Degradation of the compatible solute trehalose in Ectothiorhodospira halochloris: isolation and characterization of trehalase

Trehalase, which hydrolyzes the disaccharide trehalose to -d-glucose was isolated and partially purified (124-fold) from the phototrophic halo-alkaliphilic bacterium Ectothiorhodospira halochloris. The molecular mass was determined to be 480,000 and the isoelectric point pH 5.6. Temperature optimum was found to be 40°C and the pH-optimum 7.8–8.1. In spite of its high K _m-value of 0.5 M, trehalase of E. halochloris was shown to be specific for trehalose. Trehalase is activated by phosphate which is, however, not involved in the reaction mechanism. The enzyme is activated by the compatible solute betaine and inhibited by salts. In the presence of betaine the K _m-value is lowered from 0.5 M to 0.16 M; moreover, betaine partially protects enzymatic activity from salt inhibition. The findings indicate that betaine might regulate the trehalose level in the cells by affecting trehalase activity.     

The role of trehalose as a substitute for nitrogen-containing compatible solutes (Ectothiorhodospira halochloris)

The halophilic phototrophic bacterium Ectothiorhodospira halochloris is able to synthesize both nitrogen-containing (betaine, ectoine) and nitrogen-free (trehalose) compatible solutes. In the absence of external ammonium and under nitrogen-limited growth conditions ectoine was metabolized and trehalose partly replaced betaine. The cytoplasmic trehalose concentration did not exceeded 0.5 mol/kg water (approx. 30% of total compatible solutes). A decreasing content of betaine in cells growing under nitrogen limitation is a result of decreased biosynthesis. Apparently, the betaine pool cannot be used as a nitrogen source, not even in a situation of total nitrogen depletion.     

Utilization of sulfide and elemental sulfur by Ectothiorhodospira halochloris

Ectothiorhodospira halochloris grows photoheterotrophically with a variety of sulfur sources. During sulfide oxidation to elemental sulfur considerable amounts of polysulfides may be accumulated transiently. When grown on elemental sulfur no sulfate was produced by oxidation, but sulfide and polysulfide were formed by reduction. Only one soluble cytochrome c-551 was isolated and purified. It was a small acidic hemeprotein with a molecular weight of 6,300, an isoelectric point of 3.1 and a redox potential of-11 mV at pH 7.0. It showed three absorption maxima in the reduced state (=551 nm; =523 nm; =417 nm). The addition of various c-type cytochromes to a suspension of spheroplasts stimulated the velocity of sulfide oxidation. This stimulation was best with the small acidic cytochromes from E. halochloris or Ectothiorhodospira abdelmalekii. Sulfide oxidation was stopped by several uncoupling agents, ionophores and electron transport inhibitors. Antimycin A, rotenone and cyanide had no effect on sulfide oxidation.Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

Fate of compatible solutes during dilution stress in Ectothiorhodospira halochloris

Abstract Ectothiorhodospira halochloris reacts upon enhancement of the water activity in the environment by excreting its major compatible solute, glycine betaine, thus decreasing the osmotic pressure inside the cell. A suddenly induced dilution stress leads to an overshoot of this reaction, so that more glycine betaine than necessary to compensate the external osmotic change is released. Subsequently the cells take up glycine betaine until they reach osmotic balance with the medium. E. halochloris possesses an active transport system that allows an uptake of glycine betaine against a concentration gradient. Glycine betaine is not metabolized in E. halochloris . Ectoine, a minor compatible solute of E. halochloris , is excreted in a similar manner to that of glycine betaine during dilution stress, whereas no excretion of the third compatible solute, trehalose, was detected.     

Ectothiorhodospira halochloris sp. nov., a new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b

A new bacteriochlorophyll b containing phototrophic bacterium was isolated from extremely saline and alkaline soda lakes in Egypt. Enrichment and isolation were performed using a synthetic medium with high contents of sodium carbonate, sodium sulfate and sodium chloride. Photoautotrophic growth occurred with hydrogen sulfide as photosynthetic electron donor. During oxidation of sulfide to sulfate extracellular elemental sulfur globules appeared in the medium. Cells were also capable to grow under photoheterotrophic conditions with acetate, propionate, pyruvate, succinate, fumarate or malate as carbon sources and electron donors. Under these conditions sulfate was assimilated. Optimal growth under the applied experimental conditions occurred at a total salinity of 14–27%, a pH-range between 8.1 and 9.1 and a temperature between 47°C and 50°C. The cells were 0.5–0.6 m wide and, depending on cultural conditions, 2.5–8.0 m long; they were spiral shaped, multiplied by binary fission and were motile by means of bipolar flagella. Intercytoplasmic photosynthetic membranes were present as stacks. Bacteriochlorophyll b was the main photosynthetic pigment; small amounts of carotenoids were mainly present as glucosides of rhodopin and its methoxy derivative. The new organism is described as Ectothiorhodospira halochloris.Dedicated to Professor C. B. van Niel on the occasion of his 80th birthday     

