Accumulation of Trehalose as a Compatible Solute under Osmotic Stress in Euglena gracilis Z

ABSTRACT. The photosynthetic protozoon Euglena gracilis, accumulated a large amount of trehalose in the cells under salt or osmotic stresses. Radioactivity of [14C] paramylon, a β-1,3-polyglucan which was stored in the cells of E. gracilis. was degraded rapidly and this radioactivity was almost stoichiometrically incorporated into trehalose. The interconversion of trehalose from paramylon by salt or osmotic stresses was dependent on the concentrations or osmotic pressures, suggesting that E. gracilis accumulate trehalose as an osmoprotectant. After the removal of salt or osmotic stresses, trehalose was gradually degraded, however, it was not converted into paramylon.     

Role of Glutathione in the Response of Escherichia coli to Osmotic Stress

The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity. A mutant in -glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain. The unfavorable effect of the gsh mutation on osmoadaptation of growing E. coli cells was more pronounced at low concentrations of K⁺ in the medium. An increase in osmolarity caused an increase in the intracellular content of glutathione. Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage. The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation. Changes in the level of intracellular K⁺ were similarly biphasic: a rapid increase in the K⁺ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage. In mutant gshA cells adapted to osmotic shock, the intracellular K⁺ level was markedly higher than in the parental strain cells. The possible role of glutathione in the response of E. coli to osmotic shock is discussed.     

外源海藻糖对PEG渗透胁迫下小麦Rubisco及其活化酶的影响

以抗旱品种‘晋麦47’和干旱敏感品种‘郑引1号’为材料,通过室内水培试验研究了外源海藻糖对PEG渗透胁迫下小麦叶片净光合速率、1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)和1,5-二磷酸核酮糖羧化酶/加氧酶活化酶(RCA)含量和相关基因表达特性的影响。结果表明:(1)外源海藻糖和渗透胁迫均能显著增加2个小麦品种叶片海藻糖含量。(2)渗透胁迫显著降低了2个品种小麦叶片的净光合速率,而外源海藻糖能显著缓解受胁迫小麦叶片净光合速率的降低幅度。(3)渗透胁迫仅使‘郑引1号’Rubisco大亚基基因(rbcL)相对表达量及相应蛋白含量显著降低;渗透胁迫显著降低了小麦RCAα和β亚基基因相对表达量,并显著降低RCA蛋白含量,而外源海藻糖不能缓解RCA蛋白含量的降低;渗透胁迫显著降低了Rubisco总活性、初始活性、活化状态及RCA活性,而外源海藻糖则能显著缓解上述酶活性的下降。(4)小麦叶片净光合速率与其rbcL、RCAα和β亚基基因相对表达量及Rubisco总活性、初始活性、活化状态及RCA活性均呈极显著正相关关系。研究发现,在渗透胁迫条件下,外源海藻糖主要从翻译后层面对小麦叶片Rubisco和RCA的活性发挥显著保护作用,从而缓解了小麦净光合速率的降低。     

大肠杆菌海藻糖的代谢调控

海藻糖是一种重要的抗逆物质。大肠杆菌中otsBA操纵子编码的两种酶负责海藻糖合成。otsBA基因的表达受渗透压诱导和σs因子的调节。细胞的周质海藻糖酶(treA)将外源海藻糖分解成两个葡萄糖分子。尽管大肠杆菌中渗透压诱导合成的海藻糖并不能保护细胞抗干燥,我们将otsA单个基因通过农杆菌转入烟草时,转基因株提高了耐盐和抗干燥特性,同时在转基因烟草提取物中检测到海藻糖,证明otsA基因在烟草中表达并合成海藻糖。我们认为若将otsA基因转入其它植物,可望改善这些植物的抗干旱、耐盐碱特性和延长采摘后的保鲜期 。     

用重组大肠杆菌发酵生产人生长激素研究

通过不同培养基、不同糖浓度对重组菌Ｅ．ｃｏｌｉＤＨ１０Ｂ／ｐＩＮⅢＡ３ＨＧＨ的菌体生长与外源蛋白表达量的影响的比较，确定较为合适的培养条件，并对发酵过程中调节ｐＨ的氨水用量与外源蛋白的表达量之间的相关性作探索，得到相关性曲线，从而根据氨水用量了解细菌的生长状况。     

Temperature-Sensitive Osmotic Remedial Mutants of Escherichia coli

A collection of temperature-sensitive mutants of Escherichia coli K-12 was examined for ability to grow at the restrictive temperature when the osmotic pressure of the medium was increased. Five of the fourteen mutants were found to be osmotic remedial. Four strains containing temperature-sensitive, osmotic-remedial mutations affecting aminoacyl-transfer ribonucleic acid synthetases were found to have altered permeability characteristics which may be attributable to changes in the lipopolysaccharide layer of the cell envelope at restrictive temperatures.     

