Molecular Cloning and Construction of agp Gene Deletion-mutant in Cyanobacterium Synechocystis sp. PCC 6803

The agp gene encoding the ADP-glucose pyrophosphorylase involved in cyanobacterial glycogen synthesis was amplified by PCR. The resulting agp fragment was cloned in plasmid pUC118 to generate plasmid pUCA. Part of the fragment within the agp DNA was deleted and replaced by an erythromycin resistance cassette to generate plasmid pUCAE, which was used to transform the Synechocystis sp. PCC 6803 wild-type strain and a mutant with resistance to erythromycin was obtained. PCR analysis of the genomic DNA from the resulting mutant indicated that the appropriate deletion and insertion indeed had occurred. The cell growth and Chl a, glycogen content in the mutant showed difference from those in the wild-type strain. The obtained biomass as well as the Chl a content in the mutant strain was higher than that of the wild-type strain, which suggested that the photosynthesis efficiency in the agp(-) strain was higher than that in the wild-type strain. No glycogen was found in the mutant, providing evidence for the correction of the mutant in physiological level.

Isolation of Plasma and Thylakoid Membranes from the Heterocystous Cyanobacterium Anabaena sp. PCC 7120

利用水溶性多聚体双相法分离蓝细菌Anabaenasp .PCC 712 0质膜和类囊体膜两种膜系统。吸收光谱分析表明 ,质膜相和类囊体膜相的主要色素分别为类胡萝卜素和叶绿素。SDS_凝胶电泳显示这两种膜系统蛋白组成有很大差别。这种分离方法容易操作 ,对研究蓝细菌的膜蛋白和膜脂非常有用。

Probit Analysis of Carbamate-Pesticide-Toxicity at Soil-Water Interface to N_2-Fixing Cyanobacterium Cylindrospermum sp.

Toxicity-data of two carbamate insecticides, carbaryl and carbofuran, and three fungicides, ziram, zineb and mancozeb with rice-field N2-fixing cyanobacterium Cylindrospermum sp., obtained by in vitro growth and at soil-water interface, were analyzed by the probit method. Growth enhancing concentration, no-observed effective concentration, minimum inhibitory concentration, the highest permissive concentration and lethal concentration100 （LCloo） were determined experimentally. The LC^o values of carbaryl, carbofuran, ziram, zineb and mancozeb in N2-fixing liquid medium were 56.2, 588.8, 0.07, 4.2 and 3.4 IJg/mL, respectively, whereas the corresponding LCloo values were 100.0, 1500.0, 0.17, 25.0 and 9.0 IJg/mL, respectively. The LC50 values of these pesticides in succession in N2-fixing agar medium were 44.7, 239.9, 0.07, 1.8 and 2.3 IJg/mL, respectively, whereas the corresponding LC100 values were 100.0, 600.0, 0.17, 10.0 and 7.0 IJg/mL, respectively. Similar results with nitrate supplemented liquid and agar media indicated that nitrate supplementation had toxicity reducing effect. The LCso and LC^oo values of toxicity in the N2-fixing liquid medium at soil-water interface were 91.2 and 200.0 IJg/mL for carbaryl, 2 317 and 6 000 pg/mL for carbofuran, 0.15 and 0.50 pg/mL for ziram, 16.4 and 50.0 pg/mL for zineb, and 7.2 and 25.0 pg/mL for mancozeb, respectively. Each LC^oo value at soil- water interface with a pesticide was significantly higher than its corresponding LCloo value at liquid/agar media. It can be concluded that, under the N2-fixing conditions, the cyanobacterium tolerated higher levels of each pesticide at soil-water interface.

