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Geoffrey A. Codd - One of the best experts on this subject based on the ideXlab platform.
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Immunogold localisation of microcystins in cryosectioned cells of Microcystis.
Journal of Structural Biology, 2005Co-Authors: Fiona M. Young, James S Metcalf, Calcum Thomson, John M. Lucocq, Geoffrey A. CoddAbstract:Abstract 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.
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Investigations into the taxonomy, toxicity and ecology of benthic cyanobacterial accumulations in Myall Lake, Australia
Marine and Freshwater Research, 2005Co-Authors: Matthew Dasey, Natasha Ryan, Joanne Wilson, Glenn B. Mcgregor, Larelle Fabbro, Harri Kankaanpää, Louise F. Morrison, Brendan P. Burns, Brett A. Neilan, Geoffrey A. CoddAbstract:Large benthic accumulations of cyanobacteria occur in sheltered embayments within Myall Lake, New South Wales, Australia. The lake is shallow, with the entire bottom within the euphotic zone, and it is generally considered pristine, having low nutrient concentrations. The accumulations are highly organic and contain a mix of species mainly from the order Chroococcales, with two forms of Aphanothece being dominant. However polymerase chain reaction (PCR) analysis indicates a close similarity to Microcystis flos-aquae. The cells appear to lack aerotopes and form sticky mucilaginous amalgamations, which may enhance their benthic habit. Although Chroococcales also dominate the planktonic cyanobacterial community, the benthic species are seldom, if ever, found entrained within the water column. Some hepatotoxicity was indicated by mouse bioassay, protein phosphatase inhibition assay, enzyme-linked immuno-sorbent assay (ELISA) for microcystins, PCR and by chromatographic evidence for a microcystin. Ecological aspects of the distribution, gross morphology of the organisms and management implications for recreational water-users are discussed.
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effects of light on the microcystin content of Microcystis strain pcc 7806
Applied and Environmental Microbiology, 2003Co-Authors: Claudia Wiedner, James S Metcalf, Jutta Fastner, Petra M Visser, Geoffrey A. CoddAbstract:Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m−2 s−1 on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell−1 and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m−2 s−1, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m−2 s−1 than at 10 μmol of photons m−2 s−1 and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.
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effects of enteric bacterial and cyanobacterial lipopolysaccharides and of microcystin lr on glutathione s transferase activities in zebra fish danio rerio
Aquatic Toxicology, 2002Co-Authors: J.h. Best, James S Metcalf, Stephan Pflugmacher, F.b. Eddy, Claudia Wiegand, Geoffrey A. CoddAbstract:Abstract Cyanobacteria (blue-green algae) can produce a variety of toxins including hepatotoxins e.g. microcystins, and endotoxins such as lipopolysaccharides (LPS). The combined effects of such toxins on fish are little known. This study examines the activities of microsomal (m) and soluble (s) glutathione S-transferases (GST) from embryos of the zebra fish, Danio rerio at the prim six embryo stage, which had been exposed since fertilisation to LPS from different sources. A further aim was to see how activity was affected by co-exposure to LPS and microcystin-LR (MC-LR). LPS were obtained from Salmonella typhimurium, Escherichia coli, a laboratory culture of Microcystis CYA 43 and natural cyanobacterial blooms of Microcystis and Gloeotrichia. Following in vivo exposure of embryos to each of the LPS preparations, mGST activity was significantly reduced (from 0.50 to between 0.06 and 0.32 nanokatals per milligram (nkat mg−1) protein). sGST activity in vivo was significantly reduced (from 1.05 to between 0.19 and 0.22 nkat mg−1 protein) after exposure of embryos to each of the cyanobacterial LPS preparations, but not in response to S. typhimurium or E. coli LPS. Activities of both m- and sGSTs were reduced after co-exposure to MC-LR and cyanobacterial LPS, but only mGST activity was reduced in the S. typhimurium and E. coli LPS-treated embryos. In vitro preparations of GST from adult and prim six embryo D. rerio showed no significant changes in enzyme activity in response to the LPS preparations with the exception of Gloeotrichia bloom LPS, where mGST was reduced in adult and embryo preparations. The present study represents the first investigations into the effects of cyanobacterial LPS on the phase-II microcystin detoxication mechanism. LPS preparations, whether from axenic cyanobacteria or cyanobacterial blooms, are potentially capable of significantly reducing activity of both the s- and mGSTs, so reducing the capacity of D. rerio to detoxicate microcystins. The results presented here have wide ranging implications for both animal and human health.
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immuno crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin microcystin lr
Fems Microbiology Letters, 2000Co-Authors: James S Metcalf, Kenneth A Beattie, Stephan Pflugmacher, Geoffrey A. CoddAbstract:Immunoassays are increasingly used to investigate the production, properties and fates of the cyanobacterial hepatotoxic microcystins in vitro and in vivo. Responses of an ELISA immunoassay to microcystins have been determined using the authentic toxin antigen, microcystin-LR, and conjugation products between the toxin and glutathione, cysteine-glycine and cysteine. The antibodies against microcystin-LR crossreacted with the toxin conjugation products with similar affinities (96–112%) to that of microcystin-LR, when assayed at a concentration of 1 μg l−1. Toxicity assessment of the conjugates, in comparison to microcystin-LR, indicated a reduction according to mouse bioassay. In vitro protein phosphatase inhibition assay indicated that the conjugates possessed approximately 3–9-fold lower toxicity than microcystin-LR.
Kaarina Sivonen - One of the best experts on this subject based on the ideXlab platform.
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microcystin production in the tripartite cyanolichen peltigera leucophlebia
Molecular Plant-microbe Interactions, 2009Co-Authors: Ulla Kaasalainen, Jouni Jokela, David P Fewer, Kaarina Sivonen, Jouko RikkinenAbstract:We show that the cyanobacterial symbionts of a tripartite cyanolichen can produce hepatotoxic microcystins in situ. Microcystins were detected with high-performance liquid chromatography mass spectrometry both from cephalodia of the tripartite cyanolichen Peltigera leucophlebia and from a symbiotic Nostoc strain isolated from the same lichen specimen. Genetic identities of symbiotic Nostoc strains were studied by amplifying and sequencing the 16S rRNA gene. Also, the presence of the microcystin synthetase gene mcyE was confirmed by sequencing. Three highly toxic microcystins were detected from the lichen specimen. Several different Nostoc 16S rRNA haplotypes were present in the lichen sample but only one was found in the toxin-producing cultures. In culture, the toxin-producing Nostoc strain produced a total of 19 different microcystin variants. In phylogenetic analysis, this cyanobacterium and related strains from the lichen thallus grouped together with a previously known microcystin-producing Nostoc st...
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discovery of rare and highly toxic microcystins from lichen associated cyanobacterium nostoc sp strain io 102 i
Applied and Environmental Microbiology, 2004Co-Authors: Ilona Oksanen, Jouko Rikkinen, Jouni Jokela, David P Fewer, Matti Wahlsten, Kaarina SivonenAbstract:The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda 5 ]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda 5 ]microcystin-LR and [D-Asp 3 ,ADMAdda 5 ]microcystin-LR and a partial structure of three new [ADMAdda 5 ]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.
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quantitative real time pcr for determination of microcystin synthetase e copy numbers for Microcystis and anabaena in lakes
Applied and Environmental Microbiology, 2003Co-Authors: Jaana Vaitomaa, Anne Rantala, Katrianna Halinen, Leo Rouhiainen, Petra Tallberg, Lena Mokelke, Kaarina SivonenAbstract:Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjarvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjarvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjarvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.
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PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera
Archives of Microbiology, 2003Co-Authors: Michael Hisbergues, Guntram Christiansen, Kaarina Sivonen, Leo Rouhiainen, Thomas BörnerAbstract:Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena , Microcystis , Anabaena , Planktothrix , and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis , and Planktothrix . Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.
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effect of nitrogen and phosphorus on growth of toxic and nontoxic Microcystis strains and on intracellular microcystin concentrations
Microbial Ecology, 2002Co-Authors: C Vezie, Jaana Vaitomaa, Jarkko Rapala, J Seitsonen, Kaarina SivonenAbstract:A B S T R A C T The growth and intracellular microcystin concentration of two hepatotoxic and two nontoxic axenic Microcystis strains were measured in batch cultures with variable nitrogen (0.84-84 mg L -1 ) and phosphorus (0.05-5.5 mg L -1 ) concentrations. Growth was estimated by measuring dry weight, optical density, chlorophyll a, and cellular protein concentration. Microcystin concentrations in cells and in culture medium were measured by HPLC analysis. Both nontoxic strains needed less nutrients for their growth at low nutrient concentrations. With high nutrient concentrations the toxic strains grew better than the nontoxic strains. Growth and intracellular microcystin concentration did not correlate in the hepatotoxic strains. Multivariate regression analysis together with mathematical modeling revealed a significant interactive effect of nitrogen and phosphorus, which partly explains the controversial results obtained in previous studies. In this study we have shown that variation of nitrogen and phosphorus concentrations influence the growth and the microcystin production of Microcystis strains and that the strains differ in their response to nutrients. High levels of nitrogen and phosphorus in freshwaters may favor the growth of toxic Microcystis strains over nontoxic ones.
Petra M Visser - One of the best experts on this subject based on the ideXlab platform.
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pulsed nitrogen supply induces dynamic changes in the amino acid composition and microcystin production of the harmful cyanobacterium planktothrix agardhii
FEMS Microbiology Ecology, 2010Co-Authors: Dedmer B Van De Waal, Petra M Visser, Linda Tonk, Jef Huisman, Gonzalo Ferreruela, Ellen Van Donk, Hans C P MatthijsAbstract:Planktothrix agardhii is a widespread harmful cyanobacterium of eutrophic waters, and can produce the hepatotoxins [Asp3]microcystin-LR and [Asp3]microcystin-RR. These two microcystin variants differ in their first variable amino acid position, which is occupied by either leucine (L) or arginine (R). Although microcystins are extensively investigated, little is known about the mechanisms that determine the production of different microcystin variants. We hypothesize that enhanced nitrogen availability will increase the intracellular content of the nitrogen-rich amino acid arginine, and thereby promote the production of the variant [Asp3]microcystin-RR. To test this hypothesis, we transferred P. agardhii strain 126/3 from nitrogen-replete to nitrogen-deficient conditions, and after 2 weeks of growth under nitrogen deficiency, we added a nitrogen pulse. We found a rapid increase in the cellular nitrogen to carbon ratio and the amino acids aspartic acid and arginine, indicative of cyanophycin synthesis. This was followed by a more gradual increase of the total amino acid content connected to balanced growth. As expected, the [Asp3]microcystin-RR variant increased strongly after the nitrogen pulse, while the [Asp3]microcystin-LR increased to a much lesser extent. We conclude that sudden nitrogen enrichment affects the amino acid composition of harmful cyanobacteria, which, in turn, affects the production and composition of their microcystins.
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Microcystis genotype succession in relation to microcystin concentrations in freshwater lakes
Aquatic Microbial Ecology, 2007Co-Authors: W E A Kardinaal, Ingmar Janse, Jef Huisman, M Meima, M Kamstvan P Agterveld, J Snoek, G Zwart, Petra M VisserAbstract:Potentially toxic Cyanobacteria, like Microcystis, form a serious threat in recreational waters and drinking-water reservoirs. We monitored the population dynamics of toxic and non-toxic Microcystis strains using rRNA of the internal transcribed spacer region in combination with DGGE to determine whether there is a seasonal succession of toxic and non-toxic Microcystis genotypes in freshwater lakes and, if so, whether this succession can explain seasonal dynamics of the toxin microcystin. We studied 3 lakes in The Netherlands, all dominated by Microcystis during summer. Coexistence of several genotypes was observed in all lakes. The seasonal succession in a deep, stratified lake started with a population consisting of several toxic genotypes at the onset of the bloom, which changed into a population dominated by non-toxic genotypes at the end of the bloom. In this lake, the genotype succession clearly accounted for the observed microcystin dynamics. In 2 unstratified lakes, we also observed a seasonal replacement of Microcystis genotypes; however, the relation between genotype succession and microcystin dynamics was less conspicuous, since toxic strains dominated throughout the bloom period. A seasonal succession of different Microcystis genotypes might often be a key mechanism determining microcystin concentrations in Microcystisdominated lakes. Therefore, factors driving the succession of toxic and non-toxic genotypes deserve further study.
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competition for light between toxic and nontoxic strains of the harmful cyanobacterium Microcystis
Applied and Environmental Microbiology, 2007Co-Authors: Edwin W A Kardinaal, Linda Tonk, Ingmar Janse, Pieter Slot, Jef Huisman, Petra M VisserAbstract:The cyanobacterium Microcystis can produce microcystins, a family of toxins that are of major concern in water management. In several lakes, the average microcystin content per cell gradually declines from high levels at the onset of Microcystis blooms to low levels at the height of the bloom. Such seasonal dynamics might result from a succession of toxic to nontoxic strains. To investigate this hypothesis, we ran competition experiments with two toxic and two nontoxic Microcystis strains using light-limited chemostats. The population dynamics of these closely related strains were monitored by means of characteristic changes in light absorbance spectra and by PCR amplification of the rRNA internal transcribed spacer region in combination with denaturing gradient gel electrophoresis, which allowed identification and semiquantification of the competing strains. In all experiments, the toxic strains lost competition for light from nontoxic strains. As a consequence, the total microcystin concentrations in the competition experiments gradually declined. We did not find evidence for allelopathic interactions, as nontoxic strains became dominant even when toxic strains were given a major initial advantage. These findings show that, in our experiments, nontoxic strains of Microcystis were better competitors for light than toxic strains. The generality of this finding deserves further investigation with other Microcystis strains. The competitive replacement of toxic by nontoxic strains offers a plausible explanation for the gradual decrease in average toxicity per cell during the development of dense Microcystis blooms.
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salt tolerance of the harmful cyanobacterium Microcystis aeruginosa
Aquatic Microbial Ecology, 2007Co-Authors: Linda Tonk, Petra M Visser, Kim Bosch, Jef HuismanAbstract:Increasing salinities in freshwater ecosystems caused by agricultural practices, droughts, or rise in sea level are likely to affect the species composition of phototrophic microorganisms. Cosmopolitan freshwater cyanobacteria of the Microcystis genus can produce the toxin microcystin, and present a potential health risk in many eutrophic lakes. In this study, M. aeruginosa Strain PCC 7806 was grown in semi-continuous turbidostats to investigate the effect of increasing salinity on growth rate, microcystin cell quota, microcystin production and extracellular microcystin concentration. Specific growth rate, microcystin cell quota and microcystin production remained more or less unaffected by salinity levels up to 10 g l–1. Specific growth rate collapsed when salinity was increased beyond 10 g l–1 for several weeks. Cell size and microcystin cell quota decreased while extracellular microcystin concentrations increased at salinities above 10 g l–1, indicating leakage and/or cell lysis. Salt-shock experiments revealed that M. aeruginosa can temporarily endure salinities as high as 17.5 g l–1. These results indicate that, for a freshwater species, M. aeruginosa has a high salt tolerance. Rising salinities in freshwater ecosystems are therefore unlikely to suppress M. aeruginosa blooms, and may in fact enhance the exposure of aquatic organisms to elevated concentrations of extracellular microcystins.
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toxic and nontoxic Microcystis colonies in natural populations can be differentiated on the basis of rrna gene internal transcribed spacer diversity
Applied and Environmental Microbiology, 2004Co-Authors: Ingmar Janse, Jutta Fastner, Petra M Visser, W E A Kardinaal, M Meima, Gabriel ZwartAbstract:Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.
Jutta Fastner - One of the best experts on this subject based on the ideXlab platform.
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Microcystins (cyanobacterial toxins) in surface waters of rural Bangladesh: Pilot study
Journal of Water and Health, 2005Co-Authors: Martin Welker, Ingrid Chorus, Saleha Khan, Mahfuzul Haque, Sirajul Islam, Nurul Huda Khan, Jutta FastnerAbstract:In Bangladesh the exposure of millions of inhabitants to water from (shallow) tube wells contaminated with high geogenic loads of arsenic is a major concern. As an alternative to the costly drilling of deep wells, the return to the use of surface water as a source of drinking water is considered. In addition to the well-known hazards of water borne infectious diseases associated with the use of surface water, recently the potential public health implications of toxic cyanobacteria have been recognized. As a first step towards a risk assessment for cyanotoxins in Bangladesh surface waters, seston samples of 79 ponds were analysed in late summer 2002 for the presence of cyanobacteria and microcystins (MCYST), the most frequently detected cyanobacterial toxins worldwide. Microcystins could be detected in 39 ponds, mostly together with varying abundance of potentially microcystin-producing genera such as Microcystis, Planktothrix and Anabaena. Total microcystin concentrations ranged between 1,000 microg l(-1), and more than half of the positive samples contained high concentrations of more than 10 microg l(-1). The results clearly show that concentrations of microcystins well above the provisional WHO guideline value of 1 microg l(-1) MCYST-LR can be frequently detected in Bangladesh ponds. Thus, an increasing use of surface water for human consumption introduces a risk of replacing one health hazard by another and therefore needs to be accompanied by cyanotoxin hazard assessments.
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distribution of microcystin producing and non microcystin producing Microcystis sp in european freshwater bodies detection of microcystins and microcystin genes in individual colonies
Systematic and Applied Microbiology, 2004Co-Authors: Elke Dittmann, Jutta Fastner, Michael Hisbergues, Lorena Viaordorika, Rainer Kurmayer, Jiri Komarek, Marcel ErhardAbstract:Summary Microcystis is a well-known cyanobacterial genus frequently producing hepatotoxins named microcystins. Toxin production is encoded by microcystin genes (mcy). This study aims (i) to relate the mcy occurrence in individual colonies to the presence of microcystin, (ii) to assess whether morphological characteristics (morphospecies) are related to the occurrence of mcy genes, and (iii) to test whether there are geographical variations in morphospecies specificity and abundance of mcy genes. Individual colonies of nine different European countries were analysed by (1) morphological characteristics, (2) PCR to amplify a gene region within mcyA and mcyB indicative for microcystin biosynthesis, (3) matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to detect microcystins. Almost one hundred percent of the colonies predicted to produce microcystins by PCR analysis were found to contain microcystins. A high similarity in microcystin variants in the different colonies selected from lakes across Europe was demonstrated. The different morphospecies varied in the frequency with which they contained mcy genes. Most colonies (>75%) of M. aeruginosa and M. botrys contained the mcy genes, whereas ≤20% of the colonies identified as M. ichthyoblabe and M. viridis gave a PCR product of the mcy genes. No colonies of M. wesenbergii gave a PCR product of either mcy gene. In addition, a positive relationship was found between the size of the colony and the frequency of those containing the mcy genes. It is concluded that the analysis of morphospecies is indicative for microcystin production, although the quantitative analysis of microcystin concentrations in water remains indispensable for hazard
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toxic and nontoxic Microcystis colonies in natural populations can be differentiated on the basis of rrna gene internal transcribed spacer diversity
Applied and Environmental Microbiology, 2004Co-Authors: Ingmar Janse, Jutta Fastner, Petra M Visser, W E A Kardinaal, M Meima, Gabriel ZwartAbstract:Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.
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natural variation in the microcystin synthetase operon mcyabc and impact on microcystin production in Microcystis strains
Journal of Bacteriology, 2003Co-Authors: Bjorg Mikalsen, Jutta Fastner, Gudrun Boison, Olav M Skulberg, William Davies, Tove M Gabrielsen, Knut Rudi, Kjetill S JakobsenAbstract:Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.
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effects of light on the microcystin content of Microcystis strain pcc 7806
Applied and Environmental Microbiology, 2003Co-Authors: Claudia Wiedner, James S Metcalf, Jutta Fastner, Petra M Visser, Geoffrey A. CoddAbstract:Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m−2 s−1 on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell−1 and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m−2 s−1, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m−2 s−1 than at 10 μmol of photons m−2 s−1 and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.
Ken-ichi Harada - One of the best experts on this subject based on the ideXlab platform.
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Immunoaffinity purification method for detection and quantification of microcystins in lake water
Toxicon, 2000Co-Authors: Fumio Kondo, Kiyomi Tsuji, Hiroshi Matsumoto, Seiji Yamada, Yoshio Ueno, Ken-ichi HaradaAbstract:Abstract We have developed a new clean-up method, which consisted of solid-phase extraction on a Sep-Pak PS-2 (styrene–divinylbenzene copolymer) or Excelpak SPE-GLF (polymethacrylate) cartridge instead of conventional ODS silica gel and silica gel together with following immunoaffinity purification using anti-microcystin-LR monoclonal antibodies. This newly developed method was demonstrated to eliminate co-existing substances and to concentrate microcystins in the lake water. The recoveries from lake water (1 liter) spiked with 100 ng each of microcystins-RR, -YR and -LR were 85.5, 89.2 and 92.2%, respectively, with coefficients of variation of 3.3–7.6%. Only 3 h were required to complete the total procedures starting from the microcystin extraction, the immunoaffinity purification, and the quantification using HPLC. The detection limits for all of the 3 microcystins in lake water were 0.005 μg/l. Applicability of this method has been demonstrated by measuring the concentrations of microcystins in water samples collected from lakes where water blooms occurred, which turned out to be 0.012–0.177 μg/l of total microcystins.
