Osmotically induced intracellular trehalose, but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli

Trehalose considerably increased the tolerance of Escherichia coli to air drying, whether added as an excipient prior to drying or accumulated as a compatible solute in response to osmotic stress. The protective effect of exogenously added trehalose was concentration dependent, up to a threshold value of 350 mM. However, trehalose alone cannot explain the intrinsically greater desiccation tolerance of stationary compared to exponential phase E. coli cells, although their tolerance was also enhanced by exogenous or endogenously accumulated trehalose. In contrast, glycine betaine whether added as an excipient or accumulated intracellularly had no influence on desiccation tolerance. These data demonstrate that the protection provided by compatible solutes to cells subjected to desiccation differs from that during osmotic stress, due to the much greater reduction in available cell water. The protective effects of trehalose during desiccation appear to be due to its stabilising influence on membrane structure, its chemically inert nature and the propensity of trehalose solutions to form glasses upon drying, properties which are not shared by glycine betaine.     

Trehalose transporter from African chironomid larvae improves desiccation tolerance of Chinese hamster ovary cells

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.     

Factors affecting the survival of Bradyrhizobium applied in liquid cultures to soya bean [Glycine max (L.) Merr.] seeds

AIMS: To determine the impact of medium composition, bacterial strain, trehalose accumulation, and relative humidity during seed storage on the survival of Bradyrhizobium japonicum on soya bean [Glycine max (L.) Merr.] seeds. METHODS AND RESULTS: Bacteria in liquid cultures were applied to seeds, and the number of survivors was quantified after 2, 24, 48, or 96 h. Addition of yeast extract to a defined medium increased on-seed survival 50- to 80-fold. Addition of 40 mmol l(-1) of NaCl to the medium doubled or tripled the accumulation of trehalose in cells and increased survival several fold, and the addition of both salt and trehalose had an additive effect. There was a threefold difference among strains in survival, and survival of the various strains was significantly correlated with differences in the accumulation of trehalose. The correlation between trehalose accumulation by bacteria and survival was also highly significant in other experiments. Studies in controlled humidity environments showed 100-fold or more differences in survival. CONCLUSIONS: The consistently significant correlation of trehalose content of cells with survival on seed suggests that trehalose is an important component of the survival mechanisms. When some of the factors (salt and trehalose in the medium plus humidity control) were studied in combination, several 100-fold increases in survival of bacteria on seeds were recorded. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible by manipulation of several parameters--strain selection, salt and trehalose content of the medium, control of relative humidity--to achieve substantial improvements in survival of Bradyrhizobium on soya bean seeds.     

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.     

Intracellular trehalose is neither necessary nor sufficient for desiccation tolerance in yeast

Trehalose is thought to be important for desiccation tolerance in a number of organisms, including Saccharomyces cerevisiae, but there is limited in vivo evidence to support this hypothesis. In wild-type yeast, the degree of desiccation tolerance has been shown previously to increase in cultures after diauxic shift and also in exponential-phase cultures after exposure to heat stress. Under both these conditions, increased survival of desiccation correlates with elevated intracellular trehalose concentrations. Our data confirm these findings, but we have tested the apparent importance of trehalose using mutant strains with a deleted trehalose-6-phosphate synthase gene (tps1Delta). Although tps1Delta strains do not produce trehalose, they are nevertheless capable of desiccation tolerance, and the degree of tolerance also increases after diauxic shift or heat stress, albeit slightly less than in the wild type. Conversely, when wild-type yeast is subjected to osmotic stress, mid-exponential-phase cultures produce high concentrations of intracellular trehalose but show little improvement in desiccation tolerance. These results show that there is no consistent relationship between intracellular trehalose levels and desiccation tolerance in S. cerevisiae. Trehalose seems to be neither necessary nor sufficient for, although in some strains might quantitatively improve, survival of desiccation, suggesting that other adaptations are more important.     

Rhizobia and bradyrhizobia under salt stress: possible role of trehalose in osmoregulation

Seven rhizobium fredii strains and seven Bradyrhizobium japonicum strains were grown in defined medium with or without 20m m trehalose in the presence or absence of NaCl. Trehalose had no effect on the growth rate of the strains in the absence of NaCl, but increased the growth rate of some strains in the presence of NaCl. Bradyrhizobium japonicum strain RCR 3827 was completely inhibited by 0·08 m NaCl in absence of trehalose, but multiplied when trehalose was added. The results indicate that trehalose may act as an osmoregulator in these strains of Rhizobium and Bradyrhizobium .     

