Heat and cold shock protein synthesis in arctic and temperate strains of rhizobia.

We compared heat shock proteins (HSPs) and cold shock proteins (CSPs) produced by different species of Rhizobium having different growth temperature ranges. Several HSPs and CSPs were induced when cells of three arctic (psychrotrophic) and three temperate (mesophilic) strains of rhizobia were shifted from their optimal growth temperatures (arctic, 25 degrees C; temperate, 30 degrees C) to shock temperatures outside their growth temperature ranges. At heat shock temperatures, three major HSPs of high molecular weight (106,900, 83,100, and 59,500) were present in all strains for all shock treatments (29, 32, 36.4, 38.4, 40.7, 41.4, and 46.4 degrees C), with the exception of temperate strains exposed to 46.4 degrees C, in which no protein synthesis was detected. Cell survival of arctic and temperate strains decreased markedly with the increase of shock temperature and was only 1% at 46.4 degrees C. Under cold shock conditions, five proteins (52.0, 38.0, 23.4, 22.7, and 11.1 kDa) were always present for all treatments (-2, -5, and -10 degrees C) in arctic strains. Among temperate strains, five CSPs (56.1, 37.1, 34.4, 17.3, and 11.1 kDa) were present at temperatures down to 0 degrees C. The 34.4- and the 11.1-kDa components were present in all temperate strains at -5 degrees C and in one strain at -10 degrees C. Survival of all strains decreased with cold shock temperatures but was always higher than 50%. These results show that rhizobia can synthesize proteins at temperatures not permissive for growth. In all shock treatments, no correspondence between the number of HSPs or CSPs produced and rhizobial survival was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Symbiotic effectiveness of indigenous arctic rhizobia on a temperate forage legume: Sainfoin (Onobrychis viciifolia)

Sainfoin (Onobrychis viciifolia), a temperate perennial forage legume, can be nodulated by rhizobia isolated from 3 arctic legume species:Astragalus alpinus, oxytropis maydelliana andOxytropis arctobia. Arctic rhizobia, which are adapted to growth at low temperatures, may be useful in improving symbiotic nitrogen fixation during cold phases of the growing season, if they are effective on a temperate legume. In this study, we report on the symbiotic effectiveness of arctic rhizobia on sainfoin, as appraised by the total shoot dry matter yield obtained from 2 harvests. Under N-free conditions, 5 arctic strains at the first harvest and 8 at the second harvest were as effective as temperate standard strains. In the presence of 30 mgl⁻¹ NO₃-N, 7 arctic strains gave significantly higher yields than temperate strains at the second harvest. These results indicate that effective arctic rhizobia have a potential for use as inoculants on sainfoin. Contribution no 325 of Agriculture Canada Research Station a Sainte-Foy.     

Varying patterns of protein synthesis in bread wheat during heat shock

High temperatures during seedling growth are considered as one of the factors that can modify surviving properties in wheat (Triticum aestivum L.) plant. This work attempts to evaluate the heat shock responses of seedling of winter wheat (Bezostaya-1) using growth parameters (seedling length, embryonal root length and embryonal root number), membrane stability index (MSI) and two dimensional (2D) gel electrophoresis analysis of heat shock proteins (HSPs) during heat shock. Seedlings grown until first leaf opening at controlled conditions (23 degrees C, 200 micromol m(-2) s(-1), 16h day/8h night, 50-60% humidity) were exposed to 37 degrees C or 45 degrees C high temperatures for 2, 4 and 8 hours. While 37 degrees C did not cause any significant change, 45 degrees C heat treatments caused significant decrease in terms of seedling and root length, and leaf MSI for all exposure times. However, all the plants from 45 degrees C heat treatments continued to grow during recovery period. 2D protein analysis indicated that 37 degrees C, 8 hours exposure caused stronger and more diverse heat shock response than the other treatments, followed by 37 degrees C, 4 hours, 45 degrees C, 8 hours, 45 degrees C, 4 hours, 45 degrees C, 2 hours treatments. 5 protein spots, ranging from 6-7.8 pl (isoelectric point) and 27-31.7 kDA molecular weight, were expressed at 37 degrees C, 2 hours and continued at 37 and 45 degrees C for all exposure times. This suggests that these early proteins and other newly synthesized proteins may have protective effects at 37 and 45 degrees C and provide plants for healthy growth during the recovery period.     

Differentiation between cold shock proteins and cold acclimation proteins in a mesophilic gram-positive bacterium, Enterococcus faecalis JH2-2.

Transfer of Enterococcus faecalis to a cold temperature (8 degrees C for 4 to 30 h) led to increased expression of 11 cold shock proteins (CSPs). Furthermore, this mesophilic prokaryote synthesized 10 cold acclimation proteins, five of them distinct from CSPs, during continuous growth (4 days) at the same temperature (8 degrees C).     

Synthesis of soluble heat shock proteins in seminal root tissues of some cultivated and wild wheat genotypes

Effect of heat stress on the synthesis of soluble heat shock proteins (HSPs) and the regrowth in seminal roots of three cultivated and three wild wheat genotypes was examined. In regrowth experiments, 2-d-old etiolated seedlings were exposed to 23 (control), 32, 35, 37 and 38 degrees C for 24 h, and 35 and 37 degrees C (24 h) followed by 50 degrees C (1 h). The lengths of the seminal roots generally decreased significantly at the end of 48 and 72 h recovery growth periods at 35, 37 and 38 degrees C temperature treatments compared with control. Genotypic variability was significant level at all temperature treatments for the seminal root length. Also, genotypic differences for the number of seminal roots were determined among the wheat cultivars and between the wild wheat species and the wheat cultivars at all temperature treatments; but genotypic differences among wild wheat species were only detected at 37-->50 degrees C treatment. Acquired thermotolerance for the seminal root length is over 50% at 37-->50 degrees C treatment. The genotypic variability of soluble heat shock proteins in seminal root tissues were analyzed by two-dimensional electrophoresis (2-DE). Total number of low molecular weight (LMW) HSPs was more than intermediate-(IMW) and high- (HMW) HSPs at high temperature treatments. The most of LMW HSPs which were generally of acidic character ranged between 14.2-30.7 kDa. The genotypes had both common (43 HSP spots between at least two genotypes and 23 HSP spots between 37 and 37-->50 degrees C) and genotype-specific (72 HSP spots) LMW HSPs.     

Nodulation and nitrogen fixation in extreme environments

Biological nitrogen fixation is a phenomenon occurring in all known ecosystems. Symbiotic nitrogen fixation is dependent on the host plant genotype, theRhizobium strain, and the interaction of these symbionts with the pedoclimatic factors and the environmental conditions. Extremes of pH affect nodulation by reducing the colonization of soil and the legume rhizosphere by rhizobia. Highly acidic soils (pH<4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high concentrations of aluminium and manganese which are often toxic for both partners; nodulation is more affected than host-plant growth and nitrogen fixation. Highly alkaline soils (pH>8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduce nitrogen fixation. Nodulation and N-fixation are observed under a wide range of temperatures with optima between 20–30°C. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate Iegumes, whereas in tropical legumes nitrogen fixation efficiency is mainly affected. Furthermore, temperature changes affect the competitive ability ofRhizobium strains. Low temperatures reduce nodule formation and nitrogen fixation in temperate legumes; however, in the extreme environment of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates, indicating that both the plants and their rhizobia have successfully adapted to arctic conditions. In addition to low temperatures, arctic legumes are exposed to a short growing season, a long photoperiod, low precipitation and low soil nitrogen levels. In this review, we present results on a number of structural and physiological characteristics which allow arctic legumes to function in extreme environments.     

Low-temperature-induced changes in trehalose,mannitol and arabitol associated with enhanced tolerance to freezing in ectomycorrhizal basidiomycetes (<Emphasis Type="Italic">Hebeloma</Emphasis> spp.)

Ectomycorrhizal fungi have been shown to survive sub-zero temperatures in axenic culture and in the field. However, the physiological basis for resistance to freezing is poorly understood. In order to survive freezing, mycelia must synthesise compounds that protect the cells from frost damage, and certain fungal-specific soluble carbohydrates have been implicated in this role. Tissue concentrations of arabitol, mannitol and trehalose were measured in axenic cultures of eight Hebeloma strains of arctic and temperate origin grown at 22, 12, 6 and 2 degrees C. In a separate experiment, mycelia were frozen to -5 degrees C after pre-conditioning at either 2 degrees C or 22 degrees C. For some, especially temperate strains, there was a clear increase in specific soluble carbohydrates at lower growth temperatures. Trehalose and mannitol were present in all strains and the highest concentrations (close to 2.5% and 0.5% dry wt.) were recorded only after a cold period. Arabitol was found in four strains only when grown at low temperature. Cold pre-conditioning enhanced recovery of mycelia following freezing. In four out of eight strains, this was paralleled by increases in mannitol and trehalose concentration at low temperature that presumably contribute towards cryoprotection. The results are discussed in an ecological context with regard to mycelial overwintering in soil.     

Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli

Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.     

Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.     

The cold shock response of Lactobacillus casei: relation between HPr phosphorylation and resistance to freeze/thaw cycles

When carrying out a proteome analysis with a ptsH3 mutant of Lactobacillus casei, we found that the cold shock protein CspA was significantly overproduced compared to the wild-type strain. We also noticed that CspA and CspB of L. casei and CSPs from other organisms exhibit significant sequence similarity to the C-terminal part of EIIA(Glc), a glucose-specific component of the phosphoenolpyruvate:sugar phosphotransferase system. This similarity suggested a direct interaction of HPr with CSPs, as histidyl-phosphorylated HPr has been shown to phosphorylate EIIA(Glc) in its C-terminal part. We therefore compared the cold shock response of several carbon catabolite repression mutants to that of the wild-type strain. Following a shift from 37 degrees C to lower temperatures (20, 15 or 10 degrees C), all mutants showed significantly reduced growth rates. Moreover, glucose-grown mutants unable to form P-Ser-HPr (ptsH1, hprK) exhibited drastically increased sensitivity to freeze/thaw cycles. However, when the same mutants were grown on ribose or maltose, they were similarly resistant to freezing and thawing as the wild-type strain. Although subsequent biochemical and genetic studies did not allow to identify the form of HPr implicated in the resistance to cold and freezing conditions, they strongly suggested a direct interaction of HPr or one of its phospho-derivatives with CspA and/or another, hitherto undetected cold shock protein in L. casei.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

Differences in egg thermotolerance between tropical and temperate populations of the migratory locust Locusta migratoria (Orthoptera: Acridiidae)

The migratory locust Locusta migratoria L., which is widely distributed throughout the world, exhibits within- and between-population variation in cold tolerance. To understand physiological adaptation in populations, we studied the genetic basis of thermotolerance in Hainan (tropical) and Liaoning (temperate) populations and measured expression of Hsp70 and Hsp90 mRNA in both populations at low (0 degrees C) and high temperatures (40 degrees C). Phenotypic variation of thermotolerance is heritable. Heritable characteristics differed among different stages of locust egg development, as well as among different measures of thermotolerance. Nuclear genetic factors, rather than cytoplasmic factors, contribute to differences in cold tolerance between the tropical and temperate populations of the migratory locust; for heat tolerance, maternal effects were involved in three stages of egg development. Expression of Hsp90 mRNA was induced in temperate population after heat shock (40 degrees C x 12h), whereas expression of Hsp70 and 90 was induced in tropical population after cold shock (0 degrees C x 12h). We suggest that thermotolerance of locust eggs has a complex genetic basis and heat shock proteins may be involved in differences of thermotolerance between locust populations.     

乳酸乳球菌中冷休克蛋白基因的高效表达及其抗冻保护作用

[目的]微生物在适应外界环境急剧降温的条件下都会发生应激反应,产生一系列蛋白质被称为冷休克蛋白.冷休克蛋白对乳酸菌适应低温环境和增强抗冻能力方面发挥着重要作用.本文目的是为了研究乳酸乳球菌中冷休克蛋白CspC、CspD的作用.[方法]将冷休克蛋白CspC、CspD基因分别重组到质粒pNZ8148,转化乳酸乳球菌NZ9000后,加入Nisin诱导,对表达产物进行SDS-PAGE电泳分析,比较重组菌与空白菌在30℃条件下菌体生长差异及反复冻融活菌数的差异.[结果]得出CspC、CspD的相对分子量分别为7.0、6.2 kDa.[结论]CspC使菌体更加迅速的恢复了生长;冷休克蛋白CspD增强了菌体的抗冻存活率(增加了30～40倍).     

SDS-PAGE heat-shock protein profiles of environmental Aeromonas strains

Aeromonas microorganisms normally grow at temperatures between 5 degrees C and 45 degrees C and therefore should have high thermotolerance. Thus it was of interest to find out whether A. hydrophila, A. caviae and A. veronii biovar sobria serovars respond to abrupt temperature changes with a heat shock-like response. To this end the present study was undertaken to determine whether Aeromonas species exhibits a heat shock response to different temperatures and time factors. The response of Aeromonas serovars to 24 h and 48 h of thermal stress at 25 degrees C, 42 degrees C and 50 degrees C involved the synthesis of 12-18 heat shock proteins (HSPs) bands with molecular weights ranging between 83.5-103.9 kDa in the high HSP molecular mass and 14.5-12.0 as low molecular mass HSP. Electrophoretic analysis of the HSPs showed that the serovars do not cluster very tightly and also that they are distinct from each other.     

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15°C as well as at 37°C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B . subtilis . After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.     

Unfolding and double-stranded DNA binding of the cold shock protein homologue Cla h 8 from Cladosporium herbarum

The cloning, purification, and biophysical characterization of the first eukaryotic cold shock protein homologue, Cla h 8, expressed as single functional polypeptide is reported here. It was discovered as a minor allergen of the mold Cladosporium herbarum by phage display using a library selectively enriched for IgE-binding proteins. Based on the sequence homology of Cla h 8 with bacterial cold shock proteins (CSPs), a homology-based computer model of the allergen was computed indicating an all-beta structure of Cla h 8. This major structural feature was confirmed by CD spectroscopy. Despite the structural similarities with bacterial CSPs, the DNA-binding and unfolding behavior of Cla h 8 exhibited unique and previously undescribed characteristics. High affinities of Cla h 8 for single-stranded DNA as well as for double-stranded DNA corresponding to the human Y-box were detected. The affinity for double-stranded DNA increased significantly with decreasing temperature, which was paralleled by an increase in the beta sheet content of the protein. Temperature-dependent fluorescence anisotropy and far-UV CD measurements revealed different unfolding transitions at 28 and at 35.7 degrees C, respectively, indicating a multistate transition, which is uncommon for CSPs. The enhanced affinity for DNA at low temperatures together with the low unfolding transition refer to the functional significance of Cla h 8 at reduced temperatures.     

微生物冷休克蛋白的生理功能及其潜在应用

冷休克反应是普遍存在于微生物体内的一种适应环境温度骤降的应答机制,而氨基酸序列高度保守的冷休克蛋白则是调控冷休克反应的重要因子。越来越多的研究表明,冷休克蛋白除了调控冷休克反应外,还参与调控了生物体多个性状,如宿主正常生长和分化、病原菌的侵袭力和致病性以及宿主对多种逆境（高渗透压、抗生素）的应答。综述了冷休克蛋白及其来源、其参与调控的生理性状和基于冷休克反应及冷休克蛋白的应用,以期为发酵条件优化、食品储藏、致病菌抑制以及作物性状改良等方面提供有益参考。     

Effects of temperature stress on bean-nodulating Rhizobium strains

High soil temperatures in tropical areas limit nodulation and dinitrogen fixation by strains of Rhizobium. Several heat-tolerant bean-nodulating Rhizobium strains have been isolated previously. However, the basis of their resistance to heat remains unknown. In this study, we compared the effects of heat on symbiotic nitrogen fixation, cell survival, amino acid uptake, and protein synthesis in a heat-tolerant (CIAT899) and a heat-sensitive (CNPAF512) bean-nodulating Rhizobium strain. Acetylene reduction activity of nodulated roots excised from unstressed plants was strongly diminished at 35 or 40 degrees C when plants were nodulated either by CIAT899 or by CNPAF512. When these strains were tested under free-living conditions, survival at 40 degrees C as well as the kinetics of l-[S]methionine uptake and protein synthesis at 35 and 40 degrees C indicated the higher tolerance of CIAT899 than of CNPAF512 to thermal stress. The synthesis of heat shock proteins was detected in both strains, although at different temperatures. Increased synthesis of 14 heat shock proteins in CNPAF512 and of 6 heat shock proteins in CIAT899 was observed at 40 and 45 degrees C, respectively. A heat shock protein of approximately 21 kDa, of which the synthesis was strongest in both Rhizobium strains upon a temperature shift up, was also conserved in several other bean-nodulating rhizobia. Acquired thermotolerance in CIAT899 was shown to depend on protein synthesis.     

Changes in protein composition ofSaccharomyces brewing strains in response to heat shock and ethanol stress

Summary Heat shock and ethanol stress of brewing yeast strains resulted in the induction of a set of proteins referred to as heat shock proteins (HSPs). At least six strongly induced HSPs were identified in a lager brewing strain and four HSPs in an ale brewing strain. Four of these HSPs with molecular masses of approximately 70, 38, 26 and 23 kDa were also identified in two laboratory strains ofSaccharomyces cerevisiae. The appearance of HSPs correlated with increased survival of strains at elevated temperatures and high concentrations of ethanol. These results suggest that HSPs may play a role in the ethanol and thermotolerance of yeasts. The properties of these proteins and membrane fatty acids in relation to heat and ethanol shock are being investigated.