Utilization Of Trehalose Research Articles

Induction of the lambda receptor is essential for effective uptake of trehalose in Escherichia coli

Trehalose transport in Escherichia coli after growth at low osmolarity is mediated by enzyme IITre of the phosphotransferase system (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). The apparent Km (16 microM) of trehalose uptake is low. Since trehalose is a good source of carbon and the apparent affinity of the uptake system is high, it was surprising that the disaccharide trehalose [O-alpha-D-glucosyl(1-1)-alpha-D-glucoside] has no problems diffusing through the outer membrane at high enough rates to allow full growth, particularly at low substrate concentrations. Here we show that induction of the maltose regulon is required for efficient utilization of trehalose. malT mutants that lack expression of all maltose genes, as well as lamB mutants that lack only the lambda receptor (maltoporin), still grow on trehalose at the usual high (10 mM) trehalose concentrations in agar plates, but they exhibit the half-maximal rate of trehalose uptake at concentrations that are 50-fold higher than in the wild-type (malT+) strain. The maltose system is induced by trehalose to about 30% of the fully induced level reached when grown in the presence of maltose in a malT+ strain or when grown on glycerol in a maltose-constitutive strain [malT(Con)]. The 30% level of maximal expression is sufficient for maximal trehalose utilization, since there is no difference in the concentration of trehalose required for the half-maximal rate of uptake in trehalose-grown strains with the wild-type gene (malT+) or with strains constitutive for the maltose system [malT(Con)]. In contrast, when the expression of the lambda receptor is reduced to less than 20% of the maximal level, trehalose uptake becomes less efficient. Induction of the maltose system by trehalose requires metabolism of trehalose. Mutants lacking amylotrehalase, the key enzyme in trehalose utilization, accumulate trehalose but do not induce the maltose system.

Biotypes of the dermatophyte Microsporum distortum

During a study of the epidemiology of Microsporum canis and closely related species, sub-specific variation was demonstrated within thirteen isolates of Microsporum distortum based on the pattern of carbohydrate utilization. Strains from three continents could be differentiated by their utilization of trehalose and erythritol. Isolates from New Zealand yielded the same patterns as M. canis (Arthroderma otae (-)); examination of 120 isolates indicated that M. canis could not be biotyped by carbohydrate utilization, although limited variation had been noted. Examination of total native protein patterns by gel electrophoresis showed that variations existed within the M. distortum group; the much larger number of M. canis (A. otae (-)) strains had appeared to be homogenous.

Trehalose transport and metabolism in Escherichia coli

Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITre/IIIGlc)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EIITre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate- and IITre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.

Analysis and DNA sequence of the osmoregulated treA gene encoding the periplasmic trehalase of Escherichia coli K12.

The treA gene of Escherichia coli K12 codes for a periplasmic trehalase that is induced by growth at high osmolarity. The position of treA within a cloned chromosomal DNA fragment was identified by subcloning of restriction fragments and analysis of the gene product in minicells. The nucleotide sequence of the treA coding region as well as its upstream control region was determined. The treA gene consists of 1695 bp encoding 565 amino acids. The amino-terminus of the mature trehalase was found to begin with the amino acid Glu at position 31 of the open reading frame. The first 30 amino acids resemble a typical signal sequence, consistent with trehalase being a secreted periplasmic enzyme. Two previously isolated phoA fusions to the osmA gene were transferred by homologous recombination on to a treA-containing plasmid and found to be within treA. Analysis of the hybrid genes and their gene products aided the localization of treA and the determination of its direction of transcription within the cloned chromosomal segment. The treA-phoA fusions encoded hybrid proteins which could be found in the periplasm. We found that at high osmolarity the normal pathway for the uptake and utilization of trehalose is blocked. Therefore, the function of the periplasmic trehalase is to provide the cell with the ability to utilize trehalose at high osmolarity by splitting it into glucose molecules that can subsequently be taken up by the phosphotransferase-mediated uptake system.

