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Elke Dittmann - One of the best experts on this subject based on the ideXlab platform.
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the cyanobacterial hepatotoxin Microcystin binds to proteins and increases the fitness of microcystis under oxidative stress conditions
PLOS ONE, 2011Co-Authors: Yvonne Zilliges, Janchristoph Kehr, Keishi Ishida, Stefan Mikkat, Sven Meissner, Martin Hagemann, Aaron Kaplan, Thomas Börner, Elke DittmannAbstract:Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that Microcystin fulfils an important function within cells of its natural producer Microcystis. The Microcystin deficient mutant ΔmcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that Microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a Microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of Microcystin in the wild type. Finally, the mutant defective in Microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for Microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites.
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a mannan binding lectin is involved in cell cell attachment in a toxic strain of microcystis aeruginosa
Molecular Microbiology, 2006Co-Authors: Janchristoph Kehr, Yvonne Zilliges, Peter H. Seeberger, Andreas Springer, Matthew D. Disney, Daniel D. Ratner, Christiane Bouchier, Nicole Tandeau De Marsac, Elke DittmannAbstract:Microcystin, a hepatotoxin that represents a serious health risk for humans and livestock, is produced by the bloom-forming cyanobacterium Microcystis aeruginosa in freshwater bodies worldwide. Here we describe the discovery of a lectin, microvirin (MVN), in M. aeruginosa PCC7806 that shares 33% identity with the potent anti-HIV protein cyanovirin-N from Nostoc ellipsosporum. Carbohydrate microarrays were employed to demonstrate the high specificity of the protein for high-mannose structures containing alpha(1-->2) linked mannose residues. Lectin binding analyses and phenotypic characterizations of MVN-deficient mutants suggest that MVN is involved in cell-cell recognition and cell-cell attachment of Microcystis. A binding partner of MVN was identified in the lipopolysaccharide fraction of M. aeruginosa PCC7806. MVN is differentially expressed in mutants lacking the hepatotoxin Microcystin. Additionally, MVN-deficient mutants contain much lower amounts of Microcystin than the wild-type cells. We discuss a possible functional correlation between Microcystin and the lectin and possible implications on Microcystis morphotype formation. This study provides the first experimental evidence that Microcystins may have an impact on Microcystis colony formation that is highly important for the competitive advantage of Microcystis over other phytoplankton species.
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A mannan binding lectin is involved in cell–cell attachment in a toxic strain of Microcystis aeruginosa
Molecular microbiology, 2006Co-Authors: Janchristoph Kehr, Yvonne Zilliges, Peter H. Seeberger, Andreas Springer, Matthew D. Disney, Daniel D. Ratner, Christiane Bouchier, Nicole Tandeau De Marsac, Elke DittmannAbstract:Microcystin, a hepatotoxin that represents a serious health risk for humans and livestock, is produced by the bloom-forming cyanobacterium Microcystis aeruginosa in freshwater bodies worldwide. Here we describe the discovery of a lectin, microvirin (MVN), in M. aeruginosa PCC7806 that shares 33% identity with the potent anti-HIV protein cyanovirin-N from Nostoc ellipsosporum. Carbohydrate microarrays were employed to demonstrate the high specificity of the protein for high-mannose structures containing alpha(1-->2) linked mannose residues. Lectin binding analyses and phenotypic characterizations of MVN-deficient mutants suggest that MVN is involved in cell-cell recognition and cell-cell attachment of Microcystis. A binding partner of MVN was identified in the lipopolysaccharide fraction of M. aeruginosa PCC7806. MVN is differentially expressed in mutants lacking the hepatotoxin Microcystin. Additionally, MVN-deficient mutants contain much lower amounts of Microcystin than the wild-type cells. We discuss a possible functional correlation between Microcystin and the lectin and possible implications on Microcystis morphotype formation. This study provides the first experimental evidence that Microcystins may have an impact on Microcystis colony formation that is highly important for the competitive advantage of Microcystis over other phytoplankton species.
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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: Michael Hisbergues, Lorena Viaordorika, Rainer Kurmayer, Jutta Fastner, Elke Dittmann, 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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Microcystin biosynthesis in planktothrix genes evolution and manipulation
Journal of Bacteriology, 2003Co-Authors: Guntram Christiansen, Jutta Fastner, Marcel Erhard, Thomas Börner, Elke DittmannAbstract:Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of Microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential Microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel Microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.
Petra M Visser - One of the best experts on this subject based on the ideXlab platform.