Cultivation and Characterization of the MaMV-DC Cyanophage that Infects Bloom-forming Cyanobacterium Microcystis aeruginosa

The MaMV-DC cyanophage,which infects the bloom-forming cyanobacterium Microcystis aeruginosa,was isolated from Lake Dianchi,Kunming,China.Twenty-one cyanobacterial strains were used to detect the host range of MaMV-DC.Microcystic aeruginosa FACHB-524 and plaque purification were used to isolate individual cyanophages,and culturing MaMV-DC with cyanobacteria allowed us to prepare purified cyanophages for further analysis.Electron microscopy demonstrated that the negatively stained viral particles are tadpole-shaped with an icosahedral head approximately 70 nm in diameter and a contractile tail approximately 160 nm in length.Using one-step growth experiments,the latent period and burst size of MaMV-DC were estimated to be 24–48 hours and approximately 80infectious units per cell,respectively.Restriction endonuclease digestion and agarose gel electrophoresis were performed using purified MaMV-DC genomic DNA,and the genome size was estimated to be approximately 160 kb.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)analysis revealed four major structural proteins.These results support the growing interest in using freshwater cyanophages to control bloom-forming cyanobacterium.

Toxicity Evaluation of Freshwater Cyanobacterium Microcystis aeruginosa PCC 7806: Ⅱ Nephrotoxicity in Rats

Nephrotoxic potential of laboratory cultures of freshwater cyanobacterium (blue-green al ga) Microcystis aeruginosa PCC 7806 (Pasteur Institute) was assessed in male rats. The ani mals were injected intraperitoneally with 0. 5, 1. 0 and 2. 0 LD50 doses of lyophilized cell ex tract. Elevated plasma urea and creatinine levels were accompanied by decrease in protein and albumin levels, followed by hematuria, proteinuria and bilirubinuria. Also decrease in kidney lactate dehydrogenase and glutamic oxaloacetic transaminase indicated possible nephrotoxic po tential of the cyanobacteria. The extract also produced various hematological changes associat ed with stagnant type of hypoxia. High perfomance liquid chromatography of the culture I dentified the active principle (toxin) as Microcystin-LR

De Novo Organelle Biogenesis in the Cyanobacterium TDX16 Released from the Green Alga <i>Haematococcus pluvialis</i>

It is believed that eukaryotes arise from prokaryotes, which means that organelles can form de novo in prokaryotes. Such events, however, had not been observed previously. Here, we report the biogenesis of organelles in the endosymbiotic cyanobacterium TDX16 (prokaryote) that was released from its senescent/necrotic host cell of green alga Haematococcus pluvialis (eukaryote). Microscopic observations showed that organelle biogenesis in TDX16 initiated with cytoplasm compartmentalization, followed by de-compartmentalization, DNA allocation, and re-compartmentalization, as such two composite organelles-the primitive chloroplast and primitive nucleus sequestering minor and major fractions of cellular DNA respectively were formed. Thereafter, the eukaryotic cytoplasmic matrix was built up from the matrix extruded from the primitive nucleus;mitochondria were assembled in and segregated from the primitive chloroplast, whereby the primitive nucleus and primitive chloroplast matured into the nucleus and chloroplast respectively. While mitochondria subsequently turned into double-membraned vacuoles after matrix degradation. Results of pigment analyses, 16S rRNA and genome sequencing revealed that TDX16 is a phycocyanin-containing cyanobacterium resembling Chroococcidiopsis thermalis, which had acquired 9,017,401 bp DNAs with 10,301 genes from its host. Accordingly, we conclude that organelle biogenesis in TDX16 is achieved by hybridizing the acquired eukaryotic DNAs with its own one and expressing the hybrid genome. The formation of organelles in cyanobacterium TDX16 is the first case of organelle biogenesis in prokaryotes observed so far, which sheds an unprecedented light on eukaryotes and their connections with prokaryotes, and thus has broad implications on biology.

Isolation of a novel strain of Cyanobacterium sp.with good adaptation to extreme alkalinity and high polysaccharide yield

