微囊藻毒素缓解过氧化氢胁迫下铜绿微囊藻损伤的初步研究

过氧化氢可抑制藻类生长, 同时会导致微囊藻毒素(Microcystins, MCs)的释放, 实验设置4个处理组探讨了外源微囊藻毒素MC-LR对H2O2胁迫下铜绿微囊藻生理生化变化的影响。结果表明: 在H2O2胁迫下, 微囊藻的生长和光合活性受到显著抑制, 藻细胞存活率降低, ROS含量明显增加, SOD活性上升。与单独H2O2胁迫相比, 加入MC-LR能增加微囊藻细胞的存活率。250 mol/L H2O2处理24h和48h后, 在培养基中加入200 ng/mL MC-LR可以缓解H2O2对铜绿微囊藻光合系统PSII活性的抑制作用。当微囊藻暴露于250 mol/L H2O2环境中时, 添加了MC-LR处理组藻细胞中的ROS含量明显减少(P0.05)。在相同浓度H2O2且加入了外源MC-LR后藻细胞SOD活性下降(P0.05)。因此, 微囊藻毒素MC-LR可缓解250 mol/L H2O2引起的氧化损伤并增强微囊藻自身的生存能力。研究结果有利于阐明H2O2胁迫影响产毒蓝藻生长代谢的途径及MCs生物学意义。       

铜绿微囊藻与小球藻对低温和黑暗的响应与恢复

研究以水华蓝藻铜绿微囊藻(Microcystis aeruginosa PCC 7806)与绿藻小球藻(Chlorella sp. FACHB-31)为研究对象, 探讨低温和黑暗对其生长、色素含量、最大光化学效率(F_v/F_m)、丙二醛(MDA)含量及过氧化氢酶活性的变化。结果表明, 30d的低温和黑暗处理, 显著降低了铜绿微囊藻和小球藻的叶绿素a浓度, 增加了单位细胞类胡萝卜素含量。在低温黑暗条件下, 铜绿微囊藻的MDA含量及CAT活性均显著增加, 而小球藻变化不明显。30d低温黑暗处理, 铜绿微囊藻的存活率为54.6%, 显著高于小球藻的31.3%。当恢复正常温度与光照, 2种藻均迅速生长。这些结果表明低温黑暗影响了微囊藻和小球藻的生理特性。在低温黑暗处理下, 微囊藻的F_v/F_m显著降低, 而小球藻则保持较为恒定的F_v/F_m, 表明微囊藻通过降低自身光合活性来渡过冬季低温黑暗的条件, 而小球藻在低温黑暗条件下仍保持较高的光合活性。     

生长素吲哚乙酸对铜绿微囊藻生理生化及产毒特性的影响

为了探究生长素吲哚乙酸(IAA)对产毒铜绿微囊藻(Microcystis aeruginosa)的影响, 从生长、光合色素含量、叶绿素光诱导荧光特征、脂质氧化和微囊藻毒素合成特性等方面, 研究了IAA对M. aeruginosa CHAB6301生理生化及产毒的影响。结果表明, 在低浓度IAA(0.04和0.2 mg/L)条件下, 铜绿微囊藻生长、叶绿素含量、光合系统(PSⅡ)电子传递效率及藻毒素含量均无明显变化, 藻蓝蛋白、别藻蓝蛋白和丙二醛(MDA)含量均低于对照。高浓度IAA(1和5 mg/L)能够促进细胞生长, 提高叶绿素含量, 但是抑制藻蓝蛋白和别藻蓝蛋白含量, 降低膜脂过氧化程度和细胞内藻毒素合成。综合各指标测定结果, 低浓度IAA对M. aeruginosa CHAB6301生长和光合作用影响不明显, 而高浓度IAA可促进藻细胞生长和光合作用, 增加微囊藻水华形成几率。     