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the ecological stoichiometry of toxins produced by harmful cyanobacteria an experimental test of the carbon nutrient balance hypothesis
Ecology Letters, 2009Co-Authors: D B Van De Waal, Petra M Visser, Jolanda M H Verspagen, M Lurling, E Van Donk, Jef HuismanAbstract:The elemental composition of primary producers reflects the availability of light, carbon and nutrients in their environment. According to the carbon-nutrient balance hypothesis, this has implications for the production of secondary metabolites. To test this hypothesis, we investigated a family of toxins, known as Microcystins, produced by harmful cyanobacteria. The strain Microcystis aeruginosa HUB 5-2-4, which produces several Microcystin variants of different N:C stoichiometry, was cultured in chemostats supplied with various combinations of nitrate and CO2. Excess supply of both nitrogen and carbon yielded high cellular N:C ratios accompanied by high cellular contents of total Microcystin and the nitrogen-rich variant Microcystin-RR. Comparable patterns were found in Microcystis-dominated lakes, where the relative Microcystin-RR content increased with the seston N:C ratio. In total, our results are largely consistent with the carbon-nutrient balance hypothesis, and warn that a combination of rising CO2 and nitrogen enrichment will affect the Microcystin composition of harmful cyanobacteria.
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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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assimilation and depuration of Microcystin lr by the zebra mussel dreissena polymorpha
Aquatic Toxicology, 2004Co-Authors: L Dionisio M Pires, Petra M Visser, E Van Donk, K M Karlsson, Jussi Meriluoto, E Kardinaal, K Siewertsen, Bastiaan Willem IbelingsAbstract:Zebra mussels (Dreissena polymorpha) are an important component of the foodweb of shallow lakes in the Netherlands, amongst others in Lake IJsselmeer, an international important wetland. Large numbers of ducks feed on these mussels in autumn and winter. The mussels are filter feeders and are exposed to high densities of cyanobacteria in summer and autumn. Mussels and cyanobacteria both thrive in Lake IJsselmeer. Apparently the mussels are somehow protected against accumulation of harmful quantities of cyanobacterial toxins. In this study, we investigated the assimilation of the cyanobacterial toxin Microcystin-LR (MC-LR) in zebra mussels when fed the toxic cyanobacterium Microcystis aeruginosa as sole food or in a mixture with the eustigmatophyte Nannochloropsis limnetica. After 3 weeks of assimilation we studied the depuration of MC-LR during 3 weeks when the food of the mussels was free of cyanobacteria. These assimilation/depuration experiments were combined with grazing experiments, using the same food treatments. Microcystins were analyzed using liquid chromatography-mass spectrometry (LC-MS); in addition, covalently bound MC were analyzed using the MMPB method. The mussels showed higher clearance rates on Microcystis than on Nannochloropsis. No selective rejection of either phytoplankton species was observed in the excretion products of the mussels. Zebra mussels fed Microcystis as single food, assimilated Microcystin-LR relatively fast, and after 1 week the maximum value of free unbound Microcystin assimilation (ca. 11 microg g DW(-1)) was attained. For mussels, fed with the mixed food, a maximum of only 3.9 microg g DW(-1) was recorded after 3 weeks. Covalently bound MC never reached high values, with a maximum of approximately 62% of free MC in the 2nd week of the experiment. In the depuration period Microcystin decreased rapidly to low values and after 3 weeks only very low amounts of Microcystin were detectable. The amount of toxin that accumulated in the mussels would appear to be high enough to cause (liver) damage in diving ducks. However, death by exposure to Microcystin seems unlikely. Mussels seem efficient in minimizing the assimilation of Microcystin. If it were not for this, mass mortalities of ducks in shallow lakes in the Netherlands would presumably occur on a much more widespread scale than is currently observed.
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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, Petra M Visser, W. Edwin A. Kardinaal, Marcel Meima, Jutta Fastner, 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.
Bastiaan Willem Ibelings - One of the best experts on this subject based on the ideXlab platform.
