@article{SchuurmansBrinkmannMakoweretal.2018, author = {Schuurmans, Jasper Merijn and Brinkmann, Bregje W. and Makower, Katharina and Dittmann, Elke and Huisman, Jef and Matthijs, Hans C. P.}, title = {Microcystin interferes with defense against high oxidative stress in harmful cyanobacteria}, series = {Harmful algae}, volume = {78}, journal = {Harmful algae}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1568-9883}, doi = {10.1016/j.hal.2018.07.008}, pages = {47 -- 55}, year = {2018}, abstract = {Harmful cyanobacteria producing toxic microcystins are a major concern in water quality management. In recent years, hydrogen peroxide (H2O2) has been successfully applied to suppress cyanobacterial blooms in lakes. Physiological studies, however, indicate that microcystin protects cyanobacteria against oxidative stress, suggesting that H2O2 addition might provide a selective advantage for microcystin-producing (toxic) strains. This study compares the response of a toxic Microcystis strain, its non-toxic mutant, and a naturally non-toxic Microcystis strain to H2O2 addition representative of lake treatments. All three strains initially ceased growth upon H2O2 addition. Contrary to expectation, the non-toxic strain and non-toxic mutant rapidly degraded the added H2O2 and subsequently recovered, whereas the toxic strain did not degrade H2O2 and did not recover. Experimental catalase addition enabled recovery of the toxic strain, demonstrating that rapid H2O2 degradation is indeed essential for cyanobacterial survival. Interestingly, prior to H2O2 addition, gene expression of a thioredoxin and peroxiredoxin was much lower in the toxic strain than in its non-toxic mutant. Thioredoxin and peroxiredoxin are both involved in H2O2 degradation, and microcystin may potentially suppress their activity. These results show that microcystin-producing strains are less prepared for high levels of oxidative stress, and are therefore hit harder by H2O2 addition than non-toxic strains.}, language = {en} } @article{HackenbergHakanpaeaeCaietal.2018, author = {Hackenberg, Claudia and Hakanpaeae, Johanna and Cai, Fei and Antonyuk, Svetlana and Eigner, Caroline and Meissner, Sven and Laitaoja, Mikko and Janis, Janne and Kerfeld, Cheryl A. and Dittmann, Elke and Lamzin, Victor S.}, title = {Structural and functional insights into the unique CBS-CP12 fusion protein family in cyanobacteria}, series = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {27}, publisher = {National Acad. of Sciences}, address = {Washington}, issn = {0027-8424}, doi = {10.1073/pnas.1806668115}, pages = {7141 -- 7146}, year = {2018}, abstract = {Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine beta-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from Microcystis aeruginosa PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structurally uncharacterized N-terminal region. We provide evidence that CBS-CP12, while combining properties of both regulatory domains, behaves different from CP12 and plant CBSX. It does not form a ternary complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase. Instead, CBS-CP12 decreases the activity of PRK in an AMP-dependent manner. We propose that the novel domain architecture and oligomeric state of CBS-CP12 expand its regulatory function beyond those of CP12 in cyanobacteria.}, language = {en} } @article{HuLudsinMartinetal.2018, author = {Hu, Chenlin and Ludsin, Stuart A. and Martin, Jay F. and Dittmann, Elke and Lee, Jiyoung}, title = {Mycosporine-like amino acids (MAAs)-producing Microcystis in Lake Erie}, series = {Harmful algae}, volume = {77}, journal = {Harmful algae}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1568-9883}, doi = {10.1016/j.hal.2018.05.010}, pages = {1 -- 10}, year = {2018}, abstract = {Mycosporine-like amino acids (MAAs) are UV-absorbing metabolites found in cyanobacteria. While their protective role from UV in Microcystis has been studied in a laboratory setting, a full understanding of the ecology of MAA-producing versus non-MAA-producing Microcystis in natural environments is lacking. This study presents a new tool for quantifying MAA-producing Microcystis and applies it to obtain insight into the dynamics of MAA-producing and non-MAA-producing Microcystis in Lake Erie. This study first developed a sensitive, specific TaqMan real-time PCR assay that targets MAA synthetase gene C (mysC) of Microcystis (quantitative range: 1.7 × 101 to 1.7 × 107 copies/assay). Using this assay, Microcystis was quantified with a MAA-producing genotype (mysC+) in water samples (n = 96) collected during March-November 2013 from 21 Lake Erie sites (undetectable - 8.4 × 106 copies/ml). The mysC+ genotype comprised 0.3-37.8\% of the Microcystis population in Lake Erie during the study period. The proportion of the mysC+ genotype during high solar UV irradiation periods (mean = 18.8\%) was significantly higher than that during lower UV periods (mean = 9.7\%). Among the MAAs, shinorine (major) and porphyra (minor) were detected with HPLC-PDA-MS/MS from the Microcystis isolates and water samples. However, no significant difference in the MAA concentrations existed between higher and lower solar UV periods when the MAA concentrations were normalized with Microcystis mysC abundance. Collectively, this study's findings suggest that the MAA-producing Microcystis are present in Lake Erie, and they may be ecologically advantageous under high UV conditions, but not to the point that they exclusively predominate over the non-MAA-producers.}, language = {en} } @article{DehmKrumbholzBaunachetal.2019, author = {Dehm, Daniel and Krumbholz, Julia and Baunach, Martin and Wiebach, Vincent and Hinrichs, Katrin and Guljamow, Arthur and Tabuchi, Takeshi and Jenke-Kodama, Holger and S{\"u}ssmuth, Roderich D. and Dittmann-Th{\"u}nemann, Elke}, title = {Unlocking the spatial control of secondary metabolism uncovers hidden natural product diversity in nostoc punctiforme}, series = {ACS chemical biology}, volume = {14}, journal = {ACS chemical biology}, number = {6}, publisher = {American Chemical Society}, address = {Washington}, issn = {1554-8929}, doi = {10.1021/acschembio.9b00240}, pages = {1271 -- 1279}, year = {2019}, abstract = {Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50\% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.}, language = {en} } @article{BarchewitzGuljamowMeissneretal.2019, author = {Barchewitz, Tino and Guljamow, Arthur and Meißner, Sven and Timm, Stefan and Henneberg, Manja and Baumann, Otto and Hagemann, Martin and Dittmann, Elke}, title = {Non-canonical localization of RubisCO under high-light conditions in the toxic cyanobacterium Microcystis aeruginosa PCC7806}, series = {Environmental microbiology}, volume = {21}, journal = {Environmental microbiology}, number = {12}, publisher = {Wiley}, address = {Hoboken}, issn = {1462-2912}, doi = {10.1111/1462-2920.14837}, pages = {4836 -- 4851}, year = {2019}, abstract = {The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2 fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.}, language = {en} } @article{PancraceIshidaBriandetal.2018, author = {Pancrace, Claire and Ishida, Keishi and Briand, Enora and Pichi, Douglas Gatte and Weiz, Annika R. and Guljarmow, Arthur and Scalvenzi, Thibault and Sassoon, Nathalie and Hertweck, Christian and Dittmann, Elke and Gugger, Muriel}, title = {Unique Biosynthetic Pathway in Bloom-Forming Cyanobacterial Genus Microcystis Jointly Assembles Cytotoxic Aeruginoguanidines and Microguanidines}, series = {ACS chemical biology}, volume = {14}, journal = {ACS chemical biology}, number = {1}, publisher = {American Chemical Society}, address = {Washington}, issn = {1554-8929}, doi = {10.1021/acschembio.8b00918}, pages = {67 -- 75}, year = {2018}, abstract = {The cyanobacterial genus Microcystis is known to produce an elaborate array of structurally unique and biologically active natural products, including hazardous cyanotoxins. Cytotoxic aeruginoguanidines represent a yet unexplored family of peptides featuring a trisubstituted benzene unit and farnesylated arginine derivatives. In this study, we aimed at assigning these compounds to a biosynthetic gene cluster by utilizing biosynthetic attributes deduced from public genomes of Microcystis and the sporadic distribution of the metabolite in axenic strains of the Pasteur Culture Collection of Cyanobacteria. By integrating genome mining with untargeted metabolomics using liquid chromatography with mass spectrometry, we linked aeruginoguanidine (AGD) to a nonribosomal peptide synthetase gene cluster and coassigned a significantly smaller product to this pathway, microguanidine (MGD), previously only reported from two Microcystis blooms. Further, a new intermediate class of compounds named microguanidine amides was uncovered, thereby further enlarging this compound family. The comparison of structurally divergent AGDs and MGDs reveals an outstanding versatility of this biosynthetic pathway and provides insights into the assembly of the two compound subfamilies. Strikingly, aeruginoguanidines and microguanidines were found to be as widespread as the hepatotoxic microcystins, but the occurrence of both toxin families appeared to be mutually exclusive.