超声波对铜绿微囊藻超微结构和生理特性的影响

为了研究超声波对蓝藻细胞的影响,利用超声波（40W）处理200 mL铜绿微囊藻（Microcystis aeruginosa） 悬浮液20min,之后继续培养并于不同时间取样检测。检测悬浮藻细胞生物量发现其3d降低了97.84%;分别观察1、3、5d时沉降藻细胞超微结构变化,发现13d时细胞内脂质颗粒和藻青素颗粒增多、类囊体片层断裂、藻胆体脱落,5d时拟核区萎缩消失、细胞基础结构解体、胞质出现空洞、胞内结构颗粒降解;检测藻细胞光合放氧速率、叶绿素a （Chl.a）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性、膜透性以及跨膜ATP酶活性,发现光合放氧速率3d下降24.83%,Chl.a含量5d下降23.75%,超声组细胞SOD活性变化幅度比较大,但总体上活性降低,而CAT活性则表现为先增后减,活性始终大于对照组,同时胞内有机物渗出量增大,三种跨膜ATP酶活性（Na+/K+-ATPase、Mg2+-ATPase 和Ca2+-ATPase）均先升后降,并与膜透性变化相关。以上结果表明,超声波使铜绿微囊藻细胞沉降,并对其造成了胁迫,使部分藻细胞光合作用减弱,光合色素遭到损伤,细胞膜透性增大,甚至引起藻细胞程序性死亡。SOD活力的快速降低表明超声波使藻细胞内超氧离子（O2-）过量累积,从而对藻细胞造成氧化损伤,除此之外,超声波使藻细胞基础结构破坏、细胞内结构颗粒降解、细胞膜透性增大,这些都可能是致使部分铜绿微囊藻细胞死亡的重要原因。铜绿微囊藻细胞CAT以及跨膜ATP酶活性增大,表明藻细胞增强抗氧化酶活性以及离子调控和能量活动以抵御超声波的胁迫,而当胁迫随着时间减小后,细胞开始恢复生长和代谢,酶活力开始降低。       

两种温度条件下苯酚对铜绿微囊藻大型变种生长的影响

模拟春秋季水温 (2 2℃ )和夏季水温 (3 0℃ )环境 ,用不同浓度的苯酚处理铜绿微囊藻大型变种。结果显示 :正常水体中该藻在 3 0℃比 2 2℃生长快。浓度C≤ 2 0 0 μg/mL的苯酚对它的生长有促进作用 ,而浓度C≥ 40 0 μg/mL的苯酚则抑制其生长 ,高浓度苯酚胁迫下的藻细胞光合速率 ,可溶性蛋白含量下降 ;细胞膜透性增大 ;超氧阴离子 (O 2 )和丙二醛 (MDA)相对含量升高 ;超氧化物歧化酶 (SOD)活性降低 ,藻体自发荧光较对照减弱。表明该藻的生长更适合于夏季高温条件 ,且较高浓度苯酚对其有较强抑制作用。     

黑暗条件下不同氮源对铜绿微囊藻(Microcystis aeruginosa)生长和pH的影响

在黑暗条件下,利用不同形态的氮源(硝酸盐氮,氨氮,有机氮和硝酸盐氮,有机氮)培养蓝藻水华优势种铜绿微囊藻(Microcystis aeruginosa),分析其氮代谢和对水体pH的影响.研究结果表明,在不同氮源的培养液中铜绿微囊藻密度在最初的24 h内出现波动,之后下降.培养液中pH值在试验最初的24 h显著下降,之后趋于稳定,在硝态氮培养液中pH值下降最大,从8.18下降到7.19,其反硝化作用产生的NO-2浓度也最大.不同氮源培养液中总氮含量都有所下降,以混合氮源培养液中总氮减少量最大,说明化合态氮经过反硝化作用生成了氮气并溢出培养液,因此,在黑夜条件下藻华水体中存在反硝化作用.     

