The ultrastructure of immobilised desiccated cells of the cyanobacterium Nostoc commune UTEX 584

Cells of the cyanobacterium Nostoc commune UTEX 584 were immobilised, subjected to acute matric water stress (ψ_m = −128 MPa) and then desiccated. Their ultrastructure was investigated by the use of an anhydrous fixation procedure. Although shrunken and bleached, the integrity of the vegatative cells at the ultrastructural level was apparently preserved. The ease with which certain cyanobacterial cells can recover from desiccation may be consequent upon the maintenance of cellular organisation at the ultrastructural level.     

Sensitivity of Nostoc commune UTEX 584 (Cyanobacteria) to water stress

Cells of Nostoc commune UTEX 584 from liquid cultures expressed an upshift in nitrogenase activity when immobilised on inert supports and exposed to matric water potentials between -1.10 and -99.5 MPa. Cells incubated at 0.10 MPa (a_w=c 1.0) maintained increased activity for at least 48 h following immobilization. At water potentials below -23.1 MPa (a_w=0.85), the upshift was transitory. Nitrogenase activity decreased rapidly when immobilised cells were incubated at lower values of _m.Desiccated cells stored at -99.5 MPa (a_w=0.50) underwent an upshift in nitrogenase activity, and in the size of the intracellular ATP pool, when rewetted with either distilled water or liquid MB_o medium (_o =-0.18 MPa). The upshift in nitrogenase activity was chloramphenicol-sensitive and was preceeded by a lag. The duration of the lag depended on the time taken to equilibrate cells to-99.5 MPa, the time desiccated, and the conditions of storage and rewetting. Cells that had no, or very low, nitrogenase activity when rewetted in air, showed a marked stimulation of nitrogenase activity in the presence of 5% v/v CO₂ under both aerobic and anerobic conditions.When rewetted in the presence of 1% w/v glucose (_o =-0.14 MPa), vegetative cells remained intact, but heterocysts underwent autolysis and nitrogenase activity was not detected, even in the presence of 5% v/v CO₂.Abbreviations TTC 2,3,5-triphenyl-2-tetrazolium chloride - _m matric water potential - _o osmotic water potential - a_w water activity     

Spectroscopical and functional characterization of the hemoglobin of Nostoc commune UTEX 584 (Cyanobacteria)

Structural analysis of a monomeric hemoglobin from the cyanobacterium Nostoc commune strain UTEX 584, cyanoglobin (Potts et al. (1992) Science 256, 1690–1692), is presented. Cyanoglobin binds molecular oxygen reversibly, with high oxygen affinity and non-cooperativity. There was no evidence for decreased stability of the pigment at 37°C. Cyanoglobin-specific antibodies showed no cross-reactivity with two reference hemoglobins, leghemoglobin a and sperm whale myoglobin. The absorption spectral properties of cyanoglobin differ significantly from those of the two reference hemoglobins. The spectrum of oxy-cyanoglobin most closely resembles that of an oxy-hemoglobin from the protozoan Tetrahymena pyriformis, a hemoprotein that shares substantial amino-acid sequence identity with cyanoglobin. Met-cyanoglobin possesses spectral characteristics at pH 7.0–9.0 that resemble those of the alkaline met-hemoglobin (a putative hemichrome) of another protozoan, Paramecium caudatum. The spin-state character of met-cyanoglobin is pH-dependent. Met-cyanoglobin does not coordinate the strong-field ligands, cyanide and azide, at pH 7.0. The capacity of cyanoglobin to coordinate cyanide increased with decreasing pH. Far-UV CD spectra of cyanoglobin are indicative of a protein with a significant amount of alpha-helical structure. Data from Soret-region CD spectra suggest that the orientations of the heme moieties in cyanoglobin and leghemoglobin a are similar to one another.     