Chemical Priming with Salt and Urea Improves Germination and Seedling Growth of Black Cumin (Nigella sativa L.) under Osmotic Stress

Journal of Plant Growth Regulation - A common problem with vegetable production in saline areas is poor crop stand, but for black cumin (Nigella sativa L.) germination data are limited and...     

Trehalose transport and metabolism in Escherichia coli.

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

Enhanced Production of Recombinant Proteins in Escherichia coli by Filamentation Suppression

During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the E. coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.     

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h⁻¹. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h⁻¹ achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g.     

Osmotic Reversal of Temperature Sensitivity in Escherichia coli

Forty temperature-sensitive mutants, unable to grow on tryptone or nutrient agar at 42 C, were isolated from Escherichia coli K-12. When 0.5% NaCl was added to the medium, 32 grew at the nonpermissive temperature. Several were tested with different amounts of NaCl added to tryptone broth; all grew best when the osmolality of the medium was between 400 and 1,000 milliosmolal. One of the mutants was studied in more detail. Sucrose, inositol, KCl, and MgCl(2), as well as NaCl, permitted growth at 42 C. Glycerol, however, had no effect. When shifted from 30 to 42 C without osmotic protection, the mutant stopped growing but did not lyse, die, or leak significant amounts of intracellular material. In a similar shift experiment, a second mutant leaked all of its trichloroacetic acid-soluble pools into the medium. The majority of the mutants were hypersensitive to certain antibiotics, indicating possible cell envelope defects.     

Osmotic adaptation of T4 infected Escherichia coli

In contrast to enzymatic adaptation, osmotic adaption is possible with T4-infected Escherichia coli B cells. After an osmotic shift from 220 mOsM to 690 mOsM the intracellular content of potassium rises in infected cells as well as in uninfected cells. After osmotic shock the involved TrKA transport system shows an increased discrimination against rubidium (Rb⁺) and for potassium (K⁺).     

Effect of the Osmotic Pressure of the Growth Medium on fabB Mutants of Escherichia coli

Temperature-sensitive, unsaturated fatty acid (fabB) auxotrophs of Escherichia coli can grow at the restrictive temperature in the absence of unsaturated fatty acid in a medium with a high osmotic pressure. If a mutant culture was starved for unsaturated fatty acids and harvested just before the lysis started, the fatty acid composition of the cells was the same as that of cells grown until late log phase in a high-osmotic medium. Evidence is presented that the in vivo unsaturated fatty acid biosynthesis is significantly increased in a high osmotic medium. The increase is probably due to a partial activation of the temperature-sensitive fabB product. Besides the stimulation of the temperature-sensitive fabB product, a minimal osmotic pressure of the medium appeared to be necessary to allow growth of cells containing lipids with a changed fatty acid composition. fabA mutants are unable to grow in a high-osmotic medium in the absence of unsaturated fatty acids. No increase in the in vivo unsaturated fatty acid biosynthesis could be detected in the temperature-sensitive fabA mutants.     

Enhanced trehalose production improves growth of Escherichia coli under osmotic stress

The biosynthesis of trehalose has been previously shown to serve as an important osmoprotectant and stress protectant in Escherichia coli. Our results indicate that overproduction of trehalose (integrated lacI-Ptac-otsBA) above the level produced by the native regulatory system can be used to increase the growth of E. coli in M9-2% glucose medium at 37 degrees C to 41 degrees C and to increase growth at 37 degrees C in the presence of a variety of osmotic-stress agents (hexose sugars, inorganic salts, and pyruvate). Smaller improvements were noted with xylose and some fermentation products (ethanol and pyruvate). Based on these results, overproduction of trehalose may be a useful trait to include in biocatalysts engineered for commodity chemicals.     

Casein Micelle Dispersions under Osmotic Stress

Casein micelles dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis. This technique measured an equation of state of the dispersions over a wide range of pressures and concentrations and at different ionic strengths. Three regimes were found. i), A dilute regime in which the osmotic pressure is proportional to the casein concentration. In this regime, the casein micelles are well separated and rarely interact, whereas the osmotic pressure is dominated by the contribution from small residual peptides that are dissolved in the aqueous phase. ii), A transition range that starts when the casein micelles begin to interact through their κ-casein brushes and ends when the micelles are forced to get into contact with each other. At the end of this regime, the dispersions behave as coherent solids that do not fully redisperse when osmotic stress is released. iii), A concentrated regime in which compression removes water from within the micelles, and increases the fraction of micelles that are irreversibly linked to each other. In this regime the osmotic pressure profile is a power law of the residual free volume. It is well described by a simple model that considers the micelle to be made of dense regions separated by a continuous phase. The amount of water in the dense regions matches the usual hydration of proteins.