Three enzymes for trehalose synthesis in Bradyrhizobium cultured bacteria and in bacteroids from soybean nodules

alpha,alpha-Trehalose is a disaccharide accumulated by many microorganisms, including rhizobia, and a common role for trehalose is protection of membrane and protein structure during periods of stress, such as desiccation. Cultured Bradyrhizobium japonicum and B. elkanii were found to have three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose synthase (MOTS), and trehalose-6-phosphate synthetase. The activity level of the latter enzyme was much higher than those of the other two in cultured bacteria, but the reverse was true in bacteroids from nodules. Although TS was the dominant enzyme in bacteroids, the source of maltose, the substrate for TS, is not clear; i.e., the maltose concentration in nodules was very low and no maltose was formed by bacteroid protein preparations from maltooligosaccharides. Because bacteroid protein preparations contained high trehalase activity, it was imperative to inhibit this enzyme in studies of TS and MOTS in bacteroids. Validamycin A, a commonly used trehalase inhibitor, was found to also inhibit TS and MOTS, and other trehalase inhibitors, such as trehazolin, must be used in studies of these enzymes in nodules. The results of a survey of five other species of rhizobia indicated that most species sampled had only one major mechanism for trehalose synthesis. The presence of three totally independent mechanisms for the synthesis of trehalose by Bradyrhizobium species suggests that this disaccharide is important in the function of this organism both in the free-living state and in symbiosis.     

Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration

AIM: This work determines the efficiency of trehalose on the preservation by heat or osmotic drying of a strain of Lactobacillus delbrueckii ssp. bulgaricus. Cell recovery at different trehalose concentrations during drying correlated with the surface properties and osmotic response of cells after rehydration. METHODS AND RESULTS: Bacteria were dried in the presence of glycerol, trehalose, sucrose at 70 degrees C and at 20 degrees C. Trehalose attenuates the loss of viability at 0.25 m. At this concentration, the osmotic response and zeta potential of the bacteria were comparable with the nondried ones. CONCLUSIONS: Trehalose diminishes significantly the damage produced by dehydration both when the bacteria are dried by heating or subjected to osmotic dehydration. This effect appears related to the preservation of the permeability to water and the surface potential of the bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Dehydration occurring during heating or during osmosis appears to have similar effects. As dehydration-induced damage is in correlation with osmotic response recovery and is hindered or buffered by the presence of trehalose, it may be related to water eliminated from biological structures involved in water permeation.     

Effects of intracellular trehalose content on Streptomyces griseus spores.

The disaccharide trehalose is accumulated as a storage product by spores of Streptomyces griseus. Growth on media containing excess glucose yielded spores containing up to 25% of their dry weight as trehalose. Spores containing as little as 1% of their dry weight as trehalose were obtained during growth on media containing a limiting amount of glucose. Spores containing low levels of trehalose accumulated this sugar when incubated with glucose. The increase in trehalose content coincided with increases in spore refractility, heat resistance, desiccation resistance, and the time required for spore germination in complex media. Trehalose is accumulated by a wide variety of actinomycetes and related bacteria and may be partially responsible for their resistance properties.     

Influence of growth phases and desiccation on the degrees of unsaturation of fatty acids and the survival rates of rhizobia

The influence of growth phase on the evolution of cellular fatty acids (CFA) and survival of Sinorhizobium and Bradyrhizobium during desiccation and storage at different levels of relative humidity (R.H.) was studied. Lactobacillic, cis vaccenic and palmitic acids were the major fatty acids of S. meliloti RCR 2011, B. elkanii USDA 120 and B. japonicum 3.2, whatever the growth phase. An exchange of cis vaccenic with lactobacillic acid was observed during the course of growth. The degree of unsaturation (% unsaturated CFA/% saturated CFA = u/s ratio) was significantly higher during the mid logarithmic phase of growth. Survival rates immediately after desiccation were unaffected by the growth phase and the R.H. Furthermore, no correlation was found between survival rate and u/s ratio. During the course of desiccation, the u/s ratio of rhizobia decreased but the decrease was largely independent of the R.H. Optimum R.H. values for storage were in the range 22–67·8%, and S. meliloti was significantly more tolerant than the bradyrhizobia. Cells of rhizobia harvested in the lag phase of growth were more resistant to protracted storage than cells at other growth phases. Again, no correlation was found between u/s ratio and survival rates, despite the expected practical significance for screening for drought-tolerant micro-organisms.     