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Potential synergistic effects of Microcystins and bacterial lipopolysaccharides on life history traits of Daphnia galeata raised on low and high food levels
Aquatic Toxicology, 2011Co-Authors: L. Miguel Dionisio Pires, Dirk Sarpe, Michaela Brehm, Bastiaan Willem IbelingsAbstract:Metastudies have found no consistent effects of the cyanobacterial toxin Microcystin on Daphnia, and there are discrepancies between field observations and experiments. Confounding factors include absence or presence of alternative high quality food or the presence of bioactive compounds, other than Microcystins in cyanobacteria. Of specific interest are lipopolysaccharides (LPS) on the outer cell wall. LPS may have a number of biological effects, including reduced detoxication of Microcystins in plants and animals. When grazing seston in the field, filterfeeders take up heterotrophic bacteria attached to cyanobacteria, as well as free-living bacteria. The LPS produced by heterotrophic bacteria have been shown to be much more harmful than cyanobacterial LPS. We performed two experiments in which we tested for potential synergistic effects between bacterial LPS and Microcystins. Full-factorial experiments separated the main effects and interactions between (i) food quantity as well as food quality (addition of the green alga Scenedesmus), (ii) presence or absence of strains that vary in amount and composition of Microcystins (Microcystin free strain NIVA-CYA43, moderate Microcystin producing strain NIVA-CYA140 and high Microcystin producing strain PCC7820), and (iii) presence or absence of bacterial LPS on different life history traits of Daphnia galeata. We measured juvenile growth rate, age and size at first reproduction, death before first reproduction and standard carbon content of Daphnia. From the experiments we conclude that Microcystin-producing Microcystis had deleterious effects on the life history of D. galeata, but especially when the availability of high quality green algal food was limited in comparison to the supply of Microcystin producing strain PCC7820. In the experiment in which PCC7820 was used as Microcystin-producing strain, addition of LPS lowered SCC of Daphnia, but had no effects on other life history parameters. The interaction between Microcystis strain, Microcystis concentration and LPS was highly significant in case of PCC7820, but not in case of CYA-140, indicating that the effects of LPS and its interactions with Microcystin on Daphnia life history were strongly context dependent.
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Distribution of Microcystins in a Lake Foodweb: No Evidence for Biomagnification
Microbial Ecology, 2005Co-Authors: Bastiaan Willem Ibelings, Karsten Brüning, K. Wolfstein, L.m. Dionisio Pires, J. Postma, J. De Jonge, T. BurgerAbstract:Microcystins, toxins produced by cyanobacteria, may play a role in fish kills, although their specific contribution remains unclear. A better understanding of the eco-toxicological effects of Microcystins is hampered by a lack of analyses at different trophic levels in lake foodwebs. We present 3 years of monitoring data, and directly compare the transfer of Microcystin in the foodweb starting with the uptake of (toxic) cyanobacteria by two different filter feeders: the cladoceran Daphnia galeata and the zebra mussel Dreissena polymorpha . Furthermore foodwebs are compared in years in which the colonial cyanobacterium Microcystis aeruginosa or the filamentous cyanobacterium Planktothrix agardhii dominated; there are implications in terms of the types and amount of Microcystins produced and in the ingestion of cyanobacteria. Microcystin concentrations in the seston commonly reached levels where harmful effects on zooplankton are to be expected. Likewise, concentrations in zooplankton reached levels where intoxication of fish is likely. The food chain starting with Dreissena (consumed by roach and diving ducks) remained relatively free from Microcystins. Liver damage, typical for exposure to Microcystins, was observed in a large fraction of the populations of different fish species, although no relation with the amount of Microcystin could be established. Microcystin levels were especially high in the livers of planktivorous fish, mainly smelt. This puts piscivorous birds at risk. We found no evidence for biomagnification of Microcystins. Concentrations in filter feeders were always much below those in the seston, and yet vectorial transport to higher trophic levels took place. Concentrations of Microcystin in smelt liver exceeded those in the diet of these fish, but it is incorrect to compare levels in a selected organ to those in a whole organism (zooplankton). The discussion focuses on the implications of detoxication and covalent binding of Microcystin for the transfer of the toxin in the foodweb. It seems likely that Microcystins are one, but not the sole, factor involved in fish kills during blooms of cyanobacteria.
