中度嗜盐菌相容性溶质机制的研究进展

生活在高盐环境中的中度嗜盐菌不仅能抗衡外界的高渗透压胁迫,而且还能迅速适应短时间内的渗透冲击。为适应该环境,中度嗜盐菌依赖于一种被称为相容性溶质的物质,以执行渗透保护功能。这类物质属于极性的、易溶的和低分子量的有机化合物,其中包括糖类、氨基酸类、甜菜碱类和四氢嘧啶类等。中度嗜盐菌主要采用相容性溶质机制来适应盐环境。在此,就中度嗜盐菌的盐适应机理、相容性溶质的种类和特点,以及其作用的分子机制进行了阐述和讨论。     

Osmoadaptation of haloalkaliphilic bacteria: Role of osmoregulators and their possible practical application

The review discusses osmoadaptation of haloalkaliphilic bacteria from diverse taxonomic and physiological groups, inhabiting soda lakes. Our experimental research has confirmed the similarity of the osmoregulation strategies in neutrophilic and alkaliphilic halophiles, independent of their pH homeostasis mechanism. The external osmotic pressure is equilibrated either due to accumulation of ions from the environment, or by accumulation or synthesis of cytoplasmic osmoregulatory compounds. The alkaliphiles following the “compatible solutes” strategy contain low or moderate concentrations of salts in their cytoplasm; their proteins do not require adaptation to salts. Those that follow the “salt-in” strategy do not synthesize osmoregulators; they accumulate high levels of salts within the cell and thus equilibrate the osmotic pressures of the cell and the environment. The proteins of these bacteria contain more acidic amino acid residues compared to the proteins of neutrophiles. The functions of bacterial organic osmoregulatory compounds are discussed, as well as their characteristics of possible practical value. Applications for ectoine and betaine are discussed based on the published data.     

Osmoadaptation of haloalkaliphilic bacteria: role of osmoregulators and their possible practical application

The review discusses osmoadaptation of haloalkaliphilic bacteria from diverse taxonomic and physiological groups, inhabiting soda lakes. Our experimental research has confirmed the similarity of the osmoregulation strategies in neutrophilic and alkaliphilic halophiles, independent of their pH homeostasis mechanism. The external osmotic pressure is equilibrated either due to accumulation of ions from the environment, or by accumulation or synthesis of cytoplasmic osmoregulatory compounds. The alkaliphiles following the "compatible solutes" strategy contain low or moderate concentrations of salts in their cytoplasm; their proteins do not require adaptation to salts. Those that follow the "salt-in" strategy do not synthesize osmoregulators: they accumulate high levels of salts within the cell and thus equilibrate the osmotic pressures of the cell and the environment. The proteins of these bacteria contain more acidic amino acid residues compared to the proteins of neutrophiles. The functions of bacterial organic osmoregulatory compounds are discussed, as well as their characteristics of possible practical value. Applications for ectoine and betaine are discussed based on the published data.     

Modulation of endogenous levels of some key organic metabolites by exogenous application of glycine betaine in drought stressed plants of sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

The present study was conducted to examine the changes in some key metabolites in drought-stressed sunflower plants supplied with glycine betaine externally. Imposition of drought stress at the vegetative or reproductive growth stages decreased the plant dry matter production and increased the accumulation of organic solutes (glycine betaine, proline, soluble proteins, free amino acids and soluble sugars) in two sunflower lines, i.e., Glushan-98 and Suncross. In general, decrease in dry matter production and increase in the endogenous levels of organic solutes, were more pronounced when drought stress applied at the vegetative stage than at the reproductive stage. Glycine betaine applied as a pre-sowing seed treatment was not found to be effective in reducing the negative effects of drought stress in sunflower plants. Foliar application of GB further enhanced the leaf endogenous levels of GB, soluble proteins and total soluble sugars in drought stressed plants without exerting any negative effects on other osmotica. However, this GB-induced increase in endogenous levels of organic solutes was found to be not associated with plant dry matter production under stress conditions.     

Compatible solute addition to biological systems treating waste/wastewater to counteract osmotic and other environmental stresses: a review

This study reviews the addition of compatible solutes to biological systems as a strategy to counteract osmolarity and other environmental stresses. At high osmolarity many microorganisms accumulate organic solutes called “compatible solutes” in order to balance osmotic pressure between the cytoplasm and the environment. These organic compounds are called compatible solutes because they can function inside the cell without the need for special adaptation of the intracellular enzymes, and also serve as protein stabilizers in the presence of high ionic strength. Moreover, the compatible solutes strategy is regularly being employed by the cell, not only under osmotic stress at high salinity, but also under other extreme environmental conditions such as low temperature, freezing, heat, starvation, dryness, recalcitrant compounds and solvent stresses. The accumulation of these solutes from the environment has energetically a lower cost than de novo synthesis. Based on this cell mechanism several studies in the field of environmental biotechnology (most of them on biological wastewater treatment) employed this strategy by exogenously adding compatible solutes to the wastewater or medium in order to alleviate environmental stress. This current paper critically reviews and evaluates these studies, and examines the future potential of this approach. In addition to this, a strategy for the successful implementation of compatible solutes in biological systems is proposed.     

Overexpression and purification of halophilic proteins in Haloferax volcanii

Halophilic enzymes function optimally at high salt concentrations and are active at low water availability. Such conditions are encountered at elevated concentrations of solutes such as salts and sugars, and at high concentrations of organic solvents. However, expression in heterologous hosts such as Escherichia coli can cause problems, since halophilic proteins typically misfold and aggregate in conditions of low ionic strength. We have harnessed the sophisticated genetic tools available for the haloarchaeon Haloferax volcanii, to develop a system for the overexpression and purification of halophilic proteins under native conditions.     

The mechanism of cryoprotection of proteins by solutes

We have tested the capacity of 28 different compounds to protect lactate dehydrogenase from damage during freeze-thawing. These solutes come from very dissimilar chemical classes including sugars, polyols, amino acids, methylamines, and lyotropic salts. All the compounds tested, except NaCl, protected the enzyme, to varying degrees, from inactivation. The only characteristic that these compounds have in common, as a group, is that they have all been shown to be preferentially excluded from contact with the surface of proteins in aqueous solution. It has been demonstrated previously (via thermodynamic arguments) that this interaction of solutes with proteins leads to the stabilization of proteins in nonfrozen, aqueous systems. Conversely, those solutes, e.g., urea and guanidine HCl, that bind to proteins destabilize proteins in solution, and we have found that they also enhanced the inactivation of lactate dehydrogenase during freeze-thawing. Based on the results of our freeze-thawing experiments and a review of the theory of protein stabilization in nonfrozen, aqueous solution we propose that the cryoprotection afforded to isolated proteins by solutes can be accounted for by the fact that these solutes are preferentially excluded from contact with the protein's surface.     

Organic solute accumulation in osmotically stressed cyanobacteria

Abstract Three groups of cyanobacteria are recognized on the basis of their organic osmotica and upper salinity limit for growth. In general, the least halotolerant forms accumulate disaccharides, while cyanobacteria of intermediate halotolerance synthesize the heteroside glucosylglycerol and the most halotolerant isolates accumulate betaines in response to salt stress. However, certain strains also accumulate additional organic solutes, depending upon the growth temperature, the ambient salinity and the duration of salt stress.     

Glycinebetaine in saline conditions: an assessment of the current state of knowledge

Salt stress is one of the environmental threats that have devastating impacts on plant distribution, growth and production. Different plants are believed to have salt tolerance mechanisms that occur at the cellular level. One facet of the cellular mechanisms of adaptation to salinity stress is to accumulate either inorganic and/or organic solutes. Glycinebetaine (GB), as well as other organic solutes, has been referred to as compatible solutes, for the reason that they are innocent with essential biochemical reactions even at high concentrations. GB has been assumed to be involved in osmotic adjustment and/or osmoprotection of cellular functional macromolecules and, hence, can improve tolerance to saline conditions. However, the exact mechanism and direct evidences for such correlative data are still lacking despite many attempts to improve growth under saline conditions by exogenous application as well as genetic engineering of metabolic pathways involved in metabolism of GB. Despite the enormous amount of information accumulated in this regard, the exact function of GB in the adaptation to saline environments is not fully clear to this point, and even GB functions have been argued. Because of that, inconsistencies exist in the published data regarding GB accumulation and functions under salt stress. In this review, we provide an update on evidence supporting each of these arguments to reassess how GB affects plant growth and physiological traits under salt imposition, and whether its effects correlate with salt tolerance.     

Likely effects of salinity on acute copper toxicity to the fisheries of the lake George-Edward basin

Alkaline, saline waters are common in the Western Rift Valley of East Africa, in which the lake George-Edward basin is situated. A growing copper mining industry in the area makes it important to understand the limnology of the lakes in this basin before copper pollution occurs. The fish could possibly suffer from acute (or chronic) toxicity if copper levels increase.Abiotic factors within the alkaline, saline waters of this basin reduce the acute toxic effects of copper to fish. The most important factor is salinity, which is a measure of the total dissolved mineral salts. The relatively highly concentrations of mineral salts of these waters will to reduce the effective copper ionic activity through adsorption, precipitation, and ionic interference. The high concentrations of organic compounds in the waters, also complex and chelate the ionic Cu²⁺, thus reducing further its effective concentration. This will therefore act as a check on the copper toxicity to the fish of the lake basin.     

Solute Accumulation in Plant Cells: III. Treatments which Arrest and Restore the Course of Absorption and Growth in Cultured Carrot Explants1

Prior papers have dealt with the range of the growth responsesof carrot explants to the composition of the ambient media andhow these affected the solute concentration and compositionof the tissue. They have also dealt with the sequential eventsalong the time course of growth of the tissue explant. Thispaper presents results and conclusions derived from experimentswhich exploit changes in the growing explants when their normalcourse of growth and solute uptake is interrupted by exposingthem sequentially to different ambient media. After explants were induced to grow during an initial 6 days,they were placed in a minimal nutrient medium which lacked growthstimuli and salts, notably potassium ions. Thus the internalsalt concentrations of the cells declined as they continuedto divide and enlarge at a reduced rate and their osmotic valuewas maintained by storage of organic solutes (sugar). The subsequentresponses of these ‘low salt’ cells to differentnutrient regimes were studied. When salts were resupplied, growth was stimulated somewhat andthe osmotic value of the cells increased as salts were accumulated;with a renewed full nutrient medium and a full complement ofgrowth substances, the cultures re-embark upon their growthand attain the average cell size and composition as if theyhad not been reversibly arrested. Thus reversible trends insolute composition of cells may be superimposed upon their normaldevelopmental course by alternately withholding and restoringthe stimuli to their growth and by changing the balance betweenorganic and inorganic solutes supplied in the medium. The emphasis is on the control of osmotic value in the cellsas they enlarge and mature and this over-rides the changes inthe solutes they receive (salts or sugars) via the ambient medium.The effects here observed were induced by sequential changesin the culture medium, but these obviously relate to similarresponses of cells in the intact plant body as their growthand solute supply is modified through interactions between organsas the plant grows.     

Potential applications of stress solutes from extremophiles in protein folding diseases and healthcare

Protein misfolding, aggregation and deposition in the brain, in the form of amyloid, are implicated in the etiology of several neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and prion diseases. Drugs available on the market reduce the symptoms, but they are not a cure. Therefore, it is urgent to identify promising targets and develop effective drugs. Preservation of protein native conformation and/or inhibition of protein aggregation seem pertinent targets for drug development. Several studies have shown that organic solutes, produced by extremophilic microorganisms in response to osmotic and/or heat stress, prevent denaturation and aggregation of model proteins. Among these stress solutes, mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, diglycerol phosphate and ectoine are effective in preventing amyloid formation by Alzheimer’s Aβ peptide and/or α-synuclein in vitro. Moreover, mannosylglycerate is a potent inhibitor of Aβ and α-synuclein aggregation in living cells, and mannosylglyceramide and ectoine inhibit aggregation and reduce prion peptide-induced toxicity in human cells. This review focuses on the efficacy of stress solutes from hyper/thermophiles and ectoines to prevent amyloid formation in vitro and in vivo and their potential application in drug development against protein misfolding diseases. Current and envisaged applications of these extremolytes in neurodegenerative diseases and healthcare will also be addressed.     

Accumulation of organic and inorganic solutes in the subantarctic cruciferous species Pringlea antiscorbutica in response to saline and cold stresses

Pringlea antiscorbutica R. Br., a subantarctic endemic cruciferous species, is endangered in its natural sites by several ecological changes. This species is tolerant to salinity and a permanent cold temperature on Kerguelen and Crozet Islands. We attempted the investigation of regulating mechanisms of osmotic adjustment in this species. ¹³C NMR analyses of water-soluble compounds from leaves collected from the field revealed glucose and proline to be the main accumulated organic solutes. Colorimetric determinations in these samples showed that proline and soluble carbohydrates were present at remarkably high levels. When young plants were cultivated in growth chambers they showed a good resistance to cold and medium resistance to saline conditions. High levels of soluble carbohydrates were present in all situations. Proline was accumulated in response to a saline and a cold treatment. The quantitative variations of the pool of proline in response to saline treatments were rapid and important. The adaptive value of these responses of organic solutes in the tolerance of Pringlea antiscorbutica to various stresses is discussed. Received: 16 June 1997 / Accepted: 5 May 1998     

Glycine-rich proteins encoded by a nodule-specific gene family are implicated in different stages of symbiotic nodule development in Medicago spp

Four genes encoding small proteins with significantly high glycine content have been identified from root nodules of Medicago sativa. All of these proteins as well as their Medicago truncatula homologues carried an amino terminal signal peptide and a glycine-rich carboxy terminal domain. All except nodGRP3 lacked the characteristic repeat structure described for cell wall and stress response-related glycine-rich proteins (GRP). Expression of these GRP genes was undetectable in flower, leaf, stem, and hypocotyl cells, whereas expression was highly induced during root nodule development, suggesting that GRP genes act as nodulins. Moreover, none of these nodule-expressed GRP genes were activated by hormones or stress treatments, which are inducers of many other GRPs. In Rhizobium-free spontaneous nodules and in nodules induced by a noninfective mutant strain of Sinorhizobium meliloti, all these genes were repressed, while they were induced in Fix- nodules, unaffected in bacterial infection, but halted in bacteroid differentiation. These results demonstrated that bacterial infection but not bacteroid differentiation is required for the induction of the nodule-specific GRP genes. Differences in kinetics and localization of gene activation as well as in the primary structure of proteins suggest nonredundant roles for these GRPs in nodule organogenesis.     

Effects of ribosomes and intracellular solutes on activities and stabilities of elongation factor 2 proteins from psychrotolerant and thermophilic methanogens

Low-temperature-adapted archaea are abundant in the environment, yet little is known about the thermal adaptation of their proteins. We have previously compared elongation factor 2 (EF-2) proteins from Antarctic (Methanococcoides burtonii) and thermophilic (Methanosarcina thermophila) methanogens and found that the M. burtonii EF-2 had greater intrinsic activity at low temperatures and lower thermal stability at high temperatures (T. Thomas and R. Cavicchioli, J. Bacteriol. 182:1328-1332, 2000). While the gross thermal properties correlated with growth temperature, the activity and stability profiles of the EF-2 proteins did not precisely match the optimal growth temperature of each organism. This indicated that intracellular components may affect the thermal characteristics of the EF-2 proteins, and in this study we examined the effects of ribosomes and intracellular solutes. At a high growth temperature the thermophile produced high levels of potassium glutamate, which, when assayed in vitro with EF-2, retarded thermal unfolding and increased catalytic efficiency. In contrast, for the Antarctic methanogen adaptation to growth at a low temperature did not involve the accumulation of stabilizing organic solutes but appeared to result from an increased affinity of EF-2 for GTP and high levels of EF-2 in the cell relative to its low growth rate. Furthermore, ribosomes greatly stimulated GTPase activity and moderately stabilized both EF-2 proteins. These findings illustrate the different physiological strategies that have evolved in two phylogenetically related but thermally distinct methanogens to enable EF-2 to function satisfactorily.     

Loss of function of biomembranes and solubilization of membrane proteins during freezing

Isolated thylakoid membranes are damaged during freezing in dilute salt solutions, as shown by the inactivation of photochemical thylakoid reactions. After freezing, a number of membrane proteins were found in the particle-free supernatant. Up to 5% of the total membrane protein was solubilized by freezing, and the pattern of released proteins as seen in sodium dodecyl sulfate gel electrophoretograms was influenced by the nature of the solutes present. Membranes protected by sucrose did not release much protein during freezing. Concentrated salt solutions caused protein release also in the absence of freezing. Among the proteins released were ferredoxin—NADP⁺ reductase, plastocyanin and coupling factor CF₁. Subunits of CF₁ were found in different proportions in the supernatants of thylakoid suspensions after freezing in the presence of different salts. Cyclic photophosphorylation was largely inactivated before significant protein release could be detected.It is suggested that protein release is the final consequence of the non-specific suppression of intramembrane ionic interactions by the high ionic strength created in the vicinity of the membranes by the accumulation of salts during slow freezing. Salt effects on water structure and alterations of nonpolar membrane interactions by the incorporation of (protonated) lipophilic anions from organic salts into the membrane phase during freezing may also be involved.     

Metabolism of chloride in halophilic prokaryotes

While much understanding has been achieved on the intracellular sodium and potassium concentrations of halophilic and halotolerant microorganisms and on their regulation, we know little on the metabolism of anions. Archaea of the family Halobacteriaceae contain molar concentrations of chloride, which is pumped into the cells by cotransport with sodium ions and/or using the light-driven primary chloride pump halorhodopsin. Most halophilic and halotolerant representatives of the bacterial domain contain low intracellular ion concentrations, with organic osmotic solutes providing osmotic balance. However, some species show a specific requirement for chloride. In Halobacillus halophilus certain functions, such as growth, endospore germination, motility and flagellar synthesis, and glycine betaine transport are chloride dependent. In this organism the expression of a large number of proteins is chloride regulated. Other moderately halophilic Bacteria such as Halomonas elongata do not show a specific demand for chloride. A very high requirement for chloride was demonstrated in two groups of Bacteria that accumulate inorganic salts intracellularly rather than using organic osmotic solutes: the anaerobic Halanaerobiales and the aerobic extremely halophilic Salinibacter ruber. It is thus becoming increasingly clear that chloride has specific functions in haloadaptation in different groups of halophilic microorganisms.     

Solute Accumulation in Plant Cells: I. Reciprocal Relations Between Electrolytes and Non-Electrolytes1

This paper presents the concepts, the analytical methods, andthe experimental devices used in a reappraisal of the problemsof solute and water uptake which utilizes both quiescent andactively growing cells. The tissue used is drawn from the secondaryphloem of the carrot root and, in all experiments, it is underconditions of aseptic culture which permit both inorganic andorganic solutes to be studied for relatively long periods. The range of responses of the explanted carrot tissue has beenobserved in different media. These include simple inorganicsalt solutions (CaCl₂, KC1, NaCl, etc.), a full organic andinorganic nutrient medium and also the latter supplemented bystimuli that unleash the full ability of the otherwise restingcells to grow. The effects on both growth and composition of the cells havebeen observed with time. The high osmotic value of the maturenon-growing cells may be made up, non-specifically, by salts(KC1, NaCl) or organic solutes (sugars) which are accumulated;when growth is not primarily involved these solutes may thenbehave reciprocally in accordance with supply, in the media,and demand, in the cells. Rapidly dividing cells, on the other hand, creating vacuoles,have lower osmotic value, greater specificity for potassium,and the solutes they store are under more endogenous than exogenouscontrol. Between these extremes the solutes which are accumulated dependupon the levels of growth induced which in turn are responsesto the nutrients and stimuli furnished. These observations and their interpretation set a trend forthe papers that are to follow.     

High and low density intracellular water.

The proposal that liquid water consists of microdomains of rapidly-exchanging polymorphs of high and low density is examined for its impact upon roles of water in biology. It is assumed that the two polymorphs persist in solution and adjacent to surfaces and that solutes partition asymmetrically between them. It transpires that chaotropes are solutes which partition preferentially into low density water and displace the water equilibrium toward the high density polymorph. Kosmotropes. both ionic and non-polar, partition into high density water and induce low density water. Displacement of the water equilibrium at constant temperature and pressure has a thermodynamic cost which can be high. This appears to be a dominant factor in folding of proteins and DNA, aggregation of biopolymers and insolubility of non-polar kosmotropes. Cells control both the concentration of proteins and the selection of small solutes to produce an intracellular environment most conducive to co-ordinated enzyme function. Intracellular water has similar microdomains to bulk water, but surfaces and solutes redistribute them. Average properties, as measured by NMR are similar, but local properties on a nm scale may differ widely. Enzymes apparently use these local differences to activate cations for transport, induce movement and for synthesis.