The use of high alkaline medium is a feasible way to provide carbon source and prevent biological contamination for the outdoor cultivation of alkaliphilic microalgae and cyanobacteria.A novel cyanobacterial strain was isolated from the open pond of a marine green alga(Picochlorum sp.SCSIO-45015,Sanya,Hainan)and identified as Cyanobacterium sp.SCSIO-45682.The effects of initial sodium bicarbonate(NaHCO_(3))concentrations on the growth and biochemical composition of Cyanobacterium sp.SCSIO-45682 were investigated.The results demonstrated that Cyanobacterium sp.SCSIO-45682 had good adaptation to 16.8-g/L NaHCO_(3)(the same concentration of NaHCO_(3) used in Zarrouk medium for Spirulina).Moreover,the yields of biomass,polysaccharide,chlorophyll a(chl a),and phycocyanin increased under high NaHCO_(3) concentrations.The maximum final biomass concentration of 2.5 g/L was observed at 8.4-g/L NaHCO_(3),while the highest intracellular total saccharide content of 49.2%of dry weight(DW)and exopolysaccharide(EPS)concentration of 93 mg/L were achieved at the NaHCO_(3) concentration of 16.8 g/L.The crude protein content declined under high NaHCO_(3) concentrations,which provide a possible explanation for the accumulation of polysaccharide.This study shows a good potential of alkaliphilic Cyanobacterium sp.SCSIO-45682 as a polysaccharide feedstock.

Toxicity Evaluation of in vitro Cultures of Freshwater Cyanobacterium Microcystis aeruginosa:Ⅰ.Hepatotoxic and Histopathological Effects in Rats

Laboratory cultures of freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa PCC 7806 was cvaluated for its hepatotoxic effects in rats. The lyophilized cell extract injected intraperitoneally at 1 and 2 LD50 (15.8 and 31.6 mg/kg, respectively) produced significant increase in liver-specific enzymes viz. plasma alkaline phosphatase,γ-glutamyl transferase, lactate dehydrogenase with a concomitant decrease in hepatic glutamic pyruvic transaminase. A corresponding increase in liver body weight index and histopathological changes in liver (degeneration of hepatocytes, congestion and hemorrhage etc.) are indicative of a dose and time dependent hepatotoxic nature of the algal extract

Fractionation and characterization of polysaccharides from cyanobacterium Spirulina (Arthrospira) maxima in nitrogen-limited batch culture

By a sequentially selective extraction procedure, polysaccharides (PS) from cyanobacterium Spirulina (Arthrospira) maxima that can be bio functionally healthy components were fractionated in four parts including the culture medium (PSCM), the external layers of the cell (PSEL), the cell wall (PSCW) and storage granules (PSSG). The four fractionated parts of polysaccharides were characterized by gas liquid chromatography (GLC) equipped with FID and fused silica capillary column (15 m×0.53 mm i. d.). The contents of PSCM, PSEL, PSCW and PSSG were about 2.0%, 2.6%, 10.0% and 52.0% to cell dry matter, respectively. Glucose was almost the only monosaccharide in PSSG and PSCW and most predominant in PSEL, while in PSCM, xylose, rhamnose and glucose were the main ones. Two uronic acids represented by glucuronic acid and galacturoic acid, six neutral monosaccharides including fucose, rhamnose, xylose, mannose, galactose and glucose, and two possible unidentified sugars were found in PSEL and PSCM. These data are valuable for the selective productions of high added value sugars from Spirulina .

Protective Effects of Certain Natural and Synthetic Complexans on the Toxicity of Chromium and Tin to a N_2-Fixing Cyanobacterium, Anabaena doliolum

The effects of chromium and tin on survival, growth, carbon fixation, nitrate reduction, ammonia assimilation, and nitrogenase activity of a N_2-fixing cyanobacterium, Anabaena doliolum, and their amelioration by synthetic and natural complexans, viz., EDTA, nitrilotriacetic acid (NTA), pyridine dicarboxylic acid (PDA), and citrate, have been studied. Chromium proved to be much more toxic than tin, as it inhibited growth yield (49%), carbon fixation (53%), and nitrate reductase (79%), glutamine synthetase (30%), and nitrogenase activities (77%) at its sublethal concentration, whereas tin induced less inhibition of growth yield (42%), carbon fixation (50%). and nitrate reductase (66%), glutamine synthetase (32.4%), and nitrogenase activities (70%). Despite its inhibitory effects at 10μml^(-1), EDTA supplementation in metal-spiked medium counteracted the toxicity of chromium and tin more significantly than NTA, PDA, and citrate. When supplemented with LD_(50) of Cr, EDTA protected growth, carbon fixation, NR, GS. and Noase, respectively, by 32.6, 50.0, 33.3. 17.7. and 65.4%. However, EDTA-induced restoration of the above parameters at a sublethal concentration of tin was only 30.2, 50.0,28.1, 27.7, and 61.5%, respectively. Although NTA and citrate at 10/μgml^(-1) each were stimulatory to various processes of test cyanobacterium, they were comparatively less effective than EDTA in the amelioration of metal toxicity. On the basis of these observations, a generalized hierarchical sequence of protective efficiency of synthetic and natural cornplexing ligands may be given as EDTA > NTA > citrate > PDA. It seems plausible that the toxicity of various heavy metals may be regulated by a large array of organic complexing agents of the aquatic environment because they possess various metal binding sites. (c) 1989 Academic Press,lnc.

