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 on glycerol or methanol 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.     

Regulation of energy metabolism in yeast

Summary The recessive, nuclear gene mutation glc1, which causes glycogen deficiency in Saccharomyces cerevisiae, is highly plciotropic. Studies of the inheritance of glc1 revealed two classes of phenotypic characteristics: I. Traits invariably associated with the mutant gene and II. Traits whose expressions require the presence of glc1 and one or more additional genes. Class I traits include glycogen deficiency and the loss of capacity to accumulate trehalose in nonproliferating conditions. Traits in the second class include a decreased rate of growth on ethanol medium, a deficiency in cytochrome a.a ₃ and an enhanced accumulation of pigment, probably a metalloporphyrin. Constructed strains containing both glc1 and the constitutive maltose fermentation gene MAL4 ⁰ can accumulate trehalose but not glycogen during growth on glucose. However, accumulated trehalose is degraded when cells are exposed to nonproliferating conditions. It is proposed that the glc1 mutation affects a regulatory system, probably involving a protein kinase and/or protein phosphatase, which regulates glycogen synthase and trehalase. Independent regulation of trehalose synthesis by a system controlled by MAL4 ⁰ is indicated.     

Gene expression profiles and intracellular contents of stress protectants in Saccharomyces cerevisiae under ethanol and sorbitol stresses

In response to osmotic stress, proline is accumulated in many bacterial and plant cells. During various stresses, the yeast Saccharomyces cerevisiae induces glycerol or trehalose synthesis, but the fluctuations in gene expression and intracellular levels of proline in yeast are not yet well understood. We previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. In this study, we examined the relationships between the gene expression profiles and intracellular contents of glycerol, trehalose, and proline under stress conditions. When yeast cells were exposed to 1 M sorbitol stress, the expression of GPD1 encoding glycerol-3-phosphate dehydrogenase is induced, leading to glycerol accumulation. In contrast, in the presence of 9% ethanol, the rapid induction of TPS2 encoding trehalose-6-phosphate phosphatase resulted in trehalose accumulation. We found that intracellular proline levels did not increase immediately after addition of sorbitol or ethanol. However, the expressions of genes involved in proline synthesis and degradation did not change during exposure to these stresses. It appears that the elevated proline levels are due primarily to an increase in proline uptake from a nutrient medium caused by the induction of PUT4. These results suggest that S. cerevisiae cells do not accumulate proline in response to sorbitol or ethanol stress different from other organisms.     

Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in Saccharomyces cerevisiae

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO₂ production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.     

Behavioural and physiological responses of infective juveniles of the entomopathogenic nematode Heterorhabditis to desiccation

Infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are susceptible to a wide variety of environmental factors, including desiccation, which limit their usefulness as biocontrol agents. Although EPNs can be subjected to a gradual loss of water in their natural environment they are not full anhydrobiotes, being able to survive only moderate levels of desiccation at high relative humidities (rh). We investigated the desiccation tolerance of IJs of several Heterorhabditisspecies and strains when exposed to fast and slow desiccation regimes. We also investigated the behavioural and biochemical responses of Heterorhabditis IJs when exposed to 98% rh for 4 days. IJs of H. megidis UK211 (but not IJs of H. indica) aggregate into large clumps when desiccated at high rh, but unlike Steinernema spp., neither H. megidis nor H. indica IJs showed any tendency to coil. Preincubation of H. megidis UK211 IJs at high (98%) rh enhances their ability to survive for 150 min at 57% rh. We show that preincubation of H. megidis and H. indica at 98% rh induces the synthesis of glycerol but not of trehalose, whereas identical preincubation conditions do induce trehalose synthesis in Steinernema carpocapsae and Aphelenchus avenae. The biosynthesis of glycerol rather than trehalose by IJs of two species of Heterorhabditis in response to moderate levels of desiccation indicates that Heterorhabditis is unlikely to have the necessary metabolic responses to desiccation required to enable it to enter into a fully anhydrobiotic state.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

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.     

Phylogenetic relationships among species ofWilliopsis andSaturnospora gen. nov. as determined from partial rRNA sequences

Phylogenetic relationships among those yeast species that form saturn-shaped ascospores and which are assigned to the generaWilliopsis andPichia were estimated from their extent of nucleotide sequence divergence in three regions of ribosomal RNA. ThePichia species (P. dispora, P. saitoi, P. zaruensis andP. sp. nov.) are a closely clustered group only distantly related toWilliopsis, and it is proposed that they be reassigned toSaturnospora gen. nov. The extent of divergence amongWilliopsis species (W. californica, W. mucosa, W. pratensis, W. saturnus andW. sp. nov.) is greater than that previously observed within other ascomycetous yeast genera.     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53 % of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30 % lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.     