Physiological Changes in Rhizobia after Growth in Peat Extract May Be Related to Improved Desiccation Tolerance

Improved survival of peat-cultured rhizobia compared to survival of liquid-cultured cells has been attributed to cellular adaptations during solid-state fermentation in moist peat. We have observed improved desiccation tolerance of Rhizobium leguminosarum bv. trifolii TA1 and Bradyrhizobium japonicum CB1809 after aerobic growth in water extracts of peat. Survival of TA1 grown in crude peat extract was 18-fold greater than that of cells grown in a defined liquid medium but was diminished when cells were grown in different-sized colloidal fractions of peat extract. Survival of CB1809 was generally better when grown in crude peat extract than in the control but was not statistically significant (P > 0.05) and was strongly dependent on peat extract concentration. Accumulation of intracellular trehalose by both TA1 and CB1809 was higher after growth in peat extract than in the defined medium control. Cells grown in water extracts of peat exhibit morphological changes similar to those observed after growth in moist peat. Electron microscopy revealed thickened plasma membranes, with an electron-dense material occupying the periplasmic space in both TA1 and CB1809. Growth in peat extract also resulted in changes to polypeptide expression in both strains, and peptide analysis by liquid chromatography-mass spectrometry indicated increased expression of stress response proteins. Our results suggest that increased capacity for desiccation tolerance in rhizobia is multifactorial, involving the accumulation of trehalose together with increased expression of proteins involved in protection of the cell envelope, repair of DNA damage, oxidative stress responses, and maintenance of stability and integrity of proteins.     

Desiccation responses and survival of Sinorhizobium meliloti USDA 1021 in relation to growth phase, temperature, chloride and sulfate availability

AIMS: To identify physical and physiological conditions that affect the survival of Sinorhizobium meliloti USDA 1021 during desiccation. METHODS AND RESULTS: An assay was developed to study desiccation response of S. meliloti USDA 1021 over a range of environmental conditions. We determined the survival during desiccation in relation to (i) matrices and media, (ii) growth phase, (iii) temperature, and (iv) chloride and sulfate availability. CONCLUSIONS: This study indicates that survival of S. meliloti USDA 1021 during desiccation is enhanced: (i) when cells were dried in the stationary phase, (ii) with increasing drying temperature at an optimum of 37 degrees C, and (iii) during an increase of chloride and sulfate, but not sodium or potassium availability. In addition, we resolved that the best matrix to test survival was nitrocellulose filters. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of physical and physiological factors that determine the survival during desiccation of S. meliloti USDA 1021 may aid in (i) the strategic development of improved seed inocula, (ii) the isolation, and (iii) the development of rhizobial strains with improved ability to survive desiccation. Furthermore, this work may provide insights into the survival of rhizobia under drought conditions.     

Engineered Trehalose Permeable to Mammalian Cells

Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.     

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.
Methods and Results:  A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased.
Conclusions:  A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process.
Significance and Impact of the Study:  The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.     

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (approximately 190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.     

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (∼190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.     

Uptake of trehalose by Saccharomyces cerevisiae.

Trehalose, a storage sugar of baker's yeast, is known not to be metabolized when added to a cell suspension in water or a growth medium and to support growth only after a lag of about 10 h. However, it was transported into cells by at least two transport systems, the uptake being active, with a pH optimum at 5.5. There was no stoicheiometry with the shift of protons into cells observed at high trehalose concentrations. Trehalose remained intact in cells and was not appreciably lost to a trehalose-free medium. The uptake systems were present directly after growth on glucose, then decayed with a half-life of about 25 min but could be reactivated by aerobic incubation with trehalose, maltose, alpha-methyl-D-glucoside, glucose or ethanol. The uptake systems thus induced were different as revealed by competition experiments. At least one of the systems for trehalose uptake showed cooperative kinetics. Comparative anaysis with other disaccharides indicated the existence in Saccharomyces cerevisiae, after induction with trehalose, of at least four systems for the uptake of alpha-methyl-D-glucoside, four systems for maltose, together with the two for trehalose, variously shared by the sugars, the total of alpha-glucoside-transporting systems being five.     

Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli

Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h). No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose.     

Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress.