A glycine betaine transport system in Aphanothece halophytica and other glycine betaine-synthesising cyanobacteria

Uptake of exogenous ¹⁴C-glycine betaine has been followed in the cyanobacterium Aphanothece halophytica and other species able to synthesise glycine betaine in response to osmotic stress. At 1 mmol dm^–3 uptake was rapid (flux rate=29.50 nmol m^–2 s^–1), equilibrating at an internal concentration of 120 mmol dm^–3 within 30 min. This rapid uptake, coupled with high internal accumulation, was characteristic of glycine betaine-synthesising cyanobacteria only. The ¹⁴C-glycine betaine transported was not catabolised. Kinetic studies indicated a Michaelis-Menten type relationship (K _m=2.0 mol dm^–3, V _max=45 nmol min^–1 mm^–3 cell volume), with a pH optimum of 8.0–8.5. Darkness dramatically decreased the flux rate. Higher ¹⁴C-glycine betaine levels occurred in cells growth in medium of elevated osmotic strength, and glycine betaine uptake was sensitive to changes in external salinity. A relationship between Na⁺ availability and glycine betaine uptake was observed, with >80 mmol dm^–3 Na⁺ required for optimal stimulation of uptake in seawater-grown cells. Severe hyperosmotic stress (1000 mmol dm^–3 NaCl) reduced the rate of glycine betaine uptake but increased internal glycine betaine concentration at equilibrium. Hypo-osmotic stress caused a decline in the internal glycine betaine concentration due to an increased rate of loss, indicating that the efflux system was also sensitive to ambient salinity changes. It is envisaged that this active transport system may be an adaptive mechanism in halophilic glycine betaine-synthesising cyanobacteria.     

An efficient method for DNA extraction from cyanobacteria isolated from hypersaline and marine environments

Cyanobacteria are ancient organisms surviving on the earth due to their simple nutritional requirements and ability to produce distinct secondary metabolites that can combat detrimental environmental impacts. In order to understand these abilities of cyanobacteria at the molecular level, it is necessary to extract high‐quality genomic DNA. However, the presence of secondary metabolites and exopolysaccharides hinders the DNA extraction from these organisms, especially from hypersaline environments. Here we have developed and compared a new method with two known methods of DNA extraction from environmental isolates. The results clearly indicate that the new optimized method yielded large amount of DNA with high purity. Additionally, the extracted DNA showed reduced degradation and excellent overall quality, which can be used directly for downstream purposes such as PCR and sequencing.     

Accumulation of osmolytes and osmotic adjustment in water-stressed wheat (Triticum aestivum) and maize (Zea mays) as affected by calcium and its antagonists

Maize (Zea mays) and wheat (Triticum aestivum) were water stressed for 4 days at early vegetative growth (15-day-old) using PEG-6000 (−1.0 MPa), in the presence of 1 mM CaSO₄, 50 μM Verapamil (VP; calcium channel blocker); 50 μM Trifluoperazine (TFP; calmodulin antagonist) and then put to recovery in order to investigate the changes in osmoregulation in plants having C₃ and C₄ metabolism. Accumulation of proline (Pro) and quaternary ammonium compounds (QAC's), activities of pyrroline-5-carboxylate reductase (P5CR), proline dehydrogenase (PDH), water potential (Ψ_w), osmotic adjustment (OA), relative elongation rate (RER) and electrolyte leakage (EL) were examined during stress and recovery. Maize had significantly higher accumulation of Pro while wheat showed relatively more accumulation of QAC's. The activities of P5CR and PO were also significantly higher in maize than wheat. Maize shoots under stress showed higher Ψ_w, OA, RER and less EL than wheat shoots. Upon recovery from stress, maize regained its growth and water potential faster than wheat. Ca²⁺ elevated the accumulation of osmolytes in both the plants but OA was less sensitive to it. In the presence of Ca²⁺, wheat showed significantly more accumulation of osmolytes, higher Ψ_w, RER than maize. Ca²⁺ inhibitors partially reversed the effects of calcium indicating its involvement in governing solute accumulation. The differential sensitivity of maize and wheat towards water stress may be related to variation in endogenous calcium expression and its function.     

