Plasmid distribution among unicellular and filamentous toxic cyanobacteria

Plasmid content of 5 hepatotoxin and 2 neurotoxin producing cyanobacterial strains were analyzed. Among the hepatotoxin-producing strains, Microcystis aeruginosa PCC7820, M. aeruginosa M228 and M. aeruginosa UV027 were found to carry plasmids, whereas other hepatotoxin and neurotoxin producing strains did not harbor any plasmids. Correlations were sought between toxicity and the presence of plasmids in toxic cyanobacteria as a function of age. Aged cultures of M. aeruginosa PCC7820 exhibited toxicity and harbored plasmids. In other cyanobacterial strains, plasmids were not detected. The data add to and support the current understanding that plasmids are probably not involved in toxin production in cyanobacteria.Author for Correspondence     

Plasmids in toxic and nontoxic strains of the cyanobacteriumMicrocystis aeruginosa

The plasmid content and toxicity of nine different strains ofMicrocystis aeruginosa have been analyzed. The two toxic strains of the HUB Culture Collection were found to carry each two plasmids, pMA1 and pMA2, of 2.9 kb and 8.5 kb, respectively. In strains PCC 7813 and PCC 7820, also toxic, two different plasmids of 2.6 kb and 16 kb were detected. Hybridization experiments showed that there exists no sequence homology between the pMA plasmids and the plasmids found in the PCC strains; but the pMA plasmids hybridized to chromosomal DNA of the toxic strains PCC 7820, PCC 7813, HUB 063, and the nontoxic strain HUB 5-3. In nontoxic strains no or at most one plasmid of unstable occurrence could be detected. Only one of the toxic strains investigated, SAG 14.85 (NRC-1), contained no plasmid.     

Immunogold localisation of microcystins in cryosectioned cells of Microcystis

Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC(+)) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC(-)) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells' microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC(+)); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC(+)); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.     

藻类连续培养体系的构建与优化及其在产毒和无毒微囊藻竞争中的应用

利用发酵罐加装外置环形光源构建藻类连续培养系统, 以产毒微囊藻PCC 7806及其无毒突变株PCC 7806 mcyB–为培养材料, 通过对补料时间、接种密度和稀释率参数的优化, 获得最优培养条件, 并应用于产毒与无毒微囊藻的竞争实验中。通过优化得到连续培养的最优培养条件: 补料时间为第4天, 起始接种密度为4×106 cells/mL, 稀释率为0.15/d。在连续培养下, 光照为35 μmol/(m2·s)时, 以1﹕1的起始比例接种产毒与无毒微囊藻, 二者间的竞争会达到平衡, 并以无毒微囊藻占据优势, 且两者以不同的优势度长时间维持不变。在此基础上, 开展了不同光强对产毒与无毒微囊藻竞争影响的实验, 结果表明, 光强为35和80 μmol/(m2·s)时, 无毒株在连续培养中占据优势; 而光强为5和15 μmol/(m2·s)时, 无毒和产毒微囊藻维持起始接种比例不变。研究通过优化连续培养条件为室内藻类竞争实验提供了更为适宜的培养模式。     

Competition between a toxic and a non-toxic <Emphasis Type="Italic">Microcystis</Emphasis> strain under constant and pulsed nitrogen and phosphorus supply

The toxicity of a harmful algal bloom is strongly determined by the relative abundance of non-toxic and toxic genotypes and might therefore be regulated by competition for growth-limiting resources. Here, we studied how the toxic Microcystis aeruginosa strain PCC 7806 and a non-toxic mutant compete for nitrogen and phosphorus under constant and pulsed nutrient supply. Our monoculture and competition experiments show that these closely related genotypes have distinct nutrient physiologies and that they differ in their ability to compete for nitrogen and phosphorus. The toxic wild type won the competition under nitrogen limitation, while the non-toxic mutant dominated under phosphorus limitation. Pulses of both nitrogen and phosphorus increased the dominance of the toxic genotype, which lead to an even faster competitive exclusion of the non-toxic genotype under nitrogen pulses and to coexistence of both genotypes under phosphorus pulses. Our findings indicate that the genotype level dynamics driven by resource competition can be an important factor in determining cyanobacterial bloom toxicity.     

