Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N,?N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1?M solute were heated for 10?min at temperatures between 35–75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1?M stabilizer solutions were frozen at ?30°C and freeze-dried for 24?h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

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.     

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.     

Variations in the response of salt-stressed Rhizobium strains to betaines

A total of 15 rhizobial strains representing Rhizobium meliloti, Rhizobium japonicum, Rhizobium trifolii, Rhizobium leguminosarum, Rhizobium sp. (Sesbania rostrata) and Rhizobium sp. (Hedysarum coronarium), were studied with regard to growth rate under salt stress in defined liquid media. In the presence of inhibitory concentrations of NaCl, enhancement of growth resulting from added glycine betaine was observed for R. meliloti strains and Rhizobium sp. (Hedysarum coronarium) but not for other Rhizobium species. The concentration of glycine betaine required for maximal growth stimulation was very low (1 mM) in comparison with the osmolarity of the medium. The stimulation was shown to be independent of any specific solutes. Other related compounds like proline betaine, carnitine, choline, -butyrobetaine and pipecolate betaine were also effective compounds in restoring the growth rate of cells grown in medium of elevated osmolarity. High rate of glycine betaine uptake was demonstrated in R. meliloti cells grown in media of increased osmotic strength. The intracellular concentration of this solute was found to be 308 mM in 0.3 M NaCl-grown cells and 17 times lower in minimal medium-grown cells. Glycine betaine was used for growth under conditions of low osmolarity but could not serve as sole carbon or nitrogen source in medium of increased osmotic strength. Experiments with [¹⁴C]glycine betaine showed that this molecule was not metabolized by cells subjected to osmotic stress, whereas it was rapidly converted to dimethylglycine, sarcosine and glycine in minimal medium-grown cells.Abbreviations LAS lactate-aspartate-salts - LGS lactate-glutamate-salts - LS lactate-succinate - MSY mannitol-salts-yeast - YLS yeast-lactate-succinate     

Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool

In the coryneform Brevibacterium linens, ectoine constitutes the major intracellular solute accumulated under elevated medium osmolarity. Here we report that exogenously supplied proline, choline, glycine betaine, and even ectoine, protected bacterial cells against deleterious effects of a hyperosmotic constraint (i.e. 1.5 M NaCl). In all cases, a significant improvement of growth was observed; in parallel, intracellular osmolyte pools composed mainly of glutamate and ectoine substantially increased, either with added glycine betaine (under limiting supply) or with proline. However, these two osmoprotectants behaved differently: glycine betaine acted as a genuine osmoprotectant, whereas proline was accumulated only transiently and participated actively in the biosynthesis of glutamate, ectoine, and trehalose. The strategy developed by B. linens cells allows the proposal of a novel role for proline in the osmoprotection process through its conversion to the apparently preferred endogenous osmolyte ectoine.     

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.     

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.     

Combined Application of the Biological Product LS213 with Bacillus, Pseudomonas or Chryseobacterium for Growth Promotion and Biological Control of Soil-Borne Diseases in Pepper and Tomato

Recent works suggest that the combination of several PGPRs could be more effective than individual strains as a horticultural product. LS213 is a product formed by a combination of two PGPRs, Bacillus subtilis strain GB03 (a growth-promoting agent), B. amyloliquefaciens strain IN937a (an inducer of systemic resistance) and chitosan. The aim of this work is to establish if the combination of three PGPR, B. licheniformis CECT 5106, Pseudomonas fluorescens CECT 5398 and Chryseobacterium balustinum CECT 5399 with LS213 would have a synergistic effect on growth promotion and biocontrol on tomato and pepper against Fusarium wilt and Rhizoctonia damping off. When individual rhizobacterium and the LS213 were put together, the biometric parameters were higher than with individual rhizobacterium both in tomato and pepper, revealing a synergistic effect on growth promotion, being the most effective combination that of B. licheniformis and LS213. When P. fluorescens CECT 5398 was applied alone, it gave good results, which could be due to the production of siderophores by this strain. Biocontrol results also indicate that those treatments that combined LS213 and each of the bacteria (Treatments: T7 and T8) gave significantly higher percentages of healthy plants for both tomato (T7: 65%) and pepper (T7: 75% and T8: 70%) than the LS213 alone (45% of healthy plants for tomato and 60% for pepper) three weeks after pathogen attack. The effects in pepper were more marked than in tomato. The best treatment in biocontrol was the combination of P. fluorescens and LS213. In summary, the combination of microorganisms gives better results probably due to the different mechanisms used.     