铜绿微囊藻在不同供磷水平下对砷胁迫的响应

采用磷饥饿培养后的微囊藻细胞进行不同供磷水平的五价砷(As(V))暴露实验,考察单一胞外磷变化的情况下As(V)对滇池分离出的铜绿微囊藻FACHB905生长及产毒的影响.结果表明胞外磷浓度变化不会影响铜绿微囊藻对As(V)的耐受阈值(-10-7 mol/L).少磷条件下的半数抑制浓度(IC50)为10-2.79 mol/L,比无磷条件下的IC50(10-5.81 mol/L)高3个数量级,并且少磷条件下As(V)与细胞活性位点的结合常数要远低于无磷条件,因此胞外磷在As(V)对微囊藻的毒性效应上具有关键的作用.As(V)对微囊藻单细胞的叶绿素含量没有显著影响,但是对毒素产量具有剂量效应.在少磷条件下,As(V)浓度大于10-7 mol/L可促进微囊藻FACHB905的胞内产毒量;而在无磷条件下,所有As(V)处理组的胞内产毒量均上升78%左右.由上可知,微囊藻在产毒方面与As(V)具有协同效应,这对于全面了解滇池水华暴发期间毒素的变化规律具有一定的参考价值.     

有毒铜绿微囊藻胁迫下三角帆蚌消化系统的扫描电镜观察

通过观察暴露于不同浓度有毒铜绿微囊藻(Microcystis aeruginosa)下的三角帆蚌(Hyriopsis cumingii)的胃、前肠、中肠以及晶杆体等消化系统的扫描电镜切片,了解有毒铜绿微囊藻对三角帆蚌消化系统超显微结构的影响。结果显示,当三角帆蚌在浓度为1×107cell/L的有毒铜绿微囊藻环境下,其晶杆体随时间推移而逐渐溶解。在低浓度的铜绿微囊藻环境下,尽管其会继续滤食,但有毒铜绿微囊藻会对其消化系统造成破坏,随着接触时间的增长,造成其肠道内绒毛、纤毛数量减少,长度降低,从而降低其消化效率,对其生长造成影响。     

铜绿微囊藻和斜生栅藻非稳态营养盐限制条件下的生长竞争特性

采用“脉冲”添加方法进行了非稳态条件下铜绿微囊藻（M.）和斜生栅藻（S.）分别在氮磷单营养盐和双营养盐限制时的共培养试验。试验结果显示：当添加频率为1d时,无论何种营养盐限制,M.均成为优势藻种。氮限制条件下,氮时均浓度范围在0.3—2.4 mg/L时,M.始终具有竞争优势。磷限制条件下,磷浓度范围在0.018—0.035 mg/L时,S.只在生长初期阶段占优。氮磷双营养盐限制条件下,添加液的氮磷质量比为35:1（设定为最优比）,添加频率为8 d时,两种藻表现出共生特征;而偏离最优比时（N:P=70:1,17:1）,在不同的添加频率下均未出现共生现象,且氮的时均浓度为0.6—4.8 mg/L时（70:1）,M.具有竞争优势,而降低为0.15—0.3 mg/L时（17:1）,S.占优。随着添加频率的变化,两种藻的细胞大小也会随之改变,S.随着营养盐浓度的降低而增大,且在双营养盐限制条件下变化更显著。上述试验结果分析表明：两种藻竞争能力与添加频率相关,在藻种浓度的变化上,按照‘拾遗－机会’交替竞争理论,M.表现出机会主义者特征,而S.则表现出拾遗者的特征,两者的共生特征也符合‘中度干扰’假说。藻细胞大小变化表明,两种藻均可以改变大小实现最大限度争夺受限营养盐。在低浓度时,S.细胞大小的变化同样也变现出了“拾遗者”的特征。     