Extracellular polysaccharide of Nostoc commune (Cyanobacteria) inhibits fusion of membrane vesicles during desiccation

Cells of the cyanobacterium Nostoc commune secrete a complex, high molecular weight, extracellular polysaccharide (EPS) which accumulates to more than 60% of the dry weight of colonies. The EPS was purified from the clonal isolate N. commune DRH1. The midpoint of the membrane phase transition (T_m) of desiccated cells of N. commune CHEN was low (T_{m dry} = 8 °C) and was comparable to the T_m of rehydrated cells((T_m)_H20 = 6 °C). The EPS was not responsible for the depression of T_m. However, the EPS, at low concentrations, inhibited specifically the fusion of phosphatidylcholine membrane vesicles when they were dried in vitro at0% relative humidity (−400 MPa). Low concentrations of a trehalose:sucrose mixture, in a molar ratio which corresponded with that present in cells in vivo, together with small amounts of the EPS, were efficient in preventing leakage of carboxyfloroscein (CF) from membrane vesicles. Freeze-fracture electron microscopy resolved complex changes in the structure of the EPS and the outer membrane in response to rehydration of desiccated cells. The capacity of the EPS to prevent membrane fusion, the maintenance of a low T_{m dry} in desiccated cells, and the changes in rheological properties of the EPS in response to water availability, constitute what are likely important mechanisms for desiccation tolerance in this cyanobacterium. This revised version was published online in August 2006 with corrections to the Cover Date.     

The protein index ofNostoc commune UTEX 584 (cyanobacteria): changes induced in immobilized cells by water stress

Two-dimensional gel electrophoresis was used to analyze the effects of water stress (immobilization and rapid drying, desiccation, rewetting) on the protein index of the desiccation-tolerant cyanobacteriumNostoc commune UTEX 584. Five major landmark protein constellations were detected in the protein index of control cells (in liquid culture) and were designated A (1 protein), B (7 proteins), C (8 proteins), D (3 proteins) and E (2 proteins). These included proteins which showed different sensitivities to water stress. Upon immobilization and rapid drying of the cells at a water potential ({ie87-1}) of -99.5 MPa (a_w=0.5) for 30 min, few changes took place in the index. Four conspicuous proteins and the majority of proteins in the size range 18 to 97 K diminished in abundance while most proteins of constellations A, B and C were detected in fluorographs with the same intensity as in the control. Although protein synthesis continued during this time of drying, no novel class of proteins was detected. The level of incorporation of³⁵S in protein increased rapidly during the first 60 min of rehydration, and then decreased gradually for a further 2.5 h. Extant proteins that were hardly detectable after 24 h of drying, reappeared and increased in abundance upon rewetting of cells for 60 min while a number of proteins which disappeared after drying did not appear during this time. No novel class of proteins appeared upon rewetting. During further rehydration, extensive proteolysis was observed.ThenifH product (Fe protein of nitrogenase) was detected on Western blots — through cross-reaction with antibody — as an acidic polypeptide with a molecular mass of 33.8 K. Fe-protein was detected in immobilized cells after 30 min of drying, in desiccated material, and in rehydrated cells.Abbreviations PMSF Phenylmethylsulfonyl fluoride - IEF Isoelectric focussing     

A Structural and Utrastructural Study of Nostoc commune

Nostoc commune which belongs to blue-green algae has been observed with microscope, scanning and transmission electron microscopes. The following special structures are found: 1. The structure in thallus is spongiform with an uneven surface. 2. The filaments made up of torulose cells are tortuous with different length, some are piled up, some are branchy, and the others arrange in a circle. 3. The cell wall of torulose cell is five-layered. The outermost layer is ripply and inlays with neighbour cells. 4. Torulose cells are typical prokaryote. The thylakoids disperse in the peripheral plasma. The nucleoplasm exists in the center of the cell. There are structured granules, polyhedral and polyphosphate bodies interspersing in cytoplasm. Ribosomes and phycobilins are found on the surface of thylakoid. 5. The heterocyst is surrounded by a thicking sheath. This is an additional envelope outside the cell wall and can be called “cell envelope”. In the cell envelope exist a transparent layer and a dense electron layer with hemispherical nodule in which there is an channel interlinking up with neighbour cell. 6. The reproductive manner of N. commune is of horizontal split, and the direction of split is different.     