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assimilation and depuration of Microcystin lr by the zebra mussel dreissena polymorpha
Aquatic Toxicology, 2004Co-Authors: L Dionisio M Pires, Petra M Visser, E Van Donk, K M Karlsson, Jussi Meriluoto, E Kardinaal, K Siewertsen, Bastiaan Willem IbelingsAbstract:Zebra mussels (Dreissena polymorpha) are an important component of the foodweb of shallow lakes in the Netherlands, amongst others in Lake IJsselmeer, an international important wetland. Large numbers of ducks feed on these mussels in autumn and winter. The mussels are filter feeders and are exposed to high densities of cyanobacteria in summer and autumn. Mussels and cyanobacteria both thrive in Lake IJsselmeer. Apparently the mussels are somehow protected against accumulation of harmful quantities of cyanobacterial toxins. In this study, we investigated the assimilation of the cyanobacterial toxin Microcystin-LR (MC-LR) in zebra mussels when fed the toxic cyanobacterium Microcystis aeruginosa as sole food or in a mixture with the eustigmatophyte Nannochloropsis limnetica. After 3 weeks of assimilation we studied the depuration of MC-LR during 3 weeks when the food of the mussels was free of cyanobacteria. These assimilation/depuration experiments were combined with grazing experiments, using the same food treatments. Microcystins were analyzed using liquid chromatography-mass spectrometry (LC-MS); in addition, covalently bound MC were analyzed using the MMPB method. The mussels showed higher clearance rates on Microcystis than on Nannochloropsis. No selective rejection of either phytoplankton species was observed in the excretion products of the mussels. Zebra mussels fed Microcystis as single food, assimilated Microcystin-LR relatively fast, and after 1 week the maximum value of free unbound Microcystin assimilation (ca. 11 microg g DW(-1)) was attained. For mussels, fed with the mixed food, a maximum of only 3.9 microg g DW(-1) was recorded after 3 weeks. Covalently bound MC never reached high values, with a maximum of approximately 62% of free MC in the 2nd week of the experiment. In the depuration period Microcystin decreased rapidly to low values and after 3 weeks only very low amounts of Microcystin were detectable. The amount of toxin that accumulated in the mussels would appear to be high enough to cause (liver) damage in diving ducks. However, death by exposure to Microcystin seems unlikely. Mussels seem efficient in minimizing the assimilation of Microcystin. If it were not for this, mass mortalities of ducks in shallow lakes in the Netherlands would presumably occur on a much more widespread scale than is currently observed.
Thomas Börner - One of the best experts on this subject based on the ideXlab platform.
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the cyanobacterial hepatotoxin Microcystin binds to proteins and increases the fitness of microcystis under oxidative stress conditions
PLOS ONE, 2011Co-Authors: Yvonne Zilliges, Janchristoph Kehr, Keishi Ishida, Stefan Mikkat, Sven Meissner, Martin Hagemann, Aaron Kaplan, Thomas Börner, Elke DittmannAbstract:Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that Microcystin fulfils an important function within cells of its natural producer Microcystis. The Microcystin deficient mutant ΔmcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that Microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a Microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of Microcystin in the wild type. Finally, the mutant defective in Microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for Microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites.
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PCR-based identification of Microcystin-producing genotypes of different cyanobacterial genera
Archives of Microbiology, 2003Co-Authors: Michael Hisbergues, Guntram Christiansen, Leo Rouhiainen, Kaarina Sivonen, 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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Microcystin biosynthesis in planktothrix genes evolution and manipulation
Journal of Bacteriology, 2003Co-Authors: Guntram Christiansen, Jutta Fastner, Marcel Erhard, Thomas Börner, Elke DittmannAbstract:Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of Microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential Microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel Microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.
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role of Microcystins in poisoning and food ingestion inhibition of daphnia galeata caused by the cyanobacterium microcystis aeruginosa
Applied and Environmental Microbiology, 1999Co-Authors: Thomas Rohrlack, Elke Dittmann, Thomas Börner, Manfred Henning, Johannes-günter KohlAbstract:Several species of cyanobacteria, including the bloom-forming freshwater species Microcystis aeruginosa, are able to produce several variants of Microcystin, the most common cyanotoxin, which has been implicated in livestock poisoning and human poisoning (4, 5, 23). Recently, it was shown that these small cyclic peptides are synthesized nonribosomally by peptide synthetases (8). Numerous studies have been carried out in order to determine the ecological significance of the Microcystins. The results obtained, however, are inconsistent (11). One possible function of Microcystins is that they play a role in the defense of M. aeruginosa cells against zooplankton grazing. This hypothesis is supported by the observation that exposure to Microcystis cells reduces the life span of daphnids (3, 10, 13, 18, 24). However, the problem seems to be more complex. The data of Jungmann (13) and Jungmann and Benndorf (14), for example, suggested that an unidentified metabolite of Microcystis sp. rather than Microcystins was responsible for toxicity to Daphnia. Moreover, Nizan et al. (24) found no correlation between acute toxicity of various M. aeruginosa strains to daphnids and their quantitative Microcystin contents. In other cases, daphnids could feed on Microcystin-containing M. aeruginosa without suffering any harmful effects (22). Experiments on Microcystis toxicity have usually been performed by comparing strains which differ in Microcystin content. However, other strain-specific properties could be the source of the striking variation in the results obtained in different investigations. For example, M. aeruginosa strains differ in their content of potential toxic oligopeptides (25), which could strengthen or mask a toxic effect of Microcystins. Also strain-specific differences in ingestibility of M. aeruginosa cells by daphnids may influence the dose of an endotoxin, which is determined by the ingestion rate (amount of food taken in per time unit) and by the toxin content of the cells. Indeed, the ingestion rate of daphnids depends to a large extent on the M. aeruginosa strain offered as food. Some strains affect the ingestion rate of the animals, whereas others do not (10, 12, 15, 18, 19). Some authors (12, 19) have hypothesized that a perceptible factor (“bad taste”) is responsible for this effect. However, the possibility that Microcystins themselves are involved in the inhibition of ingestion cannot be ruled out. The experiments described in this paper were designed to study the role of Microcystins in the effect of M. aeruginosa on Daphnia galeata; both Microcystis toxicity and strain-dependent inhibition of ingestion were studied. To do this, we compared an M. aeruginosa PCC7806 mutant which was genetically engineered to knock out the production of Microcystins with the Microcystin-synthesizing wild-type strain. The two variants of strain PCC7806 used have the same genotype except that the mutant (mcy−) cells have an insertional mutation in a Microcystin synthetase gene (8).