Metabolic function of glycogen phosphorylase and trehalose phosphorylase in fruit-body formation ofFlammulina velutipes

Glycogen phosphorylase in the vegetative mycelium ofFlammulina velutipes converts glycogen to α-glucose 1-phosphate (G1P) in the colony during fruit-body development. Glycogen may contribute to the synthesis of trehalose as the starting material in the vegetative mycelium during the fruiting process of the colony, and the trehalose produced is translocated into the fruit-bodies as the main carbohydrate substrate for their development. Trehalose phosphorylase activity in the vegetative mycelium was at a relatively high level until fruit-body initiation, suggesting the turnover of this disaccharide during the vegetative stage of the colony development. Trehalose phosphorylase activity in the stipes showed a peak level at the early phase of fruit-body development, suggesting the continuing phosphorolysis of trehalose by this enzyme. The stipes also showed a high specific activity of phosphoglucomutase at a sufficient level to facilitate the conversion of G1P to α-glucose 6-phosphate (G6P). In the pilei a large amount of G1P remained until the growth of the fruit-bodies ceased. Trehalase activities in the stipes and pilei were at a very low level, and this enzyme may not contribute to the catabolism of trehalose in the fruit-body development.     

嗜盐菌中甘氨酸甜菜碱的合成途径及其生物学功能

在嗜盐菌长期的盐适应或短期的盐胁迫过程中,甘氨酸甜菜碱(又名三甲基甘氨酸,N,N,N-trimethylglycine)发挥着极为重要的作用。甘氨酸甜菜碱在嗜盐菌中的生物合成有2种途径:胆碱氧化途径和甘氨酸甲基化途径。前者以胆碱为底物,由胆碱脱氢酶(cholinedehydrogenase,BetA)和甜菜碱乙醛脱氢酶(betaine aldehyde dehydrogenase,BetB)经2次氧化生成甜菜碱;后者以甘氨酸作为底物,由甘氨酸肌醇甲基转移酶(glycine sarcosine N-methyltransferase,GSMT)和肌氨酸二甲基甘氨酸甲基转移酶(sarcosine dimethylglycine N-methyltransferase,SDMT)经3次N-甲基化生成甜菜碱。目前在JGI-IMG和EZBioCloud数据库中公布了134株嗜盐菌标准菌株的全基因组序列。其中,约56.0%的嗜盐细菌和约39.6%的嗜盐古菌拥有胆碱氧化途径所需的2个基因;约9.7%的嗜盐细菌和约0.7%的嗜盐古菌携带甲基化途径所需的2个基因。其中,8株嗜盐细菌同时拥有胆碱氧化途径和甘氨酸甲基化途径所需的全部基因。甘氨酸甜菜碱生物合成基因在典型微生物菌株或经济作物中的表达可以提高其耐盐抗逆能力,这种独特的优势已经引起科学家们强烈的兴趣,相信未来,嗜盐菌中甘氨酸甜菜碱生物合成领域内的科学理论和技术应用会有重大的突破。     

Improved long-term cryostorage of Escherichia colicompetent cells using trehalose

Trehalose added to storage buffers for chemical-induced transformations at 200 mm increased the survival of Escherichia coli competent cells 1.34.8-fold over 180 d cryostorage compared with dimethyl sulfoxide or glycerol.     

Osmoregulation in the model organismEscherichia coli: genes governing the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress tolerance

Glycine betaine is known to be the preferred osmoprotectant in many bacteria, and glycine betaine accumulation has also been correlated with increased cold tolerance. Trehalose is often a minor osmoprotectant in bacteria and it is a major determinant for desiccation tolerance in many so-called anhydrobiotic organisms such as baker's yeast(Saccharomyces cerevisiae). Escherichia coli has two pathways for synthesis of these protective molecules; i.e., a two-step conversion of UDP-glucose and glucose-6-phosphate to trehalose and a two-step oxidation of externally-supplied choline to glycine betaine. The genes governing the choline-to-glycine betaine pathway have been studied inE. coli and several other bacteria and higher plants. The genes governing UDP-glucose-dependent trehalose synthesis have been studied inE. coli andS. cerevisiae. Because of their well-documented function in stress protection, glycine betaine and trehalose have been identified as targets for metabolic engineering of stress tolerance. Examples of this experimental approach include the expression of theE. coli betA andArthrobacter globiformis codA genes for glycine betaine synthesis in plants and distantly related bacteria, and the expression of theE. coli otsA and yeastTPS1 genes for trehalose synthesis in plants. The published data show that glycine betaine synthesis protects transgenic plants and phototrophic bacteria against stress caused by salt and cold. Trehalose synthesis has been reported to confer increased drought tolerance in transgenic plants, but it causes negative side effects which is of concern. Thus, the much-used model organismE. coli has now become a gene resource for metabolic engineering of stress tolerance.     