Comparing grazing by Dreissena polymorpha on phytoplankton in the presence of toxic and non-toxic cyanobacteria

SUMMARY 1. The feeding behaviour of the zebra mussel ( Dreissena polymorpha ) was studied in the laboratory on different combinations of food, including a green alga ( Chlamydomonas reinhardtii ) and toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa .
2. The highest clearance rate of phytoplankton by zebra mussels was found when the mussels were feeding on a mixture of Chlamydomonas and non-toxic Microcystis , the lowest on a mixture of Chlamydomonas and toxic Microcystis .
3. The differences found in the clearance rates between food combinations can be partly explained by the production of pseudofaeces containing live phytoplankton cells. Zebra mussels expelled significantly more live phytoplankton cells in the presence of toxic Microcystis than in the presence of non-toxic Microcystis . The pseudofaeces contained predominantly live Chlamydomonas cells. Proportionately much less live Microcystis cells were encountered in the pseudofaeces.
4. Consequently, grazing of zebra mussels on a combination of Chlamydomonas and Microcystis may finally result in a dominance of Chlamydomonas over Microcystis . The presence of toxic Microcystis may even strengthen this shift.     

七株微囊藻系统进化关系的RAPD-PCR分析

应用RAPD-PCR的方法,选用24个随机引物,分析来自不同地区的7株微囊藻的基因组多态性。结果显示,Microcystis.viridis及M.wesenbergii明显与M.aeruginosa区分开。M.aeruginosa分为两个可视为不同种的异源分类单位。作为对照的Anabaena sp.7120与其他微囊藻株表现出完全不同的基因型及更远的遗传距离。 此项研究表明,以基因型而不是表现型为基础,分析蓝藻种内及种间区别是可能的。因此,为解决蓝藻分类问题,特别是在种和属的水平上,提供了重要的线索。结合正在进行的用特异性及准确性强的引物区分微囊藻产毒及非产毒株的方法,RAPD-PCR可望将微囊藻产毒及非产毒株进化关系澄清。     

阴离子表面活性剂LAS对产毒和无毒微囊藻生长及产毒特性的影响

文章研究了低浓度范围内不同浓度梯度的阴离子表面活性剂直链烷基苯磺酸盐(LAS)对产毒微囊藻(Microcystis aeruginosa, FACHB905)和无毒微囊藻(Microcystis wesenbergii, FACHB908)生长、光合特性、种间竞争及毒素合成的影响。结果表明,在0.05—5.0 mg/L LAS浓度梯度处理下,产毒微囊藻的生物量、产毒基因mcyD表达量和每细胞MCs含量均在培养12d后显著增加。产毒微囊藻胞内和胞外MCs含量在各LAS浓度处理后分别为0.069、0.052、0.061、0.038和0.037 fg/fg Chl.a及107.1、103.7、127.1、99.6和113.7 ng/L。即使在0.5 mg/L低浓度LAS处理条件下,上述3个参数也分别比对照组显著增加了24.2%、12.4倍和10.4%。浓度为0—0.2 mg/L LAS对无毒微囊藻的生物量无明显影响,而较高浓度的LAS(0.5—5.0 mg/L)明显抑制了无毒微囊藻的生长。在两株微囊藻混合培养时, 0.2—1.0 mg/L LAS处理组的产毒铜绿微囊藻mcy D的表达对LAS...     