}, language = {en} } @article{MeyerMainzKehretal.2017, author = {Meyer, Sabine and Mainz, Andi and Kehr, Jan-Christoph and Suessmuth, Roderich and Dittmann, Elke}, title = {Prerequisites of Isopeptide Bond Formation in Microcystin Biosynthesis}, series = {ChemBioChem : a European journal of chemical biology}, volume = {18}, journal = {ChemBioChem : a European journal of chemical biology}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1439-4227}, doi = {10.1002/cbic.201700389}, pages = {2376 -- 2379}, year = {2017}, abstract = {The biosynthesis of the potent cyanobacterial hepatotoxin microcystin involves isopeptide bond formation through the carboxylic acid side chains of d-glutamate and -methyl d-aspartate. Analysis of the in vitro activation profiles of the two corresponding adenylation domains, McyE-A and McyB-A(2), either in a didomain or a tridomain context with the cognate thiolation domain and the upstream condensation domain revealed that substrate activation of both domains strictly depended on the presence of the condensation domains. We further identified two key amino acids in the binding pockets of both adenylation domains that could serve as a bioinformatic signature of isopeptide bond-forming modules incorporating d-glutamate or d-aspartate. Our findings further contribute to the understanding of the multifaceted role of condensation domains in nonribosomal peptide synthetase assembly lines.}, language = {en} } @article{GuljamowBarchewitzGrosseetal.2021, author = {Guljamow, Arthur and Barchewitz, Tino and Große, Rebecca and Timm, Stefan and Hagemann, Martin and Dittmann, Elke}, title = {Diel Variations of Extracellular Microcystin Influence the Subcellular Dynamics of RubisCO in Microcystis aeruginosa PCC 7806}, series = {Microorganisms : open access journal}, volume = {9}, journal = {Microorganisms : open access journal}, number = {6}, publisher = {MDPI}, address = {Basel}, issn = {2076-2607}, doi = {10.3390/microorganisms9061265}, pages = {14}, year = {2021}, abstract = {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.}, language = {en} } @misc{GuljamowBarchewitzGrosseetal.2021, author = {Guljamow, Arthur and Barchewitz, Tino and Große, Rebecca and Timm, Stefan and Hagemann, Martin and Dittmann, Elke}, title = {Diel Variations of Extracellular Microcystin Influence the Subcellular Dynamics of RubisCO in Microcystis aeruginosa PCC 7806}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1154}, issn = {1866-8372}, doi = {10.25932/publishup-52128}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-521287}, pages = {16}, year = {2021}, abstract = {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.}, language = {en} } @article{BaunachChowdhuryStallforthetal.2021, author = {Baunach, Martin and Chowdhury, Somak and Stallforth, Pierre and Dittmann-Th{\"u}nemann, Elke}, title = {The landscape of recombination events that create nonribosomal peptide diversity}, series = {Molecular biology and evolution : MBE}, volume = {38}, journal = {Molecular biology and evolution : MBE}, number = {5}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0737-4038}, doi = {10.1093/molbev/msab015}, pages = {2116 -- 2130}, year = {2021}, abstract = {Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing andmatching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the A(core) domains, yet domain interfaces and the flexible A(sub) domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.}, language = {en} } @article{KoekerAkcaalanDittmannetal.2021, author = {K{\"o}ker, Latife and Ak{\c{c}}aalan, Reyhan and Dittmann, Elke and Albay, Meri{\c{c}}}, title = {Depth profiles of protein-bound microcystin in K{\"u}{\c{c}}{\"u}k{\c{c}}ekmece Lagoon}, series = {Toxicon : an international journal devoted to the exchange of knowledge on the poisons derived from the tissues of plants and animals ; official journal of the International Society on Toxinology}, volume = {198}, journal = {Toxicon : an international journal devoted to the exchange of knowledge on the poisons derived from the tissues of plants and animals ; official journal of the International Society on Toxinology}, publisher = {Elsevier}, address = {Oxford}, issn = {0041-0101}, doi = {10.1016/j.toxicon.2021.05.005}, pages = {156 -- 163}, year = {2021}, abstract = {Microcystis