纳米塑料对硫酸铜抑制铜绿微囊藻生长的影响作用

研究了水环境中聚苯乙烯(PS)纳米塑料的共存对应急杀藻剂CuSO_(4)抑制铜绿微囊藻的影响作用。带不同基团3种聚苯乙烯纳米塑料单独处理和CuSO_(4)+纳米塑料共存情况下,通过测定藻密度、叶绿素a(Chl a)含量、丙二醛(MDA)含量以及超氧化物歧化酶(SOD)活性,研究了聚苯乙烯纳米塑料与Cu^(2+)对铜绿微囊藻生长抑制的作用机制。结果表明,PS-COOH、PS、PS-NH_(2)这3种纳米塑料均能缓解CuSO_(4)对藻细胞胁迫。与空白对照组(CK)相比,CuSO_(4)、PS-COOH+CuSO_(4),PS+CuSO_(4)和PS-NH^(2+)CuSO_(4)暴露后藻密度分别抑制了42%、7%、5%、36%,Chl a含量降低了55%、6%、7%和45%,说明PS-NH_(2)与CuSO_(4)共同暴露对藻细胞生长和叶绿素合成的抑制作用与其他2种纳米塑料更为显著。相应地,PS-COOH、PS这2种纳米塑料显著缓解了CuSO_(4)对藻细胞内MDA含量、SOD活性的胁迫。与空白对照相比,PS-COOH+CuSO_(4)和PS+CuSO_(4)处理组MDA含量和SOD活性分别增加了31%、35%和7%、5%,而CuSO_(4)和PS-NH^(2+)CuSO_(4)处理后MDA含量和SOD活性分别增加了99%、66%和22%、5%。同样的,除了PS-NH_(2)外,其他2种纳米塑料均能显著降低在CuSO_(4)处理蓝藻水华过程中铜绿微囊藻胞外藻毒素(MCs)的释放。以上结果表明,带不同基团PS纳米塑料的共存在不同程度上影响CuSO_(4)的除藻效率。

太湖水华蓝藻中元素的组成及其环境意义

利用电感耦合等离子质谱仪对2008年7、9和10月采自太湖南泉水域的水华蓝藻样本中19种元素（Na、Mg、K、Ca、Zn、Mn、Fe、Al、Cu、Cr、Co、Ni、As、Se、Mo、Ag、Tl、Cd和Pb）的质量含量（w）及组成变化进行研究。结果表明,在藻华中w较高（〉1 000μg.g^-1,以干质量计）的常量元素或有益元素为Ca、K、Na、Mg、Al、Fe。其中,w（Ca）和w（K）较高（〉4 000μg.g^-1）,w（Ca）随月份变化不大,而w（K）、w（Mg）、w（Na）则逐月升高。w（Al）、w（Fe）变化较大,7月最高（〉1 000μg.g^-1）,之后迅速降低（〈400μg.g^-1）;藻华中w在10-100μg.g^-1之间变化的元素为Mn、Zn、Ni、Cu,其中,7月这4种元素w均最高,9-10月则降低。藻华中还检出痕量元素Co、Mo和Se,Tl未检出。藻华中积累的有毒元素中w（As）较高（5-15μg.g^-1）,而w（Cr）、w（Cd）、w（Pb）、w（Ag）均较低（〈5μg.g^-1）。有毒元素w最高值均出现在7月。基于上述结果,推算出2007—2009年采取物理方法除藻从太湖中移除的元素量约为：K595.89 t、Ca 544.03 t、Mg 181.18 t、Al 83.06 t、Na 63.97 t、Fe 55.45 t、Mn 2.99t、Zn 2.26 t、Ni 1.67 t、Cu 1.11 t、As 0.76 t、Cr 94.5 kg、Cd 82.70 kg、Se 45.01 kg、Pb 37.64 kg、Co 32.27 kg、Mo5.41 kg和Ag 2.46 kg。该研究不仅可为进一步寻找关键元素在蓝藻水华爆发过程中的限制作用及其机制提供基础数据,同时也可为评价利用物理除藻方法去除太湖中有毒元素和降低有可能促进藻华发展的营养元素的负荷量的有效性,从而保护和修复渔业环境提供理论依据。