The circadian rhythms of photosynthesis,ATP content and cell division in Microcystis aeruginosa PCC7820

Microcystis aeruginosa is one of the most common blue-green algae species that forms harmful water bloom, which frequently causes serious ecological pollution and poses a health hazard to animals and humans. To understand the progression of algal blooms and to provide a theoretical basis for predicting and preventing the occurrence of algal blooms and reducing the harm of algal bloom to environment, we investigated the diurnal variation of photosynthesis, ATP content and cell division in M. aeruginosa PCC7820. The results showed that the photosynthesis and ATP content of M. aeruginosa PCC7820 exhibited clear circadian rhythm with a period of approximately 24 h and that the periodic rhythms continued for at least three cycles under continuous light conditions. Furthermore, the period length showed that a temperature compensation effect and changes in light cycle or temperature could reset the phase of circadian rhythm. These results indicate that the circadian rhythms of physiological process in M. aeruginosa PCC7820 are controlled by the endogenous circadian clock. Examinations of the number, size and cytokinin content of cells also reveal that the cell division of M. aeruginosa PCC7820 with the generation time of 38.4 h exhibits robust circadian rhythms with a period close to 24 h. The circadian rhythms of cell division may be generated by a biological clock through regulation of the cell division phase of M. aeruginosa PCC7820 via a gating mechanism. The phases in which cell division slows or stop recur with a circadian periodicity of about 24 h.     

Postprandial variations in the activity of polysaccharide-degrading enzymes of fluid- and particle-associated ruminal microbial populations

Effect of an algicidal product fromOscillatoria late-virens and of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) on growth, photosynthesis, and mouse toxicity inMicrocystis PCC 7820 was examined. Their lethal concentrations abolished photosystem (PS)-II reactions and eventually bleached and detoxified the cyanobacterium. Although loss of protein, chlorophyll, and toxicity were also induced by sublethal antibiotic doses, photosynthetic activities remained unchanged and developed antibiotic tolerance. These effects could be duplicated in natural conditions, implying utility of the natural algicide in control of toxic cyanobacteria.     

东湖蓝藻水华毒性的研究 Ⅱ.季节变化及微囊藻的毒性

1984年至1986年间武汉东湖(包括湖边池塘)的水华有7种,即铜绿微囊藻、大型铜绿微囊藻、边缘微囊藻、水华鱼腥藻、卷曲鱼腥藻、美丽颤藻和束丝藻。经生物测定,除束丝藻未测、卷曲鱼腥藻和美丽颤藻未测出毒性外,其余4种皆为有毒水华。东湖的水华随着季节变化而有不同类型的更迭,其出现格局为:卷曲鱼腥藻、微囊藻、颤藻、微囊藻。微囊藻水华的毒性在不同季节也有较大差异,毒性最低在8—9月份,最高在12月份(LD_(50)=24mg/kg鼠重),随着温度的降低而提高。讨论了某些环境参数与微囊藻水华形成及其毒性变化的关系。此外,还用脑内注射和腹腔注射方法,研究了微囊藻毒素的毒性表现。     

二种淡水微囊藻rDNA16S-235基因间隔区的序列测定与分析

本研究采用PCR及序列测定的方法,对我国淡水铜绿微囊藻有毒株(M8641)和另一低毒的种类惠氏微囊藻(M574)rDNA16S-23S基因间隔区进行了序列的测定和分析,结果表明:rDNA16S-23S基因间隔区可以作为一个精细且稳定的指标,用于微囊藻的分类和鉴定。并从分子水平提出了铜绿微囊藻与惠氏微囊藻在种系发生上有较近缘的关系。本文首次对微囊藻属Microcystis rDNA基因间隔区全序列作了报道,为微囊藻属的鉴定及系统学研究提供了分子基础。       

Ecological implications of the emergence of non-toxic subcultures from toxic Microcystis strains

Two toxic, microcystin-producing, Microcystis sp. strains KLL MG-K and KLL MB-K were isolated as single colonies on agar plates from Lake Kinneret, Israel. Two non-toxic subcultures, MG-J and MB-J spontaneously succeeded the toxic ones under laboratory conditions. Southern analyses showed that MG-J and MB-J are lacking at least 34 kb of the mcy region, encoding the microcystin synthetase. Analyses of the 16S rRNA genes, the intergenic spacer region between cpcB and cpcA and the patterns of the polymerase chain reaction products of randomly amplified polymorphic DNA and highly iterated palindrome, and presence of mobile DNA elements did not allow unequivocal distinction between toxic and non-toxic subcultures. Laboratory and field experiments indicated an advantage of the toxic strain over its non-toxic successor. When grown separated by a membrane, which allowed passage of the media but not the cells, MG-K severely inhibited the growth of MG-J. Furthermore, when MG strains were placed in dialysis bags in Lake Kinneret during the season in which Microcystis is often observed, cells of MG-J lysed, whereas MG-K survived. Mechanisms whereby the non-toxic subcultures emerged and prevailed over the corresponding toxic ones under laboratory conditions, as well as a possible role of microcystin under natural conditions, are discussed.     

EVIDENCE THAT MICROCYSTIN IS A THIO-TEMPLATE PRODUCT1

The hepatotoxic cyclic heptapeptide toxins of cyanobacteria, collectively termed microcystins, are potent inhibitors of protein phosphatases PP1 and PP2A. The structure of microcystins resembles small, cyclic peptide secondary metabolites from fungi and eubacteria. Many of these metabolites are manufactured via a nonribosomal thio-template mechanism. We submit evidence that microcystin is synthesized by a similar mechanism. The organism used in this study was Microcystis aeruginosa PCC7820. Using the traditional ATP-³²PP_i exchange assay for thio-template activity, we found activity in the presence of the substrate d -amino acids occurring in microcystin. Thio-template mechanisms are known to be unaffected by protein synthesis inhibitors such as chloramphenicol. We subjected cultures in exponential and stationary growth to chloramphenicol and monitored culture health versus toxicity. Although the health of the treated cultures declined, the toxicity of the remaining cells increased. We developed an in vitro assay to measure microcystin synthesis in cell lysates in the presence of chloramphenicol. By supplementing the lysates with ATP and the substrate amino acids present in microcystin, we detected a fourfold increase in total microcystins over the course of 20 min.     

Influence of toxic and non-toxic phytoplankton on feeding and survival of Dreissena polymorpha (Pallas) larvae

Grazing and survival of larvae of the zebra mussel, Dreissena polymorpha, on a green alga and cyanobacteria were studied in laboratory experiments. Clearance rates of the larvae were determined for Chlamydomonas reinhardtii (green alga), two non-toxic and two toxic Microcystis aeruginosa strains (Cyanobacteria). Clearance rates of larvae on non-toxic Microcystis were significantly higher than on toxic Microcystis. The clearance rate on Chlamydomonas reinhardtii was in between the clearance rates on toxic and non-toxic Microcystis strains and not significantly different from them. Effects of toxicity of Microcystis on the survival of zebra mussel larvae was investigated in a short-term experiment. Survival of larvae fed toxic Microcystis was lower than that of larvae fed non-toxic Microcystis, but higher than that of starved larvae. This may imply that, for survival of zebra mussel larvae, it is better to have bad quality (toxic) food than no food.     

Phylogenetic relationships between toxic and non-toxic strains of the genus Microcystis based on 16S to 23S internal transcribed spacer sequence

16S to 23S ribosomal DNA internal transcribed spacer sequences of 47 strains of the genus Microcystis were determined. Derived maximum likelihood and DNA distance trees indicated that Microcystis can be divided into three clusters. The first cluster included toxic and non-toxic strains, the second only toxic ones, and the third only non-toxic ones. The tree topologies were not necessarily correlated with morphospecies distinction or phycobilin pigment composition, and one genotype may have more than one morphotype. Phylogenetic analysis based on intergenic spacer sequences was thought to be effective for understanding relationships among closely related species and strains.     

Gliding motility in Aphanothece halophytica: analysis of wall proteins in mot mutants.