Anhydrobiosis in Embryos of the Brine Shrimp Artemia: Characterization of Metabolic Arrest During Reductions in Cell-Associated Water

ABSTRACT Upon entry into the state of anhydrobiosis, trehalose-based energy metabolism is arrested in Artemia embryos (cysts). We have compared changes in the levels of trehalose, glycogen, some glycolytic intermediates and adenylate nucleotides in hydrated embryos observed under conditions of aerobic development with those occurring after transfer to 5·0mol 1−1 NaCl. This treatment is known to reduce cell-associated water into a range previously referred to as the ametabolic domain. The trehalose utilization and glycogen synthesis that occur during development of fully hydrated cysts are both blocked during desiccation. Upon return to 0·25 mol 1−1 NaCl both processes are resumed. Analysis of glycolytic intermediates suggests that the inhibition is localized at the trehalase, hexokinase and phosphofructokinase reactions. ATP level remains constant during the 6-h period of dehydration, as does the adenylate energy charge. An additional dehydration experiment was performed in 5·0moll−1 NaCl containing 50mmoll−1 ammonium chloride (pH9·0). The resulting level of gaseous ammonia in the medium has been shown to maintain an alkaline intracellular pH (pHi) in the embryos. The metabolic response to dehydration under these conditions was very similar to the previous dehydration series. Thus, these results are taken as strong evidence that the metabolic suppression observed during dehydration does not require cellular acidification, in contrast to the pronounced inhibitory role of low pHi during entry of hydrated embryos into the quiescent state of anaerobic dormancy. The arrest of carbohydrate metabolism seen during anhydrobiosis indeed appears to be a strict function of embryo water content.

Regulation of flight related trehalose utilization in the locust Locusta migratoria

1. 1. Trehalose dynamics were studied in haemolymph and flight muscles of flying locusts, after pulse labelling of the haemolymph pool with [U- 14C]-trehalose. 2. 2. A mathematical model was developed to enable the calculation of rate-determining constants of trehalose utilization and the time of flight needed to reach a steady-state condition of trehalose utilization. 3. 3. The results indicate that an active down regulation of trehalose utilization during flight is effected both at the sarcolemma and intracellularly in the flight muscles.

Trehalase polymorphism in Drosophila melanogaster.

The disaccharide trehalose is a major substrate for energy production and macromolecular synthesis in many insects (Friedman, 1978). As the principle hemolymph sugar, the utilization of trehalose and the maintenance of hemolymph trehalose concentrations have been implicated in a diversity of physiological processes, including flight metabolism (Clegg and Evans, 1961), cold tolerance (Wyatt, 1967), and chitin synthesis during moulting (Candy and Kilby, 1962). Catabolic utilization of trehalose in insect tissues is initiated by the enzyme trehalase (a,a-glucoside-l-glucohydrolase; EC 3.2.1.28), which catalyzes the hydrolysis of trehalose into two glucose moieties. This enzyme has a broad distribution among insect tissues and may occur as more than one isozyme (Friedman, 1975; Yanagawa, 1971). Talbot and Huber (1976) found that abdominal and thoracic trehalases in Drosophila melanogaster differed in pH optima and electrophoretic mobility, although the existence of distinct trehalase isozymes in this species has been disputed (Oliver et al., 1978; Bargiello and Grossfield, 1979). One approach to clarifying the dispute concerning multiple trehalase isozymes is to map genetically the structural gene(s) involved. Using segmental aneuploidy and trehalase activity assays, Oliver et al., (1978) found evidence for only a single trehalase structural gene (Treh) in D. melanogaster. They mapped Treh to the right arm of the second chromosome, in the region of bands 55B-E. Because no electrophoretically detectable trehalase allelic variation has been previously reported in D. melanogaster (Bargiello and

Accumulation of alpha,alpha-trehalose by Rhizobium bacteria and bacteroids.

