalose to UFA. The characteristic T(1) change is observed only for the combination of trehalose and UFA with cis double bond(s). On the other hand, from the (13)C-T(1) measurements for trehalose, the T(1) values of the C-3 (C-3') and C-6' (C-6) are found to change remarkably by addition of UFA. (1)H[bond](1)H NOESY measurements provide direct evidence for complexation of trehalose with linoleic acid. These results indicate that one trehalose molecule stoichiometrically interacts with one cis-olefin double bond of UFA. Computer modeling study indicates that trehalose forms a stable complex with an olefin double bond through OH...pi and CH...O types of hydrogen bonding. Furthermore, a significant increase in the activation energy is found for hydrogen abstraction reaction from the methylene group located between the double bonds that are both interacting with the trehalose molecules. Therefore, trehalose has a significant depression effect on the oxidation of UFA through the weak interaction with the double bond(s). This is the first study to elucidate the antioxidant function of trehalose.

Antioxidants↗
PubMed2003

Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and an altered cell wall lipid composition.

The analysis of the available Corynebacterium genome sequence data led to the proposal of the presence of all three known pathways for trehalose biosynthesis in bacteria, i.e. trehalose synthesis from UDP-glucose and glucose 6-phosphate (OtsA-OtsB pathway), from malto-oligosaccharides or alpha-1,4-glucans (TreY-TreZ pathway), or from maltose (TreS pathway). Inactivation of only one of the three pathways by chromosomal deletion did not have a severe impact on C. glutamicum growth, while the simultaneous inactivation of the OtsA-OtsB and TreY-TreZ pathway or of all three pathways resulted in the inability of the corresponding mutants to synthesize trehalose and to grow efficiently on various sugar substrates in minimal media. This growth defect was largely reversed by the addition of trehalose to the culture broth. In addition, a possible pathway for glycogen synthesis from ADP-glucose involving glycogen synthase (GlgA) was discovered. C. glutamicum was found to accumulate significant amounts of glycogen when grown under conditions of sugar excess. Insertional inactivation of the chromosomal glgA gene led to the failure of C. glutamicum cells to accumulate glycogen and to the abolition of trehalose production in a DeltaotsAB background, demonstrating that trehalose production via the TreY-TreZ pathway is dependent on a functional glycogen biosynthetic route. The trehalose-non-producing mutant with inactivated OtsA-OtsB and TreY-TreZ pathways displayed an altered cell wall lipid composition when grown in minimal broth in the absence of trehalose. Under these conditions, the mutant lacked both major trehalose-containing glycolipids, i.e. trehalose monocorynomycolate and trehalose dicorynomycolate, in its cell wall lipid fraction. The results suggest that a dramatically altered cell wall lipid bilayer of trehalose-less C. glutamicum mutants may be responsible for the observed growth deficiency of such strains in minimal medium. The results of the genetic and physiological dissection of trehalose biosynthesis in C. glutamicum reported here may be of general relevance for the whole phylogenetic group of mycolic-acid-containing coryneform bacteria.

Base Sequence↗
PubMed2004

Degradation of trehalose by rhizobia and characteristics of a trehalose-degrading enzyme isolated from Rhizobium species NGR234.

AIMS: This study was designed to examine the breakdown of trehalose by rhizobia and to characterize the trehalose-degrading enzyme isolated from Rhizobium sp. NGR234. METHODS AND RESULTS: Rhizobium sp. NGR234, Rhizobium fredii USDA257, R. phaseoli RCR3622, R. tropici CIAT899 and R. etli CE3 showed good growth in the presence of carbohydrate. Validamycin A did not prevent the growth of NGR234 on trehalose. The expression of a trehalose-degrading enzyme by NGR234 was intracellular and inducible by trehalose. The isolated enzyme digested other disaccharides, p-nitrophenyl-alpha-d-glucopyranoside and the substrate. The enzyme showed optimum activities at pH 7.0 and 30 degrees C. Its pI was 4.75 and the V(max) of the enzyme occurred at 35.7 micromol s(-1) mg(-1) protein with the K(m) of 23 mmol when trehalose was hydrolysed. CONCLUSIONS: An enzyme capable of breaking down trehalose was produced. Some of the properties of the trehalose-degrading enzyme are similar to those isolated from other organisms but, this enzyme was validamycin resistant. These rhizobia like other trehalose-degrading microbes use trehalose by enzymatic catabolic action. SIGNIFICANCE AND IMPACT OF THE STUDY: Trehalose which accumulates during legume-rhizobia symbiosis is toxic to plants. Detoxification by trehalose-degrading enzymes is important for the progress of symbiosis.