地木耳多糖的提取与纯化研究

对地木耳采用水提醇沉法获得的地木耳多糖粗提取物,采用Sevage法脱蛋白质、醇沉,干燥得粗多糖,进一步用DEAE-52纤维素柱层析分离纯化,用纸色谱和琼脂糖凝胶电泳对洗脱组分进行纯度鉴定。结果表明：Sevage法脱蛋白7次可脱除94%的蛋白质,多糖得率为13.75%。DEAE-52纤维素柱层析后得到10种组分,浓缩干燥后得到白色粉末状多糖组分,每个组分经过纯度鉴定后均为单一的多糖。选择水和NaC l溶液为洗脱剂的温和条件分离纯化多糖效果较好。     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.     

阳生和阴生地木耳生理生化特性的比较

以生长于不同光环境下的地木耳为材料,对其Fv/Fm的日变化、光合作用特性、叶绿素和类胡萝卜素的含量进行了研究,以了解其光适应的生理生化基础。同阴生地木耳相比,阳生地木耳的光饱和点、类胡萝卜素含量、类胡萝卜素和叶绿素的比值均比较高,但其P-I曲线光限制部分的斜率、暗呼吸速率、Fv/Fm、叶绿素、MAAs含量和单位面积干重较低。二者最大光合速率和光补偿点无明显差异,二者均无明显的光呼吸。同等条件的光抑制后,阳生地木耳在暗处能更快、更大程度地恢复其Fv/Fm活性。原位研究表明,阴生和阳生地木耳在雨后强光下均有不同程度的光抑制发生,但在弱光下或夜晚时会及时恢复。阳生和阴生地木耳的光合特性及色素含量显著不同,以此来适应不同环境中的光因子。     

Protein synthesis and proteolysis in immobilized cells of the cyanobacterium Nostoc commune UTEX 584 exposed to matric water stress.

Cells of the cyanobacterium Nostoc commune UTEX 584 in exponential growth were subjected to acute water stress by immobilizing them on solid supports and drying them at a matric water potential (psi m) of -99.5 MPa. Cells which had been grown in the presence of Na235SO4 before immobilization and rapid drying continued to incorporate 35S into protein for 90 min. This incorporation was inhibited by chloramphenicol. No unique proteins appeared to be synthesized during this time. Upon further drying, the level of incorporation of 35S in protein began to decrease. In contrast, there was an apparent increase in the level of certain phycobiliprotein subunits in solubilized protein extracts of these cells. Extensive proteolysis was detected after prolonged desiccation (17 days) of the cells in the light, although they still remained intact. Phycobilisomes became dissociated in both light- and dark-stored desiccated material.     

野生地木耳中次生代谢产物含量测定与分析

测定了野生地木耳（Nostoc commune Vauch.）中次生代谢产物含量,并与4种常见食用菌藻黑木耳（Auricularia auricular-judae）、银耳（Tremella fuciformis）、紫菜（Porphyra）、海带（Laminaria japonica）进行比较分析,总酚、总黄酮和缩合单宁含量测定采用分光光度法,总生物碱含量测定采用高效液相色谱法（HPLC）。结果显示,野生地木耳中总酚含量为24.255 mg/g±1.631 mg/g,总黄酮含量为5.741 mg/g±0.239 mg/g,总生物碱含量为0.768 mg/g±0.073 mg/g,缩合单宁含量为0.069 mg/g±0.009 mg/g。4种食用菌藻中次生代谢产物含量范围为：总酚（5.520～62.326 mg/g）、总黄酮（0.847～7.010 mg/g）、总生物碱（0.408～4.132 mg/g）、缩合单宁（0.063～0.233 mg/g）;比较分析结果显示,野生地木耳次生代谢产物中的总酚和总黄酮物质含量较高,且总酚是主要次生代谢产物,缩合单宁与总生物碱含量较低;总酚含量约为黑木耳和银耳的2倍、紫菜的6倍;总黄酮含量约为黑木耳的7倍,银耳的3倍。     