溶藻细菌筛选及溶藻活性物质对铜绿微囊藻生理活性的影响

从太湖水华水体中分离纯化细菌,再将细菌的LB液体和固体斜面纯培养物分别收集后感染铜绿微囊藻(Microcystis aeruginosa)细胞,从中筛选出7株具有溶藻活性的细菌,并对其中一株溶藻细菌THW7的溶藻方式及溶藻活性物质对铜绿微囊藻生理活性的影响进行了初步研究。结果表明:仅采用细菌的LB液体纯培养物进行溶藻细菌筛选会存在误筛或高估溶藻效率的风险,而采用细菌的固体斜面纯培养物进行筛选则可避免以上风险;溶藻细菌THW7通过分泌胞外活性物质的方式间接溶藻;在THW7无菌滤液胁迫下,铜绿微囊藻的生长受到显著抑制(P<0.01), 10d溶藻效率可达94.38%,光合系统活性也显著降低(P<0.01), MDA含量积累,SOD、POD、CAT活性整体呈现先升高后降低的趋势且显著高于对照组(P<0.01)。推测菌株THW7分泌的溶藻活性物质可能作用于铜绿微囊藻细胞的光合系统Ⅱ,阻碍电子传递,抑制其光合作用过程,并对藻细胞产生氧化损伤,破坏藻细胞细胞膜的完整性,从而实现溶藻作用。     

三种柑橘类果皮提取物对铜绿微囊藻生长的影响

通过分析测定生物量、叶绿素a含量以及叶绿素荧光参数,研究了3种柑橘类果皮甲醇提取液对铜绿微囊藻生长的影响。结果表明,3种提取液都能有效抑制铜绿微囊藻的生长、叶绿素a合成与光合系统Ⅱ（PSⅡ）活性,并且抑制效果随着作用浓度增加而增强。3种提取液抑制作用强弱的顺序为：蜜橘〉西柚〉脐橙。当蜜橘皮提取液浓度大于1.10g/L时,抑藻效果显著（P〈0.05）,培养9d后对铜绿微囊藻生长的抑制率达到86.4%,且在实验期间抑制作用没有减弱。当脐橙皮与西柚皮提取液的浓度大于3.31g/L时,抑藻效果显著（P〈0.05）,但培养5d后抑制作用开始减弱。据此推测,3种柑橘类果皮提取液中存在一类或几类物质,能够抑制铜绿微囊藻的叶绿素a合成,降低PSⅡ活性,从而降低其光合作用效率,导致铜绿微囊藻的生长受到抑制。且这类物质能自然降解,随着作用时间的延长,其抑藻效果也逐渐消失。     

Identification of a Ferric uptake regulator from Microcystis aeruginosa PCC7806

Ferric uptake regulator (Fur) proteins are widely recognized as repressors that in many prokaryotes regulate a large number of genes involved in iron homeostasis and oxidative stress response. In our study, we were able to identify the complete sequence of the fur gene from Microcystis aeruginosa using inverse-polymerase chain reaction. DNA sequence analysis confirmed the presence of a 183 amino-acid open reading frame that showed high identity with Fur proteins reported for cyanobacteria. The recombinant Fur protein has been purified and electrophoretical mobility shift assays shown to be active. Mn2+ and dithiothreitol enable Fur to bind to its promoter, with dithiothreitol being more potent. The expression of Fur in Microcystis was induced about twofold in iron-deficient conditions.     

Cell wall constituents of Microcystis sp. PCC 7806

Cell walls of Microcystis sp. PCC 7806 were purified from cell homogenates by sucrose density centrifugation and Triton X-100 extraction. The outer membrane contained carotenoids, two major peptidoglycan-associated proteins (Mr 49,000 and 52,000), and lipopolysaccharide (LPS) as indicated by the presence of 3-hydroxy fatty acids (3-OH-14:0, 3-OH-16:0, 3-OH-18:0), 4-oxo-18:0 fatty acid, and GlcN as lipid A components in addition to rare O-methyl sugars (2-O-methyl-6-deoxyhexoses I and II). The peptidoglycan (A1 gamma-type) was found to be covalently linked to a wall polysaccharide composed of GlcN, ManN, Man, Glc, and phosphate.     