A Highlights from MBoC Selection: Trehalose Is a Key Determinant of the Quiescent Metabolic State That Fuels Cell Cycle Progression upon Return to Growth

When conditions are unfavorable, virtually all living cells have the capability of entering a resting state termed quiescence or G0. Many aspects of the quiescence program as well as the mechanisms governing the entry and exit from quiescence remain poorly understood. Previous studies using the budding yeast Saccharomyces cerevisiae have shown that upon entry into stationary phase, a quiescent cell population emerges that is heavier in density than nonquiescent cells. Here, we show that total intracellular trehalose and glycogen content exhibits substantial correlation with the density of individual cells both in stationary phase batch cultures and during continuous growth. During prolonged quiescence, trehalose stores are often maintained in favor over glycogen, perhaps to fulfill its numerous stress-protectant functions. Immediately upon exit from quiescence, cells preferentially metabolize trehalose over other fuel sources. Moreover, cells lacking trehalose initiate growth more slowly and frequently exhibit poor survivability. Together, our results support the view that trehalose, which is more stable than other carbohydrates, provides an enduring source of energy that helps drive cell cycle progression upon return to growth.     

Levels of Glycogen and Trehalose in Mycobacterium smegmatis and the Purification and Properties of the Glycogen Synthetase

The levels of glycogen, free trehalose, and lipid-bound trehalose were compared in Mycobacterium smegmatis grown under various conditions of nitrogen limitation. In a mineral salts medium supplemented with yeast extract and containing fructose as the carbon source, the accumulation of glycogen increased dramatically as the NH(4)Cl content of the medium was lowered. However, levels of free trehalose remained relatively constant. Cells were grown in low nitrogen medium and were then shifted to medium containing high nitrogen. Under these conditions, there was a rapid accumulation of glycogen in low nitrogen, and this glycogen was rapidly depleted when cells were placed in high nitrogen medium. Again the concentration of free trehalose remained fairly constant. However, when cells were grown in low nitrogen medium with [(14)C]fructose and then transferred to high nitrogen medium with unlabeled fructose, the specific radioactivity (counts per minute per micromole) of the free trehalose fell immediately, indicating that it was being synthesized and turned over continually. On the other hand, the specific radioactivity of the glycogen and bound trehalose declined much more slowly, suggesting that these two compounds were not turning over as rapidly or were being synthesized at a much slower rate. Experiments on the incorporation of [(14)C]fructose into glycogen and trehalose indicated that cells in high nitrogen medium synthesized much less glycogen than those in low nitrogen. However, synthesis of both free trehalose and bound trehalose was the same in both cases. The specific enzymatic activities of the glycogen synthetase and the trehalose phosphate synthetase varied somewhat from one growth condition to another, but there was no correlation between enzymatic activity and the amount of glycogen or trehalose, suggesting that changes in glycogen levels were not due to increased synthetic capacity. The glycogen synthetase was purified about 35-fold and its properties were examined. This enzyme was specific for adenosine diphosphate glucose as the glucosyl donor.     

Phenol degradation by immobilized cold-adapted yeast strains of Cryptococcus terreus and Rhodotorula creatinivora

Three strains were isolated from hydrocarbon-polluted alpine habitats and were representatives of Cryptococcus terreus (strain PB4) and Rhodotorula creatinivora (strains PB7, PB12). All three strains synthesized and accumulated glycogen (both acid- and alkali-soluble) and trehalose during growth in complex medium containing glucose as carbon source and in minimal salt medium (MSM) with phenol as sole carbon and energy source. C. terreus strain PB4 showed a lower total accumulation level of storage compounds and a lower extracellular polysaccharides (EPS) production than the two R. creatinivora strains, PB7 and PB12. Biofilm formation and phenol degradation by yeast strains attached to solid carriers of zeolite or filter sand were studied at 10°C. Phenol degradation by immobilized yeast strains was always higher on zeolite compared with filter sand under normal osmotic growth conditions. The transfer of cells immobilized on both solid supports to a high osmotic environment decreased phenol degradation activity by all strains. However, both R. creatinivora PB7 and PB12 strains maintained higher ability to degrade phenol compared with C. terreus strain PB4, which almost completely lost its phenol degradation activity. Moreover, R. creatinivora strain PB7 showed the highest ability to form biofilm on both carriers under high osmotic conditions of cultivation.     