营养和环境因子对地木耳细胞生长的影响

采用BG11培养基研究了营养、温度、pH值、光照强度和光周期等对地木耳(Nostoc commune)细胞生长的影响。结果显示:(1)Ca、N和P缺失会显著降低地木耳细胞的生长速率,明显抑制胞外多糖的分泌和蛋白质的合成,定期通入体积分数为0.3%的CO2有利于细胞增殖和蛋白质的合成;(2)随着培养温度的升高,地木耳细胞的生长速率、胞外多糖和蛋白质含量先增加然后降低,并于25℃时达最大值;(3)过酸或过碱的培养环境均不利于细胞的生长,细胞生长速率和蛋白质含量于pH为7.5时最大,而胞外多糖含量于pH为8.0时最大;(4)随着光照强度的增加,细胞的生长速率和蛋白质的合成先增加然后降低,并在2000 lx时达最大值,但胞外多糖分泌量持续增加;随着光照时间逐渐缩短,胞外多糖含量持续上升、蛋白质含量持续下降,12 h光照/12 h黑暗光周期最有利于细胞的生长。结果表明,25℃、pH 7.5、光照强度为2000 lx、光周期为12 h光照/12 h黑暗是人工培养地木耳细胞比较适宜的环境条件,而Ca、N和P是地木耳细胞必需的营养元素。     

地木耳提取物水溶液对花卉营养生长及开花的影响

在温室条件下,用1%的地木耳提取物水溶液对瓜叶菊(Seneciocruentus)、万寿菊(Tageteserecta)、矮牵牛(Petuniahybrida)、香石竹(Dianthuscaryophyllus)、一品红(Euphorbiapulcherrima)、仙客来(Cyclamenpersium)、一串红(Salviasplendens)等7种花坛花进行叶面喷施后,结果表明,7种花的株高、冠径、株鲜重、叶片数目、叶长、根长比对照均有显著增加(P<0.05);万寿菊,矮牵牛,一品红,一串红的平均株干重有显著增加,而瓜叶菊,香石竹,仙客来的平均株干重增加不显著(P>0.05);除了香石竹的叶宽有显著增加外,其余6种花的叶宽对1%地木耳提取物水溶液的反应不敏感;而7种花卉的叶厚都没有显著的变化。提取物水溶液还可促进花提早开花,增加花数目,并延长花期。     

Exopolysaccharide of Nostoc muscorum (Cyanobacteria) in the aggregation of soil particles

The effects on a saline-sodic soil of exopolysaccaride isolated from Nostoc Muscorum or the addition of a cyanobacterial mass proliferation were evaluated in a greenhouse experiment. By day 180 exopolysaccharide increased soluble C by 100%, microbial activity by 366% and the amount of water-stable aggregates larger than 250μm by 12 times. Inoculation with living cyanobacterial mass increased at the end of 365 oxidizable C by 11%, soluble C by 66%, microbial activity by 73% and aggregates larger than 250 μm by66%. A slimy film 3–5 mm thick, with N. Muscorum predominating, covered all the surface of inoculated soils. The higher soil aggregate stability produced by both treatments is a consequence of increased microbial activity and concentrating the soil polysaccharide. The high percentage of clays favours the creation of firm and long-lasting slime-mineral joints. Addition of isolated exopolysaccharide produces a faster and higher increase in soil aggregate stability than cyanobacterial mass inoculation. This revised version was published online in August 2006 with corrections to the Cover Date.     

发菜磷酸葡糖胺变位酶(glmM)对干旱胁迫的响应及其基因克隆

glmM编码的磷酸葡糖胺变位酶是肽聚糖合成前体的关键酶。为探究发菜glmM响应干旱胁迫的表达调控机制及明确其分子信息,本研究对干旱胁迫条件下发菜glmM在转录水平的差异表达进行了分析,并对glmM的表达水平、磷酸化修饰、乙酰化修饰和琥珀酰化修饰水平进行了检测,克隆了发菜glmM,进行了序列分析和原核表达。结果表明,干旱胁迫条件下,发菜glmM在转录水平上的表达量先增加后减少,glmM上调表达,glmM的磷酸化修饰水平逐渐增加,乙酰化修饰水平相对稳定,琥珀酰化修饰水平有明显变化。设计特异性引物克隆glmM基因,获得全长1416 bp发菜glmM基因,与肺衣(5183)glmM的核苷酸序列同源性为95%,氨基酸同源性为97%。将glmM在大肠杆菌中表达,获得一个51.45 kD的外源蛋白,MALDI-TOF-TOF/MS分析证明该蛋白为磷酸葡糖胺变位酶。研究结果为深入研究发菜glmM的分子信息、生物学功能及其响应干旱胁迫的分子机制提供帮助。