A reliable method for spectrophotometric determination of glycine betaine in cell suspension and other systems

Glycine betaine is a quaternary ammonium compound that accumulates in a large variety of species in response to different types of stress. Glycine betaine counteracts adverse effects caused by abiotic factors, preventing the denaturation and inactivation of proteins. Thus, its determination is important, particularly for scientists focused on relating structural, biochemical, physiological, and/or molecular responses to plant water status. In the current work, we optimized the periodide technique for the determination of glycine betaine levels. This modification permitted large numbers of samples taken from a chlorophyllic cell line of the grass Bouteloua gracilis to be analyzed. Growth kinetics were assessed using the chlorophyllic suspension to determine glycine betaine levels in control (no stress) cells and cells osmotically stressed with 14 or 21% polyethylene glycol 8000. After glycine extraction, different wavelengths and reading times were evaluated in a spectrophotometer to determine the optimal quantification conditions for this osmolyte. Optimal results were obtained when readings were taken at a wavelength of 290 nm at 48 h after dissolving glycine betaine crystals in dichloroethane. We expect this modification to provide a simple, rapid, reliable, and cheap method for glycine betaine determination in plant samples and cell suspension cultures.     

Glycine betaine and polar lipid composition in halophilic archaebacteria in response to growth in different salt concentrations

Abstract Examples of halophilic archaebacteria contain low levels of between 1 and 20 mM trimethyl glycine (glycine betaine). In disrupted cell preparations, the glycine betaine is associated with the membrane fraction and is not detectable in cell supernatants. Cells of Natronococcus occultus grown in different salt concentrations show an increase in cell-associated glycine betaine along with an increase in the ratio of phosphatidyl glycerophosphate (PG) to phosphatidyl glycerol (PG) in the cell membrane.     

The compatibility of osmotica in cyanobacteria

Abstract. The solutes accumulated by cyanobacteria in response to hyper-osmotic stress include Na⁺, K⁺, sucrose, trehalose, glucosyl-glycerol, glyeine betaine and glutamate betaine. The compatibility of several of these solutes with glutamine synthetase activity has been examined using cell-free extracts from a range of freshwater, marine and halotolerant cyanobacteria. All of the solutes tested were compatible with (i.e. non-inhibitory to) enzymic activity at physiological concentrations and the results demonstrate a rank order of compatibility which correlates with the concentrations at which the organic solutes occur in cyanobacteria, i.e. glycine betaine > polyol-derivatives > disaccharides and with the upper salinity limit for growth. The protection against inhibition by NaCl (halo-protection) afforded by these solutes to enzymic activity was also examined. Only glycine betaine was found to exert a significant halo-protective effect and this may be explained by differences in the mechanism of compatible solute function between small charged molecules and sugars/polyols.     

Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine

Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7φ10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD⁺ and NADP⁺ as cofactors, but NAD⁺ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K _m values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD⁺, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress. Received: 2 May 1997 / Accepted: 2 July 1997     

Formation and breakdown of glycine betaine and trimethylamine in hypersaline environments

Glycine betaine is accumulated as a compatible solute in many photosynthetic and non-photosynthetic bacteria — the last being unable to synthesize the compound - and thus large pools of betaine can be expected to be present in hypersaline environments. A variety of aerobic and anaerobic microorganisms degrade betaine to among other products trimethylamine and methylamine, in a number of different pathways. Curiously, very few of these betaine breakdown processes have yet been identified in hypersaline environments. Trimethylamine can also be formed by bacterial reduction of trimethylamine N-oxide (also by extremely halophilic archaeobacteria). Degradation of trimethylamine in hypersaline environments by halophilic methanogenic bacteria is relatively well documented, and leads to the formation of methane, carbon dioxide and ammonia.     

Identification of structural nif genes in an iron-oxidising moderate thermophile

The effect of 500 mM NaCl on the growth, and phosphatase production of a Citrobacter sp. was investigated. Although growth was retarded, phosphatase production was enhanced by 50%. Relief from osmotic stress using the osmoprotectant glycine betaine gave normal growth, but phosphatase activity was reduced. The Citrobacter sp. ceased to grow following a shift to anaerobic conditions, but anaerobically-incubated cells continued to produce phosphatase after a transient lag.     

Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli

We present evidence that glycine betaine (betaine) which was synthesized from choline was excreted and reaccumulated in osmoregulating cells of Escherichia coli. Choline which was accumulated in bet mutants defective in betaine synthesis was shown to be excreted in response to betaine uptake. Our data suggest that E. coli has efflux systems for betaine and choline which are independent of the uptake systems for these metabolites. The ProU system of E. coli, but not that of Salmonella typhimurium, can mediate low-affinity choline uptake.     