Four strains of Rhizobium japonicum (61A76 and USDA 110, 123, and 138) were grown in eight different defined media. Regardless of the carbon or nitrogen source supplied, alpha, alpha-trehalose was the major carbohydrate (among mono- and disaccharides) accumulated by all four strains. After 7 to 9 days of growth, trehalose generally accounted for 90 to 100% of the mono- and disaccharides detected. None of the four strains would grow with trehalose as a carbon source, but the utilization of endogenous trehalose was demonstrated under carbon starvation conditions in water culture or when the carbon supply in a defined medium was exhausted. Under these conditions, a small amount of trehalose was lost from cells to the medium. In a survey of most of the serogroups of R. japonicum and several strains of other Rhizobium species, all strains tested were found to accumulate some trehalose. Trehalose concentrations varied widely; the highest concentration recorded was 41 micrograms/mg of dry weight. In all but six strains trehalose accounted for greater than 80% of the mono- and disaccharides in cells. Fast-growing strains of R. japonicum also accumulated small amounts trehalose. R. japonicum bacteroids also synthesized trehalose; the quantity in nodules varied in approximate correspondence to accumulation of trehalose by cultured bacteria. In young soybean nodules (29 days after planting), 45 to 80% of the trehalose was recovered in the cytosol. There were differences among R. japonicum strains in the retention of trehalose, and the proportion of trehalose retained by bacteroids increased with increasing plant age for all strains.

Cellular localization and proposed function of midguttrehalase in the silkworm larva, Bombyx mori

A sorbitol density gradient analysis with the aid of several marker enzymes demonstrated that midgut trehalase of the silkworm larvae. Bombyx mori, was localized in the microsomal membranes, but not in mitochondria, lysosomes and microvilli at the apical surface. Electron microscopic examination showed that trehalase-enriched membrane fraction consisted of heterogeneous mixtures of membrane vesicles derived from the endoplasmic reticulum and plasma membrane parts other than the microvillus membrane. The enzyme-histochemical stains of trehalase activity on the midgut section could be detected only at the basal surface of the epithelium against haemocoel. Such a specific localization was further confirmed by immunohistochemistry with the peroxidase-conjugated antibody technique. Thus, it is concluded that midgut trehalase of silkworm larvae is situated on the plasma membrane at the basal surface of the epithelium. An intact preparation of midgut incubated in vitro in the medium containing [14C]trehalose could hydrolyse trehalose into glucose and take it up into the cell, although some glucose was liberated into the medium when incubated for extended periods. These results suggest that midgut trehalase plays a physiological role in utilization of haemolymph trehalose not in nutrient absorption.

Enzymes of sucrose, maltose, and ?,?-trehalose catabolism in soybean root nodules

Crude, Sephadex-filtered extracts of soybean (Glycine max (L.) Merr.) root nodules contained invertase (E.C. 3.2.1.26) activity with pH optima at 5.4 and 7.8, α,α-trehalase (E.C. 3.2.1.28) activity with pH optima at 3.8 and 6.6, and maltase (E.C. 3.2.1.20) activity with a broad pH optimum between 4.5 and 5.0. Bacteroids and cytosol were separated using Percoll density gradients. Cellulase and pectinase were employed to separate protoplasts from the infected region from the nodule cortex, which remained intract. Assays of disaccharidases from these nodule fractions indicated the following localization of enzymes: (1) Bacteroids lack invertase activity (pH 5.4 and 7.8). (2) Much, if not most, of the invertase activity may be localized in the nodule cortex; this is especially likely for acid invertase. However, there was substantial invertase activity in cytosol from the infected region. (3) Most of the maltase activity (pH 5.0) and trehalase activity (pH 3.8 and 6.6) were localized in the cytosol. It is likely that most of these disaccharidase activities are in the cytosol of the infected region, in contrast to invertase. (4) Bacteroids contain maltase (pH 5.0) and trehalase (pH 3.8 and 6.6), but the amount of these enzyme activities was less than 15% of total activity in nodules. Bacteroids and nodule cortex were capable of in-vivo hydrolysis of [(14)C]trehalose and [(14)C]maltose. These disaccharides were also hydrolyzed by soybean roots and hypocotyls. Therefore, while α,α-trehalose in soybean nodules is probably synthesized by the bacteroids, the capability for utilization of trehalose was not restricted to the bacteroids.