Levels of trehalose and glycogen inArthrobacter globiformis under conditions of nutrient starvation and osmotic stress

Cells ofArthrobacter globiformis grown in carbohydrate-rich media were found to contain large quantities of low-M_r carbohydrates (800 g/mg protein) and only small amounts of amino acids, in addition to high amounts of glycogen (2 mg/mg protein). At increasing osmotic values of the medium, low-M_r carbohydrate levels increased to 1300 g/mg protein. Low-M_r pools were extracted from the cells with hot 75% ethanol, and subjected to thin layer, gel and gas-liquid chromatography. They turned out to consist mainly of ,-trehalose. Levels of trehalose inArthrobacter cells have the tendency to remain constant, both during nutrient exhaustion (resulting in glycogen consumption), and on addition of excess of carbon source to the medium (resulting in an increased glycogen content of the cells). The stress-tolerant properties ofArthrobacter (resistance to nutrient starvation, desiccation and high salt concentration) are discussed with respect to the high glycogen and trehalose contents of the cells.     

Covalent linkage of N-methyl-6-oxyquinolinium betaine to trehalose

The common route to link quinolinium and pyridinium fluorophores to biomolecules via bromoacetic acid has failed in labeling the disaccharide trehalose with N-methyl-6-oxyquinolinium betaine: the unexpected, extremely high instability of the N-carboxymethyl ester was overcome by direct N-alkylation of the quinoline derivative with trehalose triflate.     

Comparative analysis of glycogen and trehalose accumulation in methylotrophic and nonmethylotrophic yeasts

Trehalose and glycogen accumulate in certain yeast species when they are exposed to unfavorable growth conditions. Accumulations of these reserve carbohydrates in yeasts provide resistance to stress conditions. The results of this study indicate that certain Pichia species do not accumulate high levels of glycogen and trehalose under normal growth conditions. However, depending on the Pichia species, both saccharides accumulate at high levels when the Pichia cells are exposed to unfavorable or stress-inducing growth conditions. Growth in glycerol or methanol medium mostly led to trehalose accumulation in Pichia species tested in this study. It was shown that the metabolic pathways for glycogen and trehalose biosynthesis are present in Pichia species. However, it appears that the biosynthesis of trehalose and glycogen may be regulated in different manners in Pichia species than in the yeast S. cerevisiae. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 6, pp. 737–741. The text was submitted by the author in English.     

Quantification of trehalose in biological samples with a conidial trehalase from the thermophilic fungus Humicola grisea var. thermoidea

The partially-purified, thermally-stable trehalase from conidia of Humicola grisea was highly specific for trehalose and was free of potentially interfering activities. The enzyme was fully stable when stored in solution at -15°C for at least 6 months. This preparation could be used to quantify trehalose from 0.05 to 1.25 mol/ml either in carbohydrate mixtures or in complex biological materials.M.J. Neves, H.F. Terenzi and J.A. Jorge are with Departmento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirao Preto, Universidade de São Paulo, 14040-901-Ribeirão Preto, São Paulo, Brazil; F.A. Leone is with Departamento de Quimica, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901-Ribeirão Preto, São Paulo, Brazil.     

Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis

Biochemical characterization of a trehalase, detected in the mid-exponential growth phase of Candida utilis NCIM Y500, has indicated that it was a neutral trehalase and possibly the only trehalase present in this strain. Unlike Saccharomyces cerevisiae and other C. utilis strains, this strain without acid trehalase grew quite well in minimal or complete medium containing trehalose as the sole source of carbon. Both these observations were contradictory to the findings reported for acid trehalase mutants of S. cerevisiae and C. utilis. The trehalase system of the strain is suggested to be similar to that of fungi.     

Innovative crystal transformation of dihydrate trehalose to anhydrous trehalose using ethanol

The crystal transformation of dihydrate trehalose to anhydrous trehalose was investigated using ethanol and a new type of crystal particle with porous structure could be obtained. The specific surface area of the anhydrous crystal transformed at 50 degrees C was 3.3 m(2)/g, with a median pore diameter of 0.21 microm, and void volume of 0.22 mL/g. The crystal transformation was monitored by measuring the crystal moisture content. The crystal transformation rates could be correlated with the Avrami equation, using the mechanism parameter n=11.5, suggesting that the change of surface area occurred during crystal transformation from dehydrate to anhydrous trehalose. The apparent activation energy of the crystal transformation was 132 kJ/mol.     