土壤缓慢脱水对开花期小麦根系及叶片渗透调节及渗透调节物质的影响

以抗旱性不同的小麦品种为材料,在小麦的水分临界期开花期进行缓慢脱水处理,分别在脱水的不同阶段取样测定叶片及根系的渗透调节能力及渗透调节物质。结果表明：随着土壤含水量的降低,叶片与根系的饱和渗透势同步下降,表现出叶片与根系对水分胁迫反应的一到场生,但根系的渗透调节能力低于叶片。根系与叶片的渗透调节物质,一方面在物质总含量方面,表现出与渗透调节能力的一致性,另一方面各种物质的相对含量又有一定差异,叶片中可溶性糖与K＋含量及增加量都高于根系,而根系中的游离氨基酸与Ca^2 的相对增加量则大于叶片。     

干旱胁迫对不同玉米自交系苗期渗透调节的影响

以5个抗旱性不同的玉米骨干自交系黄早四、掖478、郑58、旱21、齐319为试验材料,对持续水分胁迫下玉米幼苗的叶片相对含水量、渗透调节能力(OA)、脯氨酸含量和可溶性糖含量的变化规律进行了研究。结果表明:随着干旱胁迫的加重,叶片相对含水量呈现下降的趋势,渗透调节能力和可溶性糖含量呈现先上升后又下降的趋势,脯氨酸含量呈现持续上升的趋势;在持续干旱条件下,不同自交系各个指标的变化幅度不同,说明不同种质资源对干旱胁迫的响应方式不同,渗透调节能力也有差异。在水分胁迫的前7d中,渗透调节能力逐渐增加,第7天时达到最大值,OA从大到小的排列顺序为齐319郑58掖478旱21黄早四;但此后,除旱21外,其余4个自交系的OA都随之下降。旱21和齐319以可溶性糖和脯氨酸为主要的渗透调节因子,黄早四则以脯氨酸为主要的渗透调节物质。     

Osmotic adjustment in three succulent species of Zygophyllaceae

Three species, Zygophyllum album L., Z. coccineum L. and Z. simplex L., from family Zygophyllaceae were collected from two locations in Egypt to study their response to environmental conditions. Organic solutes (amino acids, soluble proteins and soluble sugars) and inorganic solutes (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?, PO₄^3? and SO₄^2?) were estimated to study their role in osmotic adjustment under the effect of drought and salinity. The study showed that Z. coccineum is most tolerant for drought and salinity than Z. simplex. Z. coccineum was dependent on soluble proteins and soluble sugars, to increase its content of bound water, to undergo water deficit in desert. Z. simplex accumulated inorganic solutes more than Z. coccineum and less organic solutes. Amino acids content increased in Z. coccineum and Z. simplex survived in saline conditions to play a role in osmotic adjustment. Under the effect of salinity, all the studied species showed a tendency and high capacity to accumulate inorganic solutes. The main inorganic salutes were Ca²⁺, Mg²⁺ and Cl^?. The role of Na⁺ was less than Ca²⁺ and Mg²⁺. Z. album and Z. simplex preferred Mg²⁺ more than Z. coccineum which preferred Ca²⁺.     

Osmotic adjustment and the inhibition of leaf,root, stem and silk growth at low water potentials in maize

The expansion growth of plant organs is inhibited at low water potentials ( _w), but the inhibition has not been compared in different organs of the same plant. Therefore, we determined elongation rates of the roots, stems, leaves, and styles (silks) of maize (Zea mays L.) as soil water was depleted. The _w was measured in the region of cell expansion of each organ. The complicating effects of transpiration were avoided by making measurements at the end of the dark period when the air had been saturated with water vapor for 10 h and transpiration was less than 1% of the rate in the light. Growth was inhibited as the _w in the region of cell expansion decreased in each organ. The _w required to stop growth was-0.50,-0.75, and-1.00 MPa, in this order, in the stem, silks, and leaves. However, the roots grew at these _w and ceased only when _w was lower than-1.4 MPa. The osmotic potential decreased in each region of cell expansion and, in leaves, roots and stems, the decrease was sufficient to maintain turgor fully. In the silks, the decrease was less and turgor fell. In the mature tissue, the _w of the stem, leaves and roots was similar to that of the soil when adequate water was supplied. This indicated that an equilibrium existed between these tissues, the vascular system, and the soil. At the same time, the _w was lower in the expanding regions than in the mature tissues, indicating that there was a _w disequilibrium between the growing tissue and the vascular system. The disequilibrium was interpreted as a _w gradient for supplying water to the enlarging cells. When water was withheld, this gradient disappeared in the leaf because _w decreased more in the xylem than in the soil, indicating that a high flow resistance had developed in the xylem. In the roots, the gradient did not decrease because vascular _w changed about the same amount as the soil _w. Therefore, the gradient in _w favored water uptake by roots but not leaves at low _w. The data show that expansion growth responds to low _w differently in different growing regions of the plant. Because growth depends on the maintenance of turgor for extending the cell walls and the presence of _w gradients for supplying water to the expanding cells, several factors could have been responsible for these differences. The decrease of turgor in the silks and the loss of the _w gradient in the leaves probably contributed to the high sensitivity of these organs. In the leaves, the gradient loss was so complete that it would have prevented growth regardless of other changes. In the roots, the maintenance of turgor and _w gradients probably allowed growth to continue. This difference in turgor and gradient maintenance could contribute to the increase in root/shoot ratios generally observed in water-limited conditions.Symbols _s osmotic potential - _w water potential     