DNA relatedness among saturn-spored yeasts assigned to the generaWilliopsis andPichia

Saturn-spored species assigned to the generaWilliopsis andPichia were compared from extent of nuclear DNA complementarity. Of thePichia spp., four were recognized as distinct taxa:P. dispora, P. saitoi, P. zaruensis andPichia sp. nov. AmongWilliopsis spp., the following were accepted:W. californica, W. mucosa comb. nov.,W. pratensis, W. saturnus var.saturnus, W. saturnus var.mrakii comb. nov.,W. saturnus var.sargentensis comb. nov.,W. saturnus var.subsufficiens comb. nov. andWilliopsis sp. nov. The newPichia andWilliopsis species are described elsewhere. Moderate (36–68%) DNA relatedness was detected between the formerPichia sargentensis and varieties ofW. saturnus again demonstrating that nitrate assimilation is not a reliable criterion for separating yeast species.     

Trehalose 6‐phosphate phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigatus

The trehalose biosynthesis pathway is critical for virulence in human and plant fungal pathogens. In this study, we tested the hypothesis that trehalose 6‐phosphate phosphatase (T6PP) is required for Aspergillus fumigatus virulence. A mutant of the A. fumigatus T6PP, OrlA, displayed severe morphological defects related to asexual reproduction when grown on glucose (1%) minimal media. These defects could be rescued by addition of osmotic stabilizers, reduction in incubation temperature or increase in glucose levels (> 4%). Subsequent examination of the mutant with cell wall perturbing agents revealed a link between cell wall biosynthesis and trehalose 6‐phosphate (T6P) levels. As expected, high levels of T6P accumulated in the absence of OrlA resulting in depletion of free inorganic phosphate and inhibition of hexokinase activity. Surprisingly, trehalose production persisted in the absence of OrlA. Further analyses revealed that A. fumigatus contains two trehalose phosphorylases that may be responsible for trehalose production in the absence of OrlA. Despite a normal growth rate under in vitro growth conditions, the orlA mutant was virtually avirulent in two distinct murine models of invasive pulmonary aspergillosis. Our results suggest that further study of this pathway will lead to new insights into regulation of fungal cell wall biosynthesis and virulence.     

Trehalose Biosynthesis in Response to Abiotic Stresses

Trehalose is a non‐reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose‐6‐phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In‐silico expression profiling of all Arabidopsis trehalose‐6‐phosphate synthases (TPSs) and trehalose‐6‐phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

Effect of nitrogen limitation and surplus upon trehalose metabolism in wine yeast

Trehalose metabolism in yeast has been related to stress and could be used as a stress indicator. Winemaking conditions are stressful for yeast and understanding trehalose metabolism under these conditions could be useful for controlling alcoholic fermentation. In this study, we analysed trehalose metabolism of a commercial wine yeast strain during alcoholic fermentation by varying the nitrogen levels from low (below adequate) to high (excess). We determined trehalose, nitrogen, sugar consumption and expression of NTH1, NTH2 and TPS1. Our results show that trehalose metabolism is slightly affected by nitrogen availability and that the main consumption of nitrogen occurs in the first 24 h. After this period, nitrogen is hardly taken up by the yeast cells. Although nitrogen and sugar are still available, no further growth is observed in high concentrations of nitrogen. Increased expression of genes involved in trehalose metabolism occurs mainly at the end of the growth period. This could be related to an adaptive mechanism for fine tuning of glycolysis during alcoholic tumultuous fermentation, as both anabolic and catabolic pathways are affected by such expression.     

Sensing trehalose biosynthesis in plants

A most unexpected finding in research on plant carbohydrate metabolism is the recent discovery that angiosperms encode genes whose products are involved in trehalose metabolism. The presence and functionality of such genes has been elegantly shown by expressing Arabidopsis-derived trehalose phosphate synthase and trehalose phosphate phosphatase genes in yeast mutants lacking these enzymatic activities. Homologue sequences have now been cloned from a number of different plant species suggesting that the capacity to synthesise trehalose is ubiquitous in angiosperms. Except for Myrothamnus flabellifolius, trehalose biosynthesis has never been observed in tissues of higher plants, probably due to the presence of high levels of trehalase activity. The function of trehalose metabolism in plants is still a mystery. One of the postulated functions of trehalose metabolism in yeast is in the control of glucose repression and a similar function in sugar sensing can be proposed for plants as well.     

Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.     

Factors affecting accumulation and degradation of curdlan, trehalose and glycogen in cultures of Cellulomonas flavigena strain KU (ATCC 53703)

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.