Morphological and physiological changes in Microcystis aeruginosa as a result of interactions with heterotrophic bacteria

1. To reveal the role of aquatic heterotrophic bacteria in the process of development of Microcystis blooms in natural waters, we cocultured unicellular Microcystis aeruginosa with a natural Microcystis‐associated heterotrophic bacterial community. 2. Unicellular M. aeruginosa at different initial cell densities aggregated into colonies in the presence of heterotrophic bacteria, while axenic Microcystis continued to grow as single cells. The specific growth rate, the chl a content, the maximum electron transport rate (ETR_max) and the synthesis and secretion of extracellular polysaccharide (EPS) were higher in non‐axenic M. aeruginosa than in axenic M. aeruginosa after cell aggregation, whereas axenic and non‐axenic M. aeruginosa displayed the same physiological characteristic before aggregation. 3. Heterotrophic bacterial community composition was analysed by PCR–denaturing gradient gel electrophoresis (PCR–DGGE) fingerprinting. The biomass of heterotrophic bacteria strongly increased in the coinoculated cultures, but the DGGE banding patterns in coinoculated cultures were distinctly dissimilar to those in control cultures with only heterotrophic bacteria. Sequencing of DGGE bands suggested that Porphyrobacter, Flavobacteriaceae and one uncultured bacterium could be specialist bacteria responsible for the aggregation of M. aeruginosa. 4. The production of EPS in non‐axenic M. aeruginosa created microenvironments that probably served to link both cyanobacterial cells and their associated bacterial cells into mutually beneficial colonies. Microcystis colony formation facilitates the maintenance of high biomass for a long time, and the growth of heterotrophic bacteria was enhanced by EPS secretion from M. aeruginosa. 5. The results from our study suggest that natural heterotrophic bacterial communities have a role in the development of Microcystis blooms in natural waters. The mechanisms behind the changes of the bacterial community and interaction between cyanobacteria and heterotrophic bacteria need further investigations.     

Buoyant density changes in the cyanobacterium Microcystis aeruginosa due to changes in the cellular carbohydrate content

Abstract The cyanobacterium Microcystis aeruginosa was grown in light-limited chemostat cultures with various light—dark rhythms providing a total periodicity length of 24 h. The buoyant density of the cells changed in parallel with the carbohydrate content. Short incubation experiments with different light intensities, and experiments with the inhibitors iodoacetic acid and arsenate, showed that the buoyant density changes were due to variations in the cellular carbohydrate content. It seems likely that the low dark-growth yield on carbohydrate, which had been stored during the light period, served to facilitate buoyancy changes.     

Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-β-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with M_r 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a M_r of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.     

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 80 infectious 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.     

Toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa contain sequences homologous to peptide synthetase genes

Abstract Toxic strains of Microcystis aeruginosa produce cyclic heptatoxins (microcystins) that are believed to be synthesized non-ribosomally by peptide synthetases. We analysed toxin-producing and non-toxic strains of M. aeruginosa with respect to the presence of DNA sequences potentially encoding peptide synthetases. Hybridizations of genomic DNA of various M. aeruginosa strains with PCR-amplificated fragments possessing homologies to adenylate-forming domains of peptide synthetase genes provided first evidence for the existence of corresponding genes in cyanobacteria. Furthermore we isolated and sequenced from genomic libraries overlapping fragments of M. aeruginosa DNA with a total length of 2982 bp showing significant homology to genes encoding peptide synthetases and hybridizing exclusively with DNA from toxic strains. Our results indicate that both toxic and non-toxic strains of M. aeruginosa possess genes coding for peptide synthetases and that hepatotoxin-producing and non-toxic strains differ in their content of genes for specific peptide synthetases.