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.

Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation.

The amounts of glycogen and trehalose have been measured in cells of a prototrophic diploid yeast strain subjected to a variety of nutrient limitations. Both glycogen and trehalose were accumulated in cells deprived specifically of nirogen, sulfur, or phosphorus, suggesting that reserve carbohydrate accumulation is a general response to nutrient limitation. The patterns of accumulation and utilization of glycogen and trehalose were not identical under these conditions, suggesting that the two carbohydrates may play distinct physiological roles. Glycogen and trehalose were also accumulated by cells undergoing carbon and energy limitation, both during diauxic growth in a relatively poor medium and during the approach to stationary phase in a rich medium. Growth in the rich medium was shown to be carbon or energy limited or both, although the interaction between carbon source limitation and oxygen limitation was complex. In both media, the pattern of glycogen accumulation and utilization was compatible with its serving as a source of energy both during respiratory adaptation and during a subsequent starvation. In contrast, the pattern of trehalose accumulation and utilization seemed compatible only with the latter role. In cultures that were depleting their supplies of exogenous glucose, the accumulation of glycogen began at glucose concentrations well above those sufficient to suppress glycogen accumulation in cultures growing with a constant concentration of exogenous glucose. The mechanism of this effect is not clear, but may involve a response to the rapid rate of change in the glucose concentration.

Trehalases from the American cockroach,Periplaneta americana:multiple occurrence of the enzymes and partial purification of enzymes from male accessory glands

Trehalase activities were found in several tissues of the adult American cockroach, Periplaneta americana. Among these, male accessory glands, fat body, thoracic muscle, hepatic cecum, blood and mid-gut contained high trehalase activity; activity in the male accessory gland was especially high. The enzymic properties of soluble trehalases were investigated and the enzymes from the male accessory gland were highly purified. The properties of these enzymes were electrophoretically and kinetically distinct from each other. The presence of enzymes with somewhat different properties in different tissues suggests that trehalose utilization and trehalase activity may be regulated by way of a tissue-specific mechanism. The detailed properties of these enzymes are presented with a discussion of their regulation.

Salmonella typhimurium mutants lacking protease II

Mutants of Salmonella typhimurium lacking protease II, an endoprotease with trypsin-like specificity, have been isolated. These mutants can be identified by using the chromogenic substrate N-methyl-N-p-toluenesulfonyl-L-lysine beta-naphthyl ester to screen colonies growing on agar for the presence of the enzyme. All of the mutations isolated map at locus tlp (typsin-like protease) which is cotransducible (approximately 1%) using phage P1 with tre (trehalose utilization) at approximately 58 min on the Salmonella map. Double mutants lacking both protease I and protease II have been constructed. These strains grew normally. They were able to degrade abnormal proteins and to carry out protein turnover during carbon starvation at the same rate as the wild type.

Interrelationships between water and metabolism in Artemia cysts—II. carbohydrates

Abstract 1. Evidence is presented that cysts of the brine shrimp, Artemia salina, initiate the utilization of trehalose and the synthesis of glycerol and glycogen only after hydration levels of 0.65 g H2O/g CaSO4-dried cysts have been achieved. 2. The ability of embryos to emerge from their shells was also correlated with this level of hydration. 3. The viability of embryos emerging in the vapor phase of NaCl solutions was examined.

Trehalose metabolism in germinating spores of Streptomyces hygroscopicus.

Evidence is presented which indicates that utilization of trehalose is an early event in spore germination in Streptomyces hygroscopicus. Early in spore germination and before any increase in cell mass, the activity of trehalase increased more than 15-fold, while the intracellular content of trehalose fell to very low levels. On the other hand, increased activity of the trehalose phosphate synthetase or disappearance of glycogen did not occur until later in germination, during which time cell mass was also increasing.

