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Although intensification of toxic cyanobacterial blooms over the last decade is a matter of growing concern due to bloom impact on water quality, the biological role of most of the toxins produced is not known. In this critical review we focus primarily on the biological role of two toxins, microcystins and cylindrospermopsin, in inter- and intra-species communication and in nutrient acquisition. We examine the experimental evidence supporting some of the dogmas in the field and raise several open questions to be dealt with in future research. We do not discuss the health and environmental implications of toxin presence in the water body.
Cyanobacteria produce an unparalleled variety of toxins that can cause severe health problems or even death in humans, and wild or domestic animals. In the last decade, biosynthetic pathways have been assigned to the majority of the known toxin families. This review summarizes current knowledge about the enzymatic basis for the production of the hepatotoxins microcystin and nodularin, the cytotoxin cylindrospermopsin, the neurotoxins anatoxin and saxitoxin, and the dermatotoxin lyngbyatoxin. Elucidation of the biosynthetic pathways of the toxins has paved the way for the development of molecular techniques for the detection and quantification of the producing cyanobacteria in different environments. Phylogenetic analyses of related clusters from a large number of strains has also allowed for the reconstruction of the evolutionary scenarios that have led to the emergence, diversification, and loss of such gene clusters in different strains and genera of cyanobacteria. Advances in the understanding of toxin biosynthesis and evolution have provided new methods for drinking-water quality control and may inspire the development of techniques for the management of bloom formation in the future.
Cyanobacteria produce about 40 percent of the world’s primary biomass, but also a variety of often toxic peptides such as microcystin. Mass developments, so called blooms, can pose a real threat to the drinking water supply in many parts of the world. This study aimed at characterizing the biological function of microcystin production in one of the most common bloom-forming cyanobacterium Microcystis aeruginosa.
In a first attempt, the effect of elevated light intensity on microcystin production and its binding to cellular proteins was studied. Therefore, conventional microcystin quantification techniques were combined with protein-biochemical methods. RubisCO, the key enzyme for primary carbon fixation was a major microcystin interaction partner. High light exposition strongly stimulated microcystin-protein interactions. Up to 60 percent of the total cellular microcystin was detected bound to proteins, i.e. inaccessible for standard quantification procedures. Underestimation of total microcystin contents when neglecting the protein fraction was also demonstrated in field samples. Finally, an immuno-fluorescence based method was developed to identify microcystin producing cyanobacteria in mixed populations.
The high light induced microcystin interaction with proteins suggested an impact of the secondary metabolite on the primary metabolism of Microcystis by e.g. modulating the activity of enzymes. For addressing that question, a comprehensive GC/MS-based approach was conducted to compare the accumulation of metabolites in the wild-type of Microcystis aeruginosa PCC 7806 and the microcystin deficient ΔmcyB mutant. From all 501 detected non-redundant metabolites 85 (17 percent) accumulated significantly different in either of both genotypes upon high light exposition. Accumulation of compatible solutes in the ΔmcyB mutant suggests a role of microcystin in fine-tuning the metabolic flow to prevent stress related to excess light, high oxygen concentration and carbon limitation.
Co-analysis of the widely used model cyanobacterium Synechocystis PCC 6803 revealed profound metabolic differences between species of cyanobacteria. Whereas Microcystis channeled more resources towards carbohydrate synthesis, Synechocystis invested more in amino acids. These findings were supported by electron microscopy of high light treated cells and the quantification of storage compounds. While Microcystis accumulated mainly glycogen to about 8.5 percent of its fresh weight within three hours, Synechocystis produced higher amounts of cyanophycin. The results showed that the characterization of species-specific metabolic features should gain more attention with regard to the biotechnological use of cyanobacteria.
Among the bloom-forming and potentially harmful cyanobacteria, the genus Microcystis represents a most diverse taxon, on the genomic as well as on morphological and secondary metabolite levels. Microcystis communities are composed of a variety of diversified strains. The focus of this study lies on potential interactions between Microcystis representatives and the roles of secondary metabolites in these interaction processes.
The role of secondary metabolites functioning as signaling molecules in the investigated interactions is demonstrated exemplary for the prevalent hepatotoxin microcystin. The extracellular and intracellular roles of microcystin are tested in microarray-based transcriptomic approaches. While an extracellular effect of microcystin on Microcystis transcription is confirmed and connected to a specific gene cluster of another secondary metabolite in this study, the intracellularly occurring microcystin is related with several pathways of the primary metabolism. A clear correlation of a microcystin knockout and the SigE-mediated regulation of carbon metabolism is found. According to the acquired transcriptional data, a model is proposed that postulates the regulating effect of microcystin on transcriptional regulators such as the alternative sigma factor SigE, which in return captures an essential role in sugar catabolism and redox-state regulation.
For the purpose of simulating community conditions as found in the field, Microcystis colonies are isolated from the eutrophic lakes near Potsdam, Germany and established as stably growing under laboratory conditions. In co-habitation simulations, the recently isolated field strain FS2 is shown to specifically induce nearly immediate aggregation reactions in the axenic lab strain Microcystis aeruginosa PCC 7806. In transcriptional studies via microarrays, the induced expression program in PCC 7806 after aggregation induction is shown to involve the reorganization of cell envelope structures, a highly altered nutrient uptake balance and the reorientation of the aggregating cells to a heterotrophic carbon utilization, e.g. via glycolysis. These transcriptional changes are discussed as mechanisms of niche adaptation and acclimation in order to prevent competition for resources.