is the most commonly found toxic cyanobacterial genus around the world and has a negative impact on the ecosystem. As a predominant producer of the potent hepatotoxin microcystin (MC), the genus causes outbreaks in freshwaters worldwide. Standard analytical methods that are used for the detection of microcystin variants can only measure the free form of microcystin in cells. Since microcystin was found as free and proteinbound forms in the cells, a significant proportion of microcystin is underestimated with analytical methods. The aim of the study was to measure protein-bound microcystins and determine the environmental factors that affect the binding of microcystin to proteins. Samples were taken at depths of surface, 1 m, 5 m, 10 m, 15 m, and 18 m in Kucukcekmece Lagoon to analyze depth profiles of two different microcystin forms from June to September 2012 at regular monthly intervals. Our findings suggest that the most important parameter affecting proteinbound microcystin at surface water is high light. Due to favorable environmental conditions such as temperature, light, and physicochemical parameters, the higher microcystin contents, both free and protein-bound MCs, were found in summer periods.}, language = {en} } @misc{desAulnoisReveillonRobertetal.2020, author = {des Aulnois, Maxime Georges and R{\´e}veillon, Damien and Robert, Elise and Caruana, Amandine and Briand, Enora and Guljamow, Arthur and Dittmann, Elke and Amzil, Zouher and Bormans, Myriam}, title = {Salt shock responses of Microcystis revealed through physiological, transcript, and metabolomic analyses}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1130}, issn = {1866-8372}, doi = {10.25932/publishup-47240}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-472405}, pages = {20}, year = {2020}, abstract = {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.}, language = {en} } @book{DittmannThuenemann2010, author = {Dittmann-Th{\"u}nemann, Elke}, title = {Toxische Cyanobakterien auf dem Vormarsch : {\"U}berlebensk{\"u}nstler und Meister der Naturstoffsynthese : Antrittsvorlesung 2010-06-16}, publisher = {Univ.-Bibl.}, address = {Potsdam}, year = {2010}, language = {de} } @article{VossBolhuisFeweretal.2013, author = {Voss, Bj{\"o}rn and Bolhuis, Henk and Fewer, David P. and Kopf, Matthias and M{\"o}ke, Fred and Haas, Fabian and El-Shehawy, Rehab and Hayes, Paul and Bergman, Birgitta and Sivonen, Kaarina and Dittmann-Th{\"u}nemann, Elke and Scanlan, Dave J. and Hagemann, Martin and Stal, Lucas J. and Hess, Wolfgang R.}, title = {Insights into the physiology and ecology of the brackish-water-adapted cyanobacterium nodularia spumigena CCY9414 based on a genome-transcriptome analysis}, series = {PLoS one}, volume = {8}, journal = {PLoS one}, number = {3}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0060224}, pages = {22}, year = {2013}, abstract = {Nodularia spumigena is a filamentous diazotrophic cyanobacterium that dominates the annual late summer cyanobacterial blooms in the Baltic Sea. But N. spumigena also is common in brackish water bodies worldwide, suggesting special adaptation allowing it to thrive at moderate salinities. A draft genome analysis of N. spumigena sp. CCY9414 yielded a single scaffold of 5,462,271 nucleotides in length on which genes for 5,294 proteins were annotated. A subsequent strand-specific transcriptome analysis identified more than 6,000 putative transcriptional start sites (TSS). Orphan TSSs located in intergenic regions led us to predict 764 non-coding RNAs, among them 70 copies of a possible retrotransposon and several potential RNA regulators, some of which are also present in other N2-fixing cyanobacteria. Approximately 4\% of the total coding capacity is devoted to the production of secondary metabolites, among them the potent hepatotoxin nodularin, the linear spumigin and the cyclic nodulapeptin. The transcriptional complexity associated with genes involved in nitrogen fixation and heterocyst differentiation is considerably smaller compared to other Nostocales. In contrast, sophisticated systems exist for the uptake and assimilation of iron and phosphorus compounds, for the synthesis of compatible solutes, and for the formation of gas vesicles, required for the active control of buoyancy. Hence, the annotation and interpretation of this sequence provides a vast array of clues into the genomic underpinnings of the physiology of this cyanobacterium and indicates in particular a competitive edge of N. spumigena in nutrient-limited brackish water ecosystems.