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genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp
Journal of Biochemistry, 1999Co-Authors: Tomoyasu Nishizawa, Ken-ichi Harada, Munehiko Asayama, Kiyonaga Fujii, Makoto ShiraiAbstract:Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.
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Stability of microcystins from cyanobacteria-II. Effect of UV light on decomposition and isomerization
Toxicon, 1995Co-Authors: Kiyomi Tsuji, Tomohiko Watanuki, Hideaki Uchida, Sumiko Suzuki, Mariyo F Watanabe, Fumio Kondo, Hiroyuki Nakazawa, Makoto Suzuki, Ken-ichi HaradaAbstract:Microcystins are very potent hepatotoxins and strong liver tumor promoters produced by cyanobacteria, and their occurrence has been reported all over the world. They could threaten human health when toxic Microcystis occurs in water supply reservoirs. In this study, we examined the stability of microcystins during photolysis with UV light. The toxins were easily decomposed by UV light at wavelengths around the absorption maxima of the toxins and the decomposition depended on the intensity of the light. The half-life of microcystin LR by 147 μW/cm 2 UV irradiation was 10 min, and the toxin was completely decomposed by 2550 μW/cm 2 UV after 10 min. When the toxins were irradiated with weaker UV light, isomerization was also observed by a different mechanism from that during photolysis by sunlight and pigment, and several products including three geometrical isomers of the conjugated diene of Adda were detected. Microcystin RR showed almost the same behavior as that of microcystin LR under the same conditions. Since no noxious products were formed in the present study, a water treatment including UV irradiation is very possible for removing microcystins from raw water. © 1995.
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seasonal variations of Microcystis species and toxic heptapeptide microcystins in lake suwa
Environmental Toxicology & Water Quality, 1993Co-Authors: Ho-dong Park, Ken-ichi Harada, Mariyo F Watanabe, Makoto Suzuki, Hidetake Hayashi, Tokio OkinoAbstract:Seasonal changes in species composition of Microcystis and concentrations of toxic heptapeptide microcystins, were investigated in Lake Suwa from June to October in 1991. Microcystins-RR and -LR were the main components of the toxins contained in bloom samples of Microcystis and a very little quantity of - YR was detected through this period. The high amounts of microcystins were estimated during the exponential growth phase of the bloom from June 11 to July 20. The highest concentrations of microcystins-RR and -LR were estimated as 121 and 81.6 μg/100 mg cells on July 20, 1991, respectively. While the higher amounts of the toxins were estimated when M. aeruginosa predominated, the lower amounts were estimated during the predominate period of M. viridis. Since the dissolved inorganic nitrogen (DIN) concentration may well affect the dominance of M. aeruginosa and M. viridis, the production of the toxins by Microcystis may be associated with the DIN concentration. © 1993 John Wiley & Sons, Inc.
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release of heptapeptide toxin microcystin during the decomposition process of Microcystis aeruginosa
Natural Toxins, 1992Co-Authors: Mariyo F Watanabe, Kiyomi Tsuji, Ken-ichi Harada, Yasunori Watanabe, Makoto SuzukiAbstract:The decomposition process of toxic blue-green alga (cyanobacteria), Microcystis aeruginosa, under dark and aerobic condition was investigated in relation to the change of the amounts of heptapeptide toxins (microcystins YR and LR) by two experiments: one with Microcystis cells and the other with two purified microcystins. In the experiment with Microcystis cells, an increase of heterotrophic bacteria observed from the beginning of the experiment, was followed by decomposition of the algal cells and the subsequent release of microcystins into the filtrate fraction. The amounts of the toxins initially present in the cells were quantitatively detected in the filtrate fraction on the 35th day. The decomposition of microcystin YR began on the 42nd day. The decomposition rate of the two toxins was different. The decomposition rate of purified microcystins YR and LR, compared in distilled water and culture medium, respectively, indicated clearly that microcystin YR was more labile to decomposition than microcystin LR in the culture medium. At the end of the experiment (45th day) microcystin YR decreased to 58.6%, while 86.2% of microcystin LR remained. © 1992 Wiley-Liss, Inc.