Germination and outgrowth of single spores of Saccharomyces cerevisiae viewed by scanning electron and phase-contrast microscopy.

Single spores of Saccharomyces cerevisiae were examined during germination and outgrowth by scanning electron and phase-contrast microscopy. Also determined were changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. These studies reveal that development of the vegetative cell from a spore follows a definite sequence of events involving dramatic physical and chemical modifications. These changes are: initial rapid loss in cellular absorbance followed later by an abrupt gain in absorbance; reduction in cell weight and a subsequent progressive increase; modification of the spore surface with concomitant diminution in refractility; elongation of the cell and augmentation of surface irregularities; rapid decline in trehalose content of the cell accompanied by extensive formation of KOH-soluble carbohydrates; and bud formation.

Properties of an inhibitor of trehalase in trehalaseless mutants of Neurospora.

THE means whereby trehalose metabolism is controlled are of considerable interest because of the ubiquity of this sugar and its potential role as a source of energy during important developmental events. Thus, trehalose and its hydrolase, trehalase (EC 3.2.1.28, α,α-glucoside 1-glucohydrolase), are found in a wide range of organisms including fungi1,2, insects3,4 and several other groups5–7. In Neurospora crassa2 the utilization of trehalose is correlated with the breaking of dormancy of ascospores and a similar correlation exists in diapausing insects6. We have therefore studied isozymes in standard strains of Neurospora8–10 and have isolated trehalaseless mutants from which we have extracted a specific inhibitor of trehalase11.

The fuel for sustained mosquito flight

Males and females of Aedes sollicitans and Aedes taeniorhynchus were flown on a flight mill and analysed for duration and distance of flight, and for utilization of triglycerides, glycogen, and trehalose. Unfed, blood-fed, and glucose-fed mosquitoes were flown either to exhaustion or for 1 hr. Triglycerides and other lipids were not used for flight. Utilization of carbohydrate was 0·08 to 0·10 cal/1000 m or 27 to 37 cal/hr per g, whether calculated from the utilization of glycogen (in unfed and blood-fed mosquitoes) or from the production of carbon dioxide (in glucose-U- 14C fed mosquitoes). The maximum metabolic rate during flight was four to five times that of non-fed controls. In sugar-fed mosquitoes, glycogen and triglycerides accumulated during flight. Glycogen was not utilized as a flight substrate as long as sugar was available. Starved mosquitoes and mosquitoes flown to exhaustion could resume vigorous flight immediately after a sugar meal. Trehalose did not change during vigorous flight and made a negligible contribution to exhaustive flight. In unfed mosquitoes, the flight following a rest period after exhaustion was sustained by residual glycogen; no increase in glycogen took place while resting. The significance of these results to migration and dispersal in mosquitoes is discussed.

The utilization of trehalose during flight by the housefly, Musca domestica

The male housefly, Musca domestica, utilizes trehalose during flight. However, the rate of utilization of treahlose is most rapid during the first few minutes of continuous flight (i.e. during the first 5 min of flight, the rate of utilization of trehalose is 187 μg/thorax per hr; this results in a thoracic trehalose level of one-third of that of the unflown fly, after 5 min of flight). However, as the period of flight is extended, the apparent rate decreases very rapidly, so that the thoracic trehalose level actually continues to rise with increasing duration of flight period. It is concluded that, following initial rapid utilization of trehalose, a secondary metabolic pool becomes implicated, so as to restore (and maintain) the thoracic trehalose levels at as high as 50 per cent of that of unflown flies, for thoraces of flies which have been permitted to fly for as long as 4·5 hr. Flight-exhausted flies, when fed on a solution of glucose, fructose, maltose, sucrose, and trehalose, resumed flight, without external stimulation, but feeding galactose, mannose, and cellobiose failed to do so. However, injection of solutions of glucose, fructose, sucrose, and trehalose did not initiate flight in such flight-exhausted flies. These data indicate that a complex, metabolic route is normally involved in the energizing of flight.