The unicellular cyanobacterium Aphanothece halophytica (PCC 7418) is motile, and spontaneous nonmotile (mot) mutants accumulate when the organism is subcultured. Analysis of mot mutants suggests that a glycoprotein in the cell wall is involved in the motility mechanism. Proteins from the wall fraction of the wild type and five mot clones were analyzed by gradient sodium dodecyl sulfate-acrylamide gel electrophoresis. Four clones were similar to the wild type, and one clone, mot-3, was missing a high-molecular-weight protein (approximately 200,000) and had at least one new polypeptide (160,000). The high-molecular-weight protein stained with periodic acid-Schiff reagent, suggesting that it was a glycoprotein. The absence of the protein in mot-3 did not affect the mechanical strength of the wall, since both mot-3 and wild-type cells were broken at the same rate by controlled cavitation. Several other cyanobacteria were also screened for the presence of glycoproteins. All motile strains have such proteins, although none had an apparent molecular weight as high as that in Aphanothece sp. Some motile strains, such as Oscillatoria limnetica and Phormidium sp., showed very large amounts of glycoproteins; whereas some nonmotile strains, such as Synechococcus sp. (UTEX 625) and Microcystis sp. (PCC 7820), showed no high-molecular-weight glycoproteins.     

The effect of a mixotrophic chrysophyte on toxic and colony-forming cyanobacteria

1. In order to test the effect of Ochromonas sp. , a mixotrophic chrysophyte, on cyanobacteria, grazing experiments were performed under controlled conditions. We studied grazing on three Microcystis aeruginosa strains, varying in toxicity and morphology, as well as on one filamentous cyanobacterium, Pseudanabaena sp. Furthermore, we analysed the co-occurrence of Ochromonas and Microcystis in natural systems in relation to various environmental parameters (TP, TN, DOC, temperature, pH), using data from 460 Norwegian lakes.
2.  Ochromonas was able to feed on all four cyanobacterial strains tested, and grew quickly on all of them. The chrysophyte caused net growth reductions in all three Microcystis strains (the very toxic single-celled strain PCC 7806; the less toxic colony-forming Bear AC and the less toxic single-celled Spring CJ). The effect of Ochromonas was strongest on the Spring CJ strain. Although the effect of Ochromonas grazing on the growth of Pseudanabaena was relatively smaller, it also reduced the net growth of this cyanobacterium significantly.
3. After 4 days of incubation with Ochromonas the total amount of cyanotoxins in the three Microcystis strains was reduced by 91.1–98.7% compared with the controls.
4.  Ochromonas occurred in similar densities across all 460 Norwegian lakes. Microcystis occurred only at higher TN, TP, temperature and pH values, although its density was often several orders of magnitude higher than that of Ochromonas . Ochromonas co-occurred in 94% of the samples in which Microcystis was present.
5. From our study it is not clear whether Ochromonas could control Microcystis blooms in natural lakes. However, our study does demonstrate that Ochromonas usually occurs in lakes with Microcystis , and our small scale experiments show that Ochromonas can strongly reduce the biomass of Microcystis and its toxin content.     

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.     

STRONG PROBABILITY OF LETHAL TOXICITY IN THE BLUE-GREEN ALGA MICROCYSTIS VIRIDIS LEMMERMANN1

Lethal toxicity (intraperitoneal, mouse) was examined in relation to Species composition of samples containing bloom-forming Microcystis populations from natural waters and correlated with toxicity of laboratory strains of four Microcystis formas and species. Toxicity was not always associated with the presence of M. aeruginosa f . aeruginosa Elenkin. A sample with almost all cells of M. aeruginosa f . aeruginosa showed no toxicity, However samples comprised of a high percentage of M. viridis Lemmermann often showed lethal toxicity. Toxicity tests were done on culture strains M. aeruginosa f aeruginosa, M. aeruginosa f flos-aquae Elenkin , M. viridis and M. wesenbergii Kamárek. All five cultured strains of M. viridis were found to be toxic, while only one out of nine strains of M. aeruginosa f . aeruginosa was toxic. Six strains of M. wesenbergii showed no toxicity, It is recommended that attention should be paid to the occurrences and possibility of toxic bloom of M. viridis from the standpoint of water management and public health .