钝顶螺旋藻中藻蓝蛋白提取纯化新工艺

用０３ｍｍｏｌ／Ｌ的十二烷基苯磺酸钠提取钝顶螺旋藻中的藻蓝蛋白，提取率达到了９８％。再在硅藻土５４５柱上分级洗脱，进一步经ＤＥＡＥ—纤维素柱纯化，其纯度达到了４１（指可见区最大吸收与２８０ｎｍ处之比），最大吸收峰在６１９ｎｍ，室温荧光发射峰在６４３ｎｍ。

控制蓝藻“水华”的C段紫外光实验教学装置

介绍一种能让学生进行蓝藻"水华"形成、控制和监测的实验教学装置。在此装置中以念珠藻7120为受试藻种,通过念珠藻生长曲线监测蓝藻"水华"形成,通过浸没式UV C技术控制此蓝藻"水华",通过显微镜观察浸没式UV C对蓝藻细胞形态畸变。因此通过此教学装置所进行的蓝藻"水华"形成、控制和监测教学实验,能使学生对蓝藻"水华"形成、控制和监测达到比较清楚的认识,具有良好的教学效果。

6株蓝藻伪空胞的临界破裂压力研究

通过改进的压力毛细管法，研究了6株蓝藻伪空胞的临界破裂压力，并研究了过滤（真空度为0．02MPa）和离心（离心力低于500r／min）2种细胞浓缩方法对细胞内伪空胞含量测定的影响．结果表明，对于分散的单细胞的微囊藻，无论是采用过滤还是离心的方法，都难以达到理想的浓缩效果；对于群体形态的微囊藻，可以采用离心的方法进行浓缩，对于丝状的浮游蓝丝藻，宜采用过滤的方法进行浓缩．此2种浓缩方法对细胞伪空胞含量的测定影响很小，伪空胞破裂率〈7％．6株蓝藻均为浅水湖泊藻类，由于长期自然选择的结果，6株蓝藻伪空胞的临界破裂压力比较接近．5株微囊藻伪空胞的临界破裂压力为0．64～0．67MPa之间；孟氏浮游蓝丝藻伪空胞的临界破裂压力为0．715MPa，与深水湖泊或水库的蓝藻相比，6株蓝藻的伪空胞平均临界破裂压力均较小；在相同光照和温度条件下，单细胞微囊藻的膨压比群体微囊藻的膨压略大．

Fe(Ⅲ)对太湖铜绿微囊藻和四尾栅藻竞争的影响

采用批量培养的方法研究Fe(Ⅲ)及其不同化学形态与EDTA,柠檬酸和羟基等配体作用对铜绿微囊藻和四尾栅藻的生长和光合作用的影响.结果表明,当Fe(Ⅲ)浓度(18μmol/L)高于强有机配体EDTA浓度(2.69μmol/L)时,Fe(Ⅲ)主要以胶体和沉淀形态存在,但Fe3+及Fe(Ⅲ)的羟基配合物等藻类易于利用的化学态能维持较高的浓度,铜绿微囊藻和四尾栅藻都生长良好;Fe(Ⅲ)浓度(≤1.0μmol/L)低于强有机配体的浓度(2.69μmol/L)时,有机配体可以与Fe(Ⅲ)形成稳定的鳌合物;可利用态的Fe(Ⅲ)浓度较低,铜绿微囊藻和四尾栅藻的生长、单位细胞叶绿素a质量、最大光合作用速率、呼吸速率、光限制部分P-I曲线斜率都下降,但铜绿微囊藻变化较快,表明Fe(Ⅲ)与强有机配体络合造成的限制有利于四尾栅藻占据优势.