}, language = {en} } @article{MeissnerFastnerDittmannThuenemann2013, author = {Meissner, Sven and Fastner, Jutta and Dittmann-Th{\"u}nemann, Elke}, title = {Microcystin production revisited conjugate formation makes a major contribution}, series = {Environmental microbiology}, volume = {15}, journal = {Environmental microbiology}, number = {6}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1462-2912}, doi = {10.1111/1462-2920.12072}, pages = {1810 -- 1820}, year = {2013}, abstract = {The impact of environmental stimuli on the production of the widespread cyanobacterial hepatotoxin microcystin (MC) is under debate. Whereas transcriptional studies of the biosynthetic genes suggest a clear influence of light conditions on toxin production the data for the metabolite itself are inconsistent and highly strain-specific. Here, we have reassessed the MC content by using two immunological detection techniques that allow a parallel quantification of MC in the methanolic extracts and the residual pellet fraction that contains high molecular weight proteins. Our results show a significant proportion of MC in the protein bound fraction in strains of Microcystis and Planktothrix and of the related toxin nodularin (NOD) in Nodularia. Moreover, we could show a very strong increase of MC after high light illumination in the protein fraction contributing to a significant overall increase in MC production under these conditions that is not seen in extracts analysed by LC-MS and ELISA. The fact that a considerable portion of MC is neglected with current analysis techniques was also confirmed for selected field samples. Immunofluorescence studies suggest strain-specific differences in the amount of MC conjugate formation.}, language = {en} } @article{ZiemertIshidaWeizetal.2010, author = {Ziemert, Nadine and Ishida, Keishi and Weiz, Annika and Hertweck, Christian and Dittmann-Th{\"u}nemann, Elke}, title = {Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides}, issn = {0099-2240}, doi = {10.1128/AEM.02858-09}, year = {2010}, abstract = {Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium Microcystis via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene mdnA encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in Microcystis aeruginosa strain NIES843 and heterologously produced variants.}, language = {en} } @misc{NeilanPearsonMuenchhoffetal.2013, author = {Neilan, Brett A. and Pearson, Leanne A. and M{\"u}nchhoff, Julia and Moffitt, Michelle C. and Dittmann-Th{\"u}nemann, Elke}, title = {Environmental conditions that influence toxin biosynthesis in cyanobacteria}, series = {Environmental microbiology}, volume = {15}, journal = {Environmental microbiology}, number = {5}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1462-2912}, doi = {10.1111/j.1462-2920.2012.02729.x}, pages = {1239 -- 1253}, year = {2013}, abstract = {Over the past 15 years, the genetic basis for production of many cyanobacterial bioactive compounds has been described. This knowledge has enabled investigations into the environmental factors that regulate the production of these toxins at the molecular level. Such molecular or systems level studies are also likely to reveal the physiological role of the toxin and contribute to effective water resource management. This review focuses on the environmental regulation of some of the most relevant cyanotoxins, namely the microcystins, nodularin, cylindrospermopsin, saxitoxins, anatoxins and jamaicamides.}, language = {en} } @article{FerirVermeireHuskensetal.2011, author = {Ferir, Geoffrey and Vermeire, Kurt and Huskens, Dana and Balzarini, Jan and Van Damme, Els J. M. and Kehr, Jan-Christoph and Dittmann-Th{\"u}nemann, Elke and Swanson, Michael D. and Markovitz, David M. and Schols, Dominique}, title = {Synergistic in vitro anti-HIV type 1 activity of tenofovir with carbohydrate-binding agents (CBAs)}, series = {Antiviral research}, volume = {90}, journal = {Antiviral research}, number = {3}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0166-3542}, doi = {10.1016/j.antiviral.2011.03.188}, pages = {200 -- 204}, year = {2011}, abstract = {Tenofovir, a well-known and highly prescribed anti-HIV-1 drug for the treatment of HIV/AIDS infections, has recently also shown its effectiveness as a potential microbicide drug in the prevention of HIV transmission. Here, we evaluated the combination of tenofovir with various members of the class of carbohydrate-binding agents (CBAs) targeting the glycans on the viral envelope gp120 for their anti-HIV efficacy. The tenofovir/CBA combinations predominantly showed synergistic antiviral activity using the median effect principle. These findings illustrate that combination of tenofovir with CBAs may increase the antiviral potency of the individual drugs and reducing the risk on potential side-effects.