Changes in intracellular composition in response to hyperosmotic stress of NaCl, sucrose or glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum

Changes in intracellular composition after hyperosmotic shock were studied in the lysine-producing mutant Brevibacterium lactofermentum NRRL B-11470 and the wild-type Corynebacterium glutamicum ATCC 13032. Both strains accumulated betaine, proline, glutamic acid, glutamine and trehalose in response to stress. The accumulated amino acids were synthesized by the cells, while betaine and trehalose were taken up from the medium. The contribution of synthesized osmoregulators was highest in C. glutamicum. In a sucrose-limited continuous culture, the increased outer osmotic pressure was balanced within 15 min for C. glutamicum and somewhat later in B. lactofermentum. The rapid regulation was due to both accumulation of osmoregulators, and shrinkage of cell and cytoplasmic volume. Immediately after shock, glutamine and glutamic acid were the dominating osmolytes. During the adaptation process, glutamine was replaced by the better osmoprotectant proline. In betaine-enriched cultures, betaine accumulation increased at the expense of glutamic acid, glutamine and trehalose. The total intracellular concentration of osmolytes increased linearly with increasing stress for all stress factors.     

Comparative cryopreservation of avian spermatozoa: benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival

A comparative approach was used to evaluate the cryosurvival of turkey and crane sperm frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants and ATP. A range (6-26%) of DMA concentrations was used alone or in combination with ATP (30, 60 or 118mM) or one of the following osmoprotectants: (1) sucrose (turkey, 8.0%; crane, 5.0%); (2) 5.0% sucrose and 5.0% trehalose; or (3) betaine hydrochloride (0.1, 0.2 or 0.4mM). The viability of thawed sperm was assessed using the nigrosin-eosin stain and sperm motility was determined using the hanging-drop technique. For semen frozen only with DMA, post-thaw sperm motility was greatest (P<0.05) for the 6.0%, 10.0% and 18% concentrations, regardless of species. Turkey sperm frozen with the sucrose/trehalose combination had greater (P<0.05) post-thaw motility for all DMA treatments compared to DMA alone. The lowest concentration of the osmoprotectant betaine hydrochloride substantially improved turkey sperm viability post-thaw in all treatments compared to DMA alone (P<0.05). The post-thaw motility of crane sperm was improved (P<0.05) with a combination of 18.0%, 24.0% or 26.0% DMA and 30mM ATP. Moreover, in the presence of osmoprotectants, crane sperm motility decreased as the osmoprotectant concentration increased. The lowest concentration of ATP also improved crane sperm viability post-thaw, especially for DMA concentrations 18% or greater. The combination of sucrose and trehalose improved (P<0.05) crane sperm viability only with 6% and 10% DMA. These data affirm that there are avian-specific differences in sperm survival after cryopreservation and suggest that post-thaw survival can be enhanced by including species-based osmoprotectant/ATP combinations in a cryodiluent where DMA is the cryoprotectant.     

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N, N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1 M solute were heated for 10 min at temperatures between 35-75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1 M stabilizer solutions were frozen at -30°C and freeze-dried for 24 h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

Enhancing viability of two biocontrol yeasts in liquid formulation by applying sugar protectant combined with antioxidant

Survivals of Cryptococcus laurentii and Pichia membranaefaciens in liquid formulations with sugar protectants (trehalose and galactose) and L-ascorbic acid (Vc) were investigated during storage at 4°C and 25°C. When galactose or trehalose was used alone as protectant, C. laurentii maintained relatively high viability in potassium phosphate buffer. Addition of Vc to trehalose improved its protective effect. P. membranaefaciens maintained viability >60% after 90 days at 4°C when 5% galactose served as a protectant, and its combination with Vc was the most effective at maintaining viability. Moreover, liquid formulation kept higher viability of the two yeasts at 4°C than at 25°C. Biocontrol efficiency of the two yeasts was maintained after formulation and storage. The results indicate that trehalose is considered as a suitable protectant for liquid formulation of C. laurentii, while galactose is better for P. membranaefaciens. Combining Vc with the sugars improves the protective efficiency.     

Proline betaine is a highly effective osmoprotectant for Staphylococcus aureus

Proline betaine is an osmoprotectant that is at least as effective as glycine betaine, and more effective than L-proline, for various strains of Staphylococcus aureus, and Staphylococcus epidermidis and Staphylococcus saprophyticus. ¹³C NMR studies revealed that proline betaine accumulated to high levels in osmotically stressed S. aureus, but was also detected in organisms grown in its presence in the absence of osmotic stress. Competition experiments indicated that proline betaine was taken up by the proline transport systems of S. aureus, but not by the high affinity glycine betaine transport system.Abbreviations PYK Peptode - Yeast extract K₂HPO₄     

Mycelial growth of strains of the genera <Emphasis Type="Italic">Suillus</Emphasis> and <Emphasis Type="Italic">Boletinus</Emphasis> in media with a wide range of concentrations of carbon and nitrogen sources