高盐环境可培养细菌噬菌体的研究进展

病毒广泛存在于各类环境中并担负着重要的生态功能,其中包括高盐环境。对高盐环境病毒的研究已成为极端环境微生物研究领域的新热点,目前已被报道的100多株盐病毒中,90多株感染古菌,仅有14株感染细菌。本文综述了目前已知的14株高盐环境细菌噬菌体的形态特征、盐度响应及基因组学的研究进展,并分析了高盐噬菌体的形态多样性、生存策略以及包含在基因组中的进化和起源信息,分析结果表明:高盐噬菌体以有尾噬菌体为主;它们具有广盐性(Euryhaline)的特征,盐度极大地影响其吸附和增殖;它们与非高盐环境噬菌体可能具有共同的起源。高盐噬菌体虽然历经近30年的研究历程,但仅有14株被分离与培养,所以其分离纯化是今后重要工作之一,且结合免培养技术揭示高盐噬菌体的多样性与生态功能是其研究的发展方向。     

Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments

All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure is essential since it is generally considered as the driving force for cell expansion. Exposure of microorganisms to high-osmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. To counteract the outflow of water, microorganisms increase their intracellular solute pool by amassing large amounts of organic osmolytes, the so-called compatible solutes. These osmoprotectants are highly congruous with the physiology of the cell and comprise a limited number of substances including the disaccharide trehalose, the amino acid proline, and the trimethylammonium compound glycine betaine. The intracellular amassing of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya. Furthermore, the nature of the osmolytes that are accumulated during water stress is maintained across the kingdoms, reflecting fundamental constraints on the kind of solutes that are compatible with macromolecular and cellular functions. Generally, compatible solutes can be amassed by microorganisms through uptake and synthesis. Here we summarise the molecular mechanisms of compatible solute accumulation in Escherichia coli and Bacillus subtilis, model organisms for the gram-negative and gram-positive branches of bacteria. Received: 12 May 1998 / Accepted: 24 July 1998     

Effects of Mineral Nutrition on Water Status and Osmotic Adjustment in Spring wheat under Different Moisture Conditions

The Ψw and RWC of the leaves of spring wheat at low level of nutrition were higher than those of the spring wheat leaves at high level during the slow drought period. Therefore, the plants at high nutrition level were more sensitive to drought. High mineral nutrition did not enhance the maximum osmotic adjustment but changed the regulatory process of osmotic adjustment under drought condition. During moderate to nearly severe drought period higher osmotic adjustment was shown in the leaves of spring wheat at high nutrition level. The major solute contributed to osmotic adjustment in the leaves of the spring wheat was soluble sugar, mostly reducing sugar.     

Osmotic adjustment in indoor grown cassava in response to water stress

The water content-water potential relation in stressed and unstressed cassava ( Man-ihot species) was examined to ascertain (i) the magnitude of osmotic adjustment in response to water stress and (ii) the mechanisms of such adjustments.
Water stress resulted in a displacement of the water content-potential relation such that at any leaf water potential the water content was higher in the stressed plants. The osmotic potentials of turgid leaves (100% relative water content) were -0.97 and -1.00 MPa in the unstressed cultivars CMC 9 and MCOL 113 respectively. In the stressed plants, the values were-1.13 MPa (CMC 9) and-1.14 MPa (MCOL 113). The 0.14 to 0.16 MPa osmotic potential difference between the stressed and unstressed plants suggests that a stress-induced osmotic adjustment occurred in both cultivars. The biiSk volumetric elastic moduli at turgor pressures above 0.10 MPa were 9.84 MPa (CMC 9) and 13.58 MPa (MCOL 113) in the unstressed plants. Tbe higher values found in the stressed plants, 14.56 MPa in CMC 9 and 16.91 MPa in MCOL 113, suggest a stress-induced decrease in cell wall elasticity. Hence, the observed shift in the wafer content-potential relations in the cassava involved both an osmotic adjustment and a decrease in cell wall elasticity. Increasing the number of stress cycles per plant did not cause a further displacement of the water content-potential curves.