Transport and metabolism of trehalose in Escherichia coli and Salmonella typhimurium

The metabolism of trehalose in wild type cells of Escherichia coli and Salmonella typhimurium has been investigated. Intact cells of Escherichia coli (grown on trehalose) accumulated [¹⁴C]-trehalose as [¹⁴C]-trehalose 6-phosphate. Toluene-treated cells catalyzed the synthesis of the [¹⁴C]-sugar phosphate from [¹⁴C]-trehalose and phosphoenolpyruvate; ATP did not serve as phosphoryl donor. Trehalose 6-phosphate could subsequently be hydrolyzed by trehalose 6-phosphate hydrolase, an enzyme which catalyzes the hydrolysis of the disaccharide phosphate into glucose and glucose 6-phosphate. Both Escherichia coli and Salmonella typhimurium induced this enzyme when they grew on trehalose.These findings suggest that trehalose is transported in these bacteria by an inducible phosphoenolpyruvate:trehalose phosphotransferase system.The presence of a constitutive trehalase was also detected.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate: glycose phosphotransferase system - O.D. optical density     

Effects of salinity on growth and compatible solutes of callus induced from <Emphasis Type="Italic">Populus euphratica</Emphasis>

Summary The present study aimed to evaluate the response to salinity of Populus euphratica, which is more salt-resistant than other poplar cultivars, at the cellular level. To this purpose, callus was induced from shoot segments of P. euphratica on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ (2.2 μM) 6-benzyladenine (BA) and 0.5 mg l⁻¹ (2.7 μM 1-naphthaleneacetic acid (NAA). Callus was transferred to MS medium supplemented with 0.25 mg l⁻¹ (1.1 μM) BA and 0.5 mg l⁻¹ NAA. The relative growth rate of callus reached a maximum in the presence of 50 mmol l⁻¹ NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, glycine betaine, and total soluble sugars increased with increasing salt concentrations. The results indicate that the response of the callus of P. euphratica to salt stress is similar to that of the whole plant.     

Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans

Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose), and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures — highly homogeneous with regard to the respective cell form produced — contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5–2% to the biomass of M. rouxii and 1.5–6% to that of C. albicans; the values for trehalose ranged from 0.2–11% in the former and 1–3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions — regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.     

Structure of Porriolide,a New Metabolite from Alternaria porri

Compounds from a soil isolate of Penicillium sp. MG-11 showed remarkable convulsive activity against silkworms. Activity-based fractionation of an acetone extract of the fermentation products resulted in the isolation of four compounds. One was identified as verruculogen, which was an active principle. Another compound, acetoxydehydroaustin, was new and was structurally characterized by spectral data and a single-crystal X-ray analysis. The two other compounds were identified as dehydroaustin and austin on the basis of the spectral data. Although the latter three compounds were not active themselves, they enhanced the convulsive activity of verruculogen in a silkworm bioassay. Three additional compounds with no identifiable activity were also isolated. Two of them were identified as austinol and dehydroaustinol, and the third was determined to be a new related compound named neoaustin by an X-ray analysis of crystals.     

Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phosphorylase (TP) gene from <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

This study generated transgenic tobacco plants expressing trehalose phosphorylase of Pleurotus sajor-caju (PsTP) constitutively under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Sixteen transgenic lines were selected by genomic Southern blot analysis for further study. Unlike yeast TPS1-transformed or Escherichia coli TPS1-transformed tobacco or potato, all of the PsTP transgenic tobacco lines showed normal growth phenotypes both in the culture tubes and soil mixture. The study measured the trehalose contents of PsTP-transformed tobacco plants as well as the wild type and empty vector-transformed control plants. Results showed that the PsTP transformant contained 6.3molg^–1 of plant tissues, while the wild type and the control plants had only minimal levels of trehalose. Because this study detected a significant amount of trehalose in PsTP transgenic tobacco plants, it decided to carry out a bioanalysis of the PsTP transgenic tobacco plants by drought treatment by not watering the plants for over 10days. A significant difference in drought resistance was observed from the second nonwatering day between the transgenic and the control tobacco plants. The transgenic tobacco plants had normal growth and did not wither, while the wild type and the only empty vector-transformed control plants withered severely. Among all the transgenic lines, line 10-4 showed the strongest resistance to drought stress. It did not wither even after 10days without watering. In addition, when the drought resistance of PsTP transgenic tobacco plants was tested using detached leaves, most transgenic plants, except one line, showed better capacity to retain water than the empty vector-transformed transgenic plant.