}, language = {en} } @article{MeissnerSteinhauserDittmannThuenemann2015, author = {Meissner, Sven and Steinhauser, Dirk and Dittmann-Th{\"u}nemann, Elke}, title = {Metabolomic analysis indicates a pivotal role of the hepatotoxin microcystin in high light adaptation of Microcystis}, series = {Environmental microbiology}, volume = {17}, journal = {Environmental microbiology}, number = {5}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1462-2912}, doi = {10.1111/1462-2920.12565}, pages = {1497 -- 1509}, year = {2015}, abstract = {Microcystis is a freshwater cyanobacterium frequently forming nuisance blooms in the summer months. The genus belongs to the predominant producers of the potent hepatotoxin microcystin. The success of Microcystis and its remarkable resistance to high light conditions are not well understood. Here, we have compared the metabolic response of Microcystis aeruginosaPCC7806, its microcystin-deficient mcyB mutant (Mut) and the cyanobacterial model organism SynechocystisPCC6803 to high light exposure of 250molphotonsm(-2)s(-1) using GC/MS-based metabolomics. Microcystis wild type and Mut show pronounced differences in their metabolic reprogramming upon high light. Seventeen percent of the detected metabolites showed significant differences between the two genotypes after high light exposure. Whereas the microcystin-producing wild type shows a faster accumulation of glycolate upon high light illumination, loss of microcystin leads to an accumulation of general stress markers such as trehalose and sucrose. The study further uncovers differences in the high light adaptation of the bloom-forming cyanobacterium Microcystis and the model cyanobacterium Synechocystis. Most notably, Microcystis invests more into carbon reserves such as glycogen after high light exposure. Our data shed new light on the lifestyle of bloom-forming cyanobacteria, the role of the widespread toxin microcystin and the metabolic diversity of cyanobacteria.}, language = {en} } @article{HuVoellerSussmuthetal.2015, author = {Hu, Chenlin and V{\"o}ller, Ginka and Sussmuth, Roderich and Dittmann-Th{\"u}nemann, Elke and Kehr, Jan-Christoph}, title = {Functional assessment of mycosporine-like amino acids in Microcystis aeruginosa strain PCC 7806}, series = {Environmental microbiology}, volume = {17}, journal = {Environmental microbiology}, number = {5}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1462-2912}, doi = {10.1111/1462-2920.12577}, pages = {1548 -- 1559}, year = {2015}, abstract = {The biological role of the widespread mycosporine-like amino acids (MAAs) in cyanobacteria is under debate. Here, we have constructed and characterized two mutants impaired in MAA biosynthesis in the bloom-forming cyanobacterium Microcystis aeruginosaPCC 7806. We could identify shinorine as the sole MAA type of the strain, which is exclusively located in the extracellular matrix. Bioinformatic studies as wells as polymerase chain reaction screening revealed that the ability to produce MAAs is sporadically distributed within the genus. Growth experiments and reactive oxygen species quantification with wild-type and mutant strains did not support a role of shinorine in protection against UV or other stress conditions in M.aeruginosaPCC 7806. The shinorine content per dry weight of cells as well as transcription of the mys gene cluster was not significantly elevated in response to UV-A, UV-B or any other stress condition tested. Remarkably, both mutants exhibited pronounced morphological changes compared with the wild type. We observed an increased accumulation and an enhanced hydrophobicity of the extracellular matrix. Our study suggests that MAAs in Microcystis play a negligible role in protection against UV radiation but might be a strain-specific trait involved in extracellular matrix formation and cell-cell interaction.