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insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium microcystis aeruginosa pcc 7806
Molecular Microbiology, 1997Co-Authors: Elke Dittmann, Brett Anthony Neilan, Hans Von Dohren, Marcel Erhard, Thomas BörnerAbstract:Summary Several bloom-forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called Microcystins. Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non-ribosomally by peptide synthetases. We identified putative peptide synthetase genes in the Microcystin-producing strain Microcystis aeruginosa PCC 7806. Non-hepatotoxic strains of M. aeruginosa lack these genes. Strain PCC 7806 was transformed to chloramphenicol resistance. The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification. This is the first report of genetic transformation and mutation, by homologous recombination, of a bloomforming cyanobacterium. Chemical and enzymatic analyses, including high-performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of Microcystin while maintaining their ability to synthesize other small peptides. The disrupted gene therefore encodes a peptide synthetase (Microcystin synthetase) that is specifically involved in the biosynthesis of Microcystins. Our results confirm that Microcystins are synthesized non-ribosomally and that a basic difference between toxic and nontoxic strains of M. aeruginosa is the presence of one or more genes coding for Microcystin synthetases.
Christian Mougin - One of the best experts on this subject based on the ideXlab platform.
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Soil irrigation with toxic cyanobacterial Microcystins increases soil nitrification potential
Environmental Chemistry Letters, 2015Co-Authors: Sylvain Corbel, Noureddine Bouaïcha, Fabrice Martin, Olivier Crouzet, Christian MouginAbstract:Microcystins are cyclic heptapeptide hepatotoxins produced by aquatic cyanobacteria such as Microcystis aeruginosa. The wide occurrence of toxic Microcystins in freshwater is a threat to the quality of water, agriculture, and human and animal health. There is actually little knowledge on the impact of Microcystins on soil biomass. Here, an agricultural soil was daily irrigated with a cyanobacterial extract diluted at environmental concentrations of Microcystin–leucine–arginine, from 0.005 to 0.1 mg equivalent MC-LR L−1, for 90 days. We measured soil enzymatic activities, nitrification potential activity of the soil microbial community, abundances of ammonia-oxidizing bacteria, and ammonia-oxidizing archaea amoA genes. Our results show an increase in potential nitrification for Microcystin levels ranging between 0.005 and 0.02 mg eq. MC-LR L−1. Global enzymatic activities were unchanged. Abundances of total bacteria, archaea, and ammonia-oxidizing functional groups were not modified and could not explain the increase in nitrification.
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Soil irrigation with water and toxic cyanobacterial Microcystins accelerates tomato development
Environmental Chemistry Letters, 2015Co-Authors: Sylvain Corbel, Noureddine Bouaïcha, Sylvie Nelieu, Christian MouginAbstract:Microcystins are cyclic heptapeptides hepatotoxins produced by aquatic cyanobacteria such as Microcystis aeruginosa. The wide occurrence of toxic Microcystins in freshwater is a threat to water quality and health of living organisms. Here, we irrigated an agricultural soil daily with a cyanobacterial extract diluted at environmental concentrations of Microcystin–leucine–arginine, from 0.005 to 0.1 mg equivalent MC-LR L−1, for 90 days. We analyzed the impact on the growth and physiology of tomato, Solanum lycopersicum cultivar MicroTom. Our results show a stimulation of the tomato plant development, in terms of inflorescence and blooming, after exposure to the lowest concentration, of 0.005 mg eq. MC-LR L−1, during the 40 first days post-germination. That effect was not apparently associated with physiological disturbances of the tomato plants.