Microcystins do not necessarily lower the sensitivity of Microcystis aeruginosa to tannic acid
(2016)
Different phytoplankton strains have been shown to possess varying sensitivities towards macrophyte allelochemicals, yet the reasons for this are largely unknown. To test whether microcystin (MC) is responsible for strain-specific sensitivities of Microcystis aeruginosa to macrophyte allelochemicals, we compared the sensitivity of 12 MC- and non-MC-producing M. aeruginosa strains, including an MC-deficient mutant and its wild type, to the polyphenolic allelochemical tannic acid (TA). Non-MC-producing strains showed a significantly higher sensitivity to TA than MC-producing strains, both in Chlorophyll a concentrations and quantum yields of photosystem II. In contrast, an MC-deficient mutant displayed a higher fitness against TA compared to its wild type. These results suggest that the resistance of M. aeruginosa to polyphenolic allelochemicals is not primarily related to MCs per se, but to other yet unknown protective mechanisms related to MCs.
Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains.
Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains.
The transfer of Microcystis aeruginosa from freshwater to estuaries has been described worldwide and salinity is reported as the main factor controlling the expansion of M. aeruginosa to coastal environments. Analyzing the expression levels of targeted genes and employing both targeted and non-targeted metabolomic approaches, this study investigated the effect of a sudden salt increase on the physiological and metabolic responses of two toxic M. aeruginosa strains separately isolated from fresh and brackish waters, respectively, PCC 7820 and 7806. Supported by differences in gene expressions and metabolic profiles, salt tolerance was found to be strain specific. An increase in salinity decreased the growth of M. aeruginosa with a lesser impact on the brackish strain. The production of intracellular microcystin variants in response to salt stress correlated well to the growth rate for both strains. Furthermore, the release of microcystins into the surrounding medium only occurred at the highest salinity treatment when cell lysis occurred. This study suggests that the physiological responses of M. aeruginosa involve the accumulation of common metabolites but that the intraspecific salt tolerance is based on the accumulation of specific metabolites. While one of these was determined to be sucrose, many others remain to be identified. Taken together, these results provide evidence that M. aeruginosa is relatively salt tolerant in the mesohaline zone and microcystin (MC) release only occurs when the capacity of the cells to deal with salt increase is exceeded.
The transfer of Microcystis aeruginosa from freshwater to estuaries has been described worldwide and salinity is reported as the main factor controlling the expansion of M. aeruginosa to coastal environments. Analyzing the expression levels of targeted genes and employing both targeted and non-targeted metabolomic approaches, this study investigated the effect of a sudden salt increase on the physiological and metabolic responses of two toxic M. aeruginosa strains separately isolated from fresh and brackish waters, respectively, PCC 7820 and 7806. Supported by differences in gene expressions and metabolic profiles, salt tolerance was found to be strain specific. An increase in salinity decreased the growth of M. aeruginosa with a lesser impact on the brackish strain. The production of intracellular microcystin variants in response to salt stress correlated well to the growth rate for both strains. Furthermore, the release of microcystins into the surrounding medium only occurred at the highest salinity treatment when cell lysis occurred. This study suggests that the physiological responses of M. aeruginosa involve the accumulation of common metabolites but that the intraspecific salt tolerance is based on the accumulation of specific metabolites. While one of these was determined to be sucrose, many others remain to be identified. Taken together, these results provide evidence that M. aeruginosa is relatively salt tolerant in the mesohaline zone and microcystin (MC) release only occurs when the capacity of the cells to deal with salt increase is exceeded.
The ubiquitous freshwater cyanobacterium Microcystis is remarkably successful, showing a high tolerance against fluctuations in environmental conditions. It frequently forms dense blooms which can accumulate significant amounts of the hepatotoxin microcystin, which plays an extracellular role as an infochemical but also acts intracellularly by interacting with proteins of the carbon metabolism, notably with the CO2 fixing enzyme RubisCO. Here we demonstrate a direct link between external microcystin and its intracellular targets. Monitoring liquid cultures of Microcystis in a diel experiment revealed fluctuations in the extracellular microcystin content that correlate with an increase in the binding of microcystin to intracellular proteins. Concomitantly, reversible relocation of RubisCO from the cytoplasm to the cell’s periphery was observed. These variations in RubisCO localization were especially pronounced with cultures grown at higher cell densities. We replicated these effects by adding microcystin externally to cultures grown under continuous light. Thus, we propose that microcystin may be part of a fast response to conditions of high light and low carbon that contribute to the metabolic flexibility and the success of Microcystis in the field.
The ubiquitous freshwater cyanobacterium Microcystis is remarkably successful, showing a high tolerance against fluctuations in environmental conditions. It frequently forms dense blooms which can accumulate significant amounts of the hepatotoxin microcystin, which plays an extracellular role as an infochemical but also acts intracellularly by interacting with proteins of the carbon metabolism, notably with the CO2 fixing enzyme RubisCO. Here we demonstrate a direct link between external microcystin and its intracellular targets. Monitoring liquid cultures of Microcystis in a diel experiment revealed fluctuations in the extracellular microcystin content that correlate with an increase in the binding of microcystin to intracellular proteins. Concomitantly, reversible relocation of RubisCO from the cytoplasm to the cell’s periphery was observed. These variations in RubisCO localization were especially pronounced with cultures grown at higher cell densities. We replicated these effects by adding microcystin externally to cultures grown under continuous light. Thus, we propose that microcystin may be part of a fast response to conditions of high light and low carbon that contribute to the metabolic flexibility and the success of Microcystis in the field.