基因工程灭蚊幼蓝藻的现场初步观察

将转入球形芽孢杆菌毒蛋白基因表达质粒的工程藻Ａｎａｂａｅｎａｓｕｂｔｒｏｐｉｃ（ｐＤＣ２６）大量培养后，应用于山东省平阴县进行现场观察。结果表明：工程藻在９．４１×１０５ｃｅｌｌｓ／ｍｌ浓度以上时，４８ｈ可杀死９９～１００％蚊幼虫；在９．１３×１０４ｃｅｌｌｓ／ｍｌ浓度时，４ｄ可杀死９９％以上蚊幼虫，连续观察２个多月，工程藻仍有明显杀蚊效果，实验组最多１４条／勺幼虫，而对照组２１０条／勺左右。观察还发现，工程藻对库蚊幼虫杀灭效果最好，其次为伊蚊，但高浓度时，对伊蚊仍有明显杀灭效果；由于按蚊幼虫密度太低，未能观察。此结果比单独使用球形芽孢杆菌杀灭蚊幼虫有明显提高，持续时间也延长，这将为蚊虫的持续控制提供一个新的有力武器。

重金属及微量元素诱导蓝藻形成液泡

鱼腥藻 5 95 (Anabaenasp 5 95 )、织线藻 2 4 6 (Plectonemaboryanum 2 4 6 )和伪枝藻2 4 8(Scynetonemahofmanni 2 4 8)在两种重金属汞、镉和两种微量元素铜、锌诱导下 ,藻丝细胞均能发生膨大、液泡化现象。不同重金属及微量元素对 3种蓝藻液泡化诱导作用强弱程度不同。汞的诱导作用明显强于镉、铜和锌 ,0 1μmol/L汞可诱导 3种蓝藻发生液泡化。采用压片法均观察到诱导形成的液泡 ,液泡在相差显微镜下显示为圆球形 。

海洋固氮蓝藻Calothrixsp.与Lyngbyasp.固氮生理的研究

研究盐度、昼夜变化、温度及阿特拉津(光合系统Ⅱ抑制剂)对2种海洋固氮蓝藻Calothrixsp.strain(代号为MCT1)和Lyngbyasp.strain(代号为MCT6)固氮活性的影响。结果表明,MCT1在盐度10—48范围内具有相对较高的固氮活性,在盐度为30时固氮活性最高,达到0.687 2μmolC2H2.(g.h)-1;而MCT6随盐度改变固氮活性变化幅度较大,盐度为24时具有最高固氮活性,其活性为0.876 8μmolC2H2.(g.h)-1。MCT1和MCT6固氮活性的昼夜变化明显不同,具有异型胞的海洋固氮蓝藻MCT1白天的固氮活性明显高于夜晚;而无异型胞的MCT6最高固氮活性发生在晚上,白天固氮活性较低。一定浓度的阿特拉津通过抑制光合作用阻断能量和还原剂的提供,使藻体在较短时间内丧失固氮能力。加入阿特拉津后2种藻体的固氮活性与对照组相比有明显的变化,实验第3天开始MCT1的所有经阿特拉津处理的样品固氮活性丧失;MCT6经(50—1 000)×10-6阿特拉津处理的样品从实验第3天开始固氮活性完全消失。

滇池水华蓝藻干粉制剂的生物脱毒实验

滇池属长江流域的一个内陆湖泊 ,近年来水体富营养化日益严重 ,每年夏季都出现大面积的水华。但品种单一 ,主要为蓝藻门 ,其中微囊藻是优势种 ,产生的微囊藻毒素具有很强的毒性。本实验以富营养化湖泊滇池中的水华蓝藻作为主要原料 ,将其制成干粉后 ,利用双协菌株在 2 8～ 3 2℃条件下对其进行固态发酵 ,培养基质初始水份为 65 %。发酵培养 2d后 ,培养基质中微囊藻毒素含量明显降低 ,6d后 ,基质中微囊藻毒素含量未检出 (最低浓度检测限 1μg/mg)。同时对实验所获得时间序列进行动力学研究 。