Culture Media↗
PubMed1989

Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae.

The properties of yeast trehalose-6-phosphate synthase were reinvestigated in relation with the recent claim made by Panek et al. [Panek, A. C., de Araujo, P. S., Moura-Neto, V. and Panek, A. D. (1987) Curr. Genet. II, 459-465] that the enzyme would be stimulated by ATP and partially inactivated by cAMP-dependent protein kinase. Trehalose-6-phosphate synthase activity was measured by the sum of [14C]trehalose 6-phosphate and [14C]trehalose formed from UDP-[14C]glucose and glucose 6-phosphate. The activity measured in an extract of Saccharomyces cerevisiae was not affected by any treatment of the cells, such as incubation in the presence of glucose or of dinitrophenol, which are known to greatly increase the intracellular concentration of cAMP, nor by preincubation of the extract in the presence of ATP-Mg, cAMP and bovine heart cAMP-dependent protein kinase. The activity was also not significantly different in several mutants affected in the cAMP system. The kinetic properties of the partially purified enzyme were investigated; no effect of ATP could be detected but Pi acted as a potent noncompetitive inhibitor (Ki = 2 mM). The activity of trehalose-6-phosphate phosphatase was measured by the amount of [14C]trehalose formed from [14C]trehalose 6-phosphate. The enzyme could be separated from other phosphatases and appeared to be highly specific for trehalose 6-phosphate. It was Mg dependent and its kinetics for trehalose 6-phosphate was hyperbolic. Studies performed with intact cells, crude extracts or the purified enzyme did not reveal any cAMP-dependent change in its activity. Remarkably, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase copurified in the course of different chromatographic procedures, suggesting that they are part of a single bifunctional protein. A 50-fold purification of the two enzymes could be achieved with a yield of only 2% by chromatography on Mono S followed by gel filtration on Superose 6B.

Chromatography↗
PubMed1994

Synthesis of 4,6:2',3':4',6'-tri-O-cyclohexylidene-alpha,alpha'-trehalose 2-palmitate: an intermediate for the synthesis of mycobacterial 2,3-di-O-acyl-alpha,alpha'-trehalose antigens.

The aim was to 'triprotect' trehalose by placing various acetals, or related protecting groups, across the 4,6-, 2',3'-, and 4',6'-positions, leaving the 2,3-positions free for subsequent acylation. Isopropylidene and ethylidene acetals were studied, with the formation of a small amount of 4,6:2',3':4',6'-tri-O-isopropylidene-alpha,alpha'-trehalose. 4,6:4',6'-Di-O-benzylidene-2',3'-O-(tetraphenyldisiloxane-1,3-d iyl)-alpha, alpha'-trehalose 2,3-diacetate was prepared in low yield. 1,1-Dimethoxycyclohexane reacted with methyl alpha-D-glucopyranoside to afford the 4,6-O-cyclohexylidene derivative, isolated as the diacetate; mild acid cleavage of the acetal gave the 2,3-diacetate. 4,6:2',3':4',6'-Tri-O-cyclohexylidene-alpha,alpha'-trehalose is the major product of the reaction between alpha,alpha'-trehalose and 1,1-dimethoxycyclohexane. 2,3:4,6:2',3':4',6'-Tetra-O-cyclohexylidene-, 4,6:4',6'-di-O-cyclohexylidene-, and 4,6-O-cyclohexylidene-alpha,alpha'-trehaloses were also isolated in lower yields, all acetals being characterised as their peracetates. The proportions of the different trehalose acetals were dependent upon the molar ratio of 1,1-dimethoxycyclohexane and particularly on the reaction temperature. The triprotected trehalose acetal was acylated with palmitic acid, with excellent regioselectivity, affording the 2-O-palmitoyl ester. This 2-monoacylated, triprotected trehalose is a key intermediate for the synthesis of 2,3-di-O-acyl-alpha,alpha'-trehalose glycolipid antigens, isolated from Mycobacterium fortuitum and Mycobacterium tuberculosis.