Suillus and Boletinus were studied using Ohta medium. In media with glucose or trehalose, all tested strains grew well. With mannose and cellobiose, strains generally grew well, except for one strain of Suillus. Utilization of dextrin and soluble starch differed with each strain, and that of sucrose and glycerol was low for all strains. Utilization of four amino acids, arginine, glutamic acid, aspartic acid, and alanine, was similar to that of ammonium tartrate for Suillus strains, but mycelial growth with amino acids was clearly lower than with ammonium tartrate for the Boletinus strain. The effect of glucose and ammonium tartrate concentrations for nine strains of the genera Suillus and Boletinus was studied with ranges for glucose of 1–100 and 200g/l, respectively, and for ammonium tartrate of 0.2–5 and 20g/l, respectively. Six strains showed maximal growth at a glucose concentration greater than 25g/l, and one strain showed maximal growth at 70g/l. The results indicate that these fungi are adapted to relatively high concentrations of carbon sources. In general, glucose concentration at mycelial growth maximum decreased as ammonium tartrate concentration increased, and at higher concentrations of glucose, mycelial growth decreased more rapidly in higher concentrations of ammonium tartrate.     

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.     

Salinity stress responses in the plant growth promoting rhizobacteria,Azospirillum spp

In order to adapt to the fluctuations in soil salinity/osmolarity the bacteria of the genusAzospirillum accumulate compatible solutes such as glutamate, proline, glycine betaine, trehalose, etc. Proline seems to play a major role in osmoadaptation. With increase in osmotic stress the dominant osmolyte inA. brasilense shifts from glutamate to proline. Accumulation of proline inA. brasilense occurs by both uptake and synthesis. At higher osmolarityA. brasilense Sp7 accumulates high intracellular concentration of glycine betaine which is taken up via a high affinity glycine betaine transport system. A salinity stress induced, periplasmically located, glycine betaine binding protein (GBBP) of ca. 32 kDa size is involved in glycine betaine uptake inA. brasilense Sp7. Although a similar protein is also present inA. brasilense Cd it does not help in osmoprotection. It is not known ifA. brasilense Cd can also accumulate glycine betaine under salinity stress and if the GBBP-like protein plays any role in glycine betaine uptake. This strain, under salt stress, seems to have inadequate levels of ATP to support growth and glycine betaine uptake simultaneously. ExceptA. halopraeferens, all other species ofAzospirillum lack the ability to convert choline into glycine betaine. Mobilization of thebet ABT genes ofE. coli intoA. brasilense enables it to use choline for osmoprotection. Recently, aproU-like locus fromA. lipoferum showing physical homology to theproU gene region ofE. coli has been cloned. Replacement of this locus, after inactivation by the insertion of kanamycin resistance gene cassette, inA. lipoferum genome results in the recovery of mutants which fail to use glycine betaine as osmoprotectant.     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of <Emphasis Type="Italic">Paecilomyces fumosoroseus</Emphasis> and <Emphasis Type="Italic">Encarsia formosa</Emphasis> on the control of the greenhouse whitefly: preliminary assessment of a compatability study

The effect of separate and combined activity of Paecilomyces fumosoroseus Wize (Brown and Smith) Trinidadian strain T11 and the parasitoid, Encarsia formosa Gahan, was assessed on populations of the greenhouse whitefly, Trialeurodes vaporariorum (Westwood), infesting Phaseolus vulgaris L. (French bean) and Pelargonium x domesticum (regal geranium) plants in replicate experiments. When infested bean and geranium plants were exposed to E. formosa for 2 days, and 4 days later sprayed with P. fumosoroseus blastospores, whitefly percent mortality was 99.5% and 75.5%, 94.6% and 59.4% for experiments 1 and 2, respectively. Treatment of infested bean plants with either E. formosa or P. fumosoroseus resulted in 87.8% and 78.7%, 73.1% and 97.0% whitefly mortality for experiments 1 and 2, respectively, while similar treatment of infested geranium plants resulted in 9.2% and 52.8%, 34.3% and 64.5% whitefly mortality for experiments 1 and 2, respectively. Our results support the use of E. formosa and P. fumosoroseus in combination in Experiment 1 for the treatment of whitefly infested P. vulgaris plants since a significant difference in mortality is observed than when either E. formosa or P. fumosoroseus is applied alone. However, in experiment 2, the combination treatment on P. vulgaris was no more effective than spraying P. fumosoroseus alone. On P. x domesticum plants, only P. fumosoroseus alone is needed for efficient control of the whitefly compared to the combination treatment. The relative timing of parasitoid oviposition and fungal infection are critical in determining the outcome of the interaction and are plant host dependent.