Acylation↗
PubMed1994

The role of trehalose synthesis for the acquisition of thermotolerance in yeast. I. Genetic evidence that trehalose is a thermoprotectant.

In the yeast Saccharomyces cerevisiae, accumulation of the non-reducing disaccharide trehalose is triggered by various stimuli that activate the heat-schock response. Several studies have shown a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. In this study, we have examined mutants defective in genes coding for key enzymes involved in trehalose metabolism with respect to the heat-induced and stationary-phase-induced accumulation of trehalose and the acquisition of thermotolerance. Inactivation of either TPS1 or TPS2, encoding subunits of the trehalose-6-phosphate synthase/phosphatase complex, caused an inability to accumulate trehalose upon a mild heat-shock or upon initiation of the stationary phase and significantly reduced the levels of heat-induced and stationary-phase-induced thermotolerance. Deletion of NTH1, the gene coding for the neutral trehalase, resulted in a defect in trehalose mobilization during recovery from a heat shock which was paralleled by an abnormally slow decrease of thermotolerance. Our results provide strong genetic evidence that heat-induced synthesis of trehalose is an important factor for thermotolerance induction. In an accompanying study [Hottiger, T., De Virgilio, C., Hall, M. N., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 187-193], we present evidence that the function of heat-induced trehalose accumulation may be to increase the thermal stability of proteins.

Acclimatization↗
PubMed2004

Role of trehalose phosphate synthase and trehalose during hypoxia: from flies to mammals.

Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage. The best known and most widely distributed pathway of trehalose synthesis involves the transfer of glucose from UDP-glucose to glucose 6-phosphate to form trehalose-6-phosphate and UDP via the trehalose-6-phosphate synthase (TPS1). Trehalose-6-phosphate phosphatase (TPS2) then converts trehalose-6-phosphate to free trehalose. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates and plants, and because of its particular physical features, trehalose is able to protect the integrity of cells against a variety of environmental stresses such as desiccation, dehydration, heat, cold and oxidation. Our current studies described here indicate that trehalose protects Drosophila and mammalian cells from hypoxic and anoxic injury. The mechanism of this protection is probably related to a decrease in protein denaturation through protein-trehalose interactions.

Animals↗
PubMed1999

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.

Journal Article↗
PubMed2001

Overexpression of trehalose synthase and accumulation of intracellular trehalose in 293H and 293FTetR:Hyg cells.

A humanized clone containing the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase (otsA/B) has been constructed. Using the Gateway Cloning System (Invitrogen, Inc.), the otsA/B genes have been placed under the control of the CMV promoter (pEXPcmv-otsA/B) or the CMV promoter and the tet operator (pEXP cmv TetO-otsA/B). The pEXPcmv-otsA/B clone has been introduced into 293H cells using LIPOFECTAMINE 2000 and the intracellular concentration of trehalose has been evaluated. The 293H cells accumulate 4-5 microg trehalose/mg dry weight and this concentration increases to 7-10 microg trehalose/mg dry weight if trehalose is included in the growth medium. The pEXPcmv TetO-otsA/B clone has been transfected into 293FTetR:Hyg cells which contain the tet repressor integrated into the genome. When these transfected cells are grown in the absence of tetracycline, no intracellular trehalose is detected. Inclusion of 0.3 microg/ml tetracycline in the growth medium results in the accumulation of 11-14 microg trehalose/mg dry weight, a value which increases to 19-20 microg trehalose/mg dry weight if trehalose is included in the growth medium. The data for the 293FTetR:Hyg cells indicate that intracellular trehalose accumulates in response to the addition of tetracycline. This system will allow us to manipulate the intracellular concentration of trehalose and to evaluate the desiccation tolerance of these cells as a function of intracellular trehalose concentration.

Cell Line↗