}, language = {en} } @misc{KehrPicchiDittmannThuenemann2011, author = {Kehr, Jan-Christoph and Picchi, Douglas Gatte and Dittmann-Th{\"u}nemann, Elke}, title = {Natural product biosyntheses in cyanobacteria a treasure trove of unique enzymes}, series = {Beilstein journal of organic chemistry}, volume = {7}, journal = {Beilstein journal of organic chemistry}, number = {2}, publisher = {Beilstein-Institut zur F{\"o}rderung der Chemischen Wissenschaften}, address = {Frankfurt, Main}, issn = {1860-5397}, doi = {10.3762/bjoc.7.191}, pages = {1622 -- 1635}, year = {2011}, abstract = {Cyanobacteria are prolific producers of natural products. Investigations into the biochemistry responsible for the formation of these compounds have revealed fascinating mechanisms that are not, or only rarely, found in other microorganisms. In this article, we survey the biosynthetic pathways of cyanobacteria isolated from freshwater, marine and terrestrial habitats. We especially emphasize modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways and highlight the unique enzyme mechanisms that were elucidated or can be anticipated for the individual products. We further include ribosomal natural products and UV-absorbing pigments from cyanobacteria. Mechanistic insights obtained from the biochemical studies of cyanobacterial pathways can inspire the development of concepts for the design of bioactive compounds by synthetic-biology approaches in the future.}, language = {en} } @article{WeizIshidaMakoweretal.2011, author = {Weiz, Annika R. and Ishida, Keishi and Makower, Katharina and Ziemert, Nadine and Hertweck, Christian and Dittmann-Th{\"u}nemann, Elke}, title = {Leader Peptide and a Membrane Protein Scaffold Guide the Biosynthesis of the Tricyclic Peptide Microviridin}, series = {Chemistry \& biology}, volume = {18}, journal = {Chemistry \& biology}, number = {11}, publisher = {Cell Press}, address = {Cambridge}, issn = {1074-5521}, doi = {10.1016/j.chembiol.2011.09.011}, pages = {1413 -- 1421}, year = {2011}, abstract = {Microviridins are unique protease inhibitors from bloom-forming cyanobacteria that have both ecological and pharmacological relevance. Their peptide backbones are produced ribosomally, and ATP grasp ligases introduce omega-ester and omega-amide bonds to yield rare cage-like structures. Bioinformatic analysis of the microviridin biosynthesis gene cluster suggests a novel type of processing machinery, which could rationalize the challenging in vivo/in vitro reconstitution of the pathway. In this work, we report the establishment of a minimal expression system for microviridins. Through bioinformatics and mutational analysis of the MdnA leader peptide we identified and characterized a strictly conserved binding motif that is specific for microviridin ligases. Furthermore, we showed that the ABC transporter MdnE is crucial for cyclization and processing of microviridins and demonstrated that MdnE is essential for stability of the microviridin biosynthesis complex.}, language = {en} } @article{GattePicchiWeizIshidaetal.2014, author = {Gatte-Picchi, Douglas and Weiz, Annika and Ishida, Keishi and Hertweck, Christian and Dittmann-Th{\"u}nemann, Elke}, title = {Functional analysis of environmental DNA-derived microviridins provides new insights into the diversity of the tricyclic peptide family}, series = {Applied and environmental microbiology}, volume = {80}, journal = {Applied and environmental microbiology}, number = {4}, publisher = {American Society for Microbiology}, address = {Washington}, issn = {0099-2240}, doi = {10.1128/AEM.03502-13}, pages = {1380 -- 1387}, year = {2014}, abstract = {Microviridins represent a unique family of ribosomally synthesized cage-like depsipeptides from cyanobacteria with potent protease-inhibitory activities. The natural diversity of these peptides is largely unexplored. Here, we describe two methodologies that were developed to functionally characterize cryptic microviridin gene clusters from metagenomic DNA. Environmental samples were collected and enriched from cyanobacterial freshwater blooms of different geographical origins containing predominantly Microcystis sp. Microviridins were produced either directly from fosmid clones or after insertion of environmental DNA-derived gene cassettes into a minimal expression platform in Escherichia coli. Three novel microviridin variants were isolated and tested against different serine-type proteases. The comparison of the bioactivity profiles of the new congeners allows deduction of further structure-function relationships for microviridins. Moreover, this study provides new insights into microviridin processing and gene cluster organization.}, language = {en} } @article{ZilligesKehrMeissneretal.2011, author = {Zilliges, Yvonne and Kehr, Jan-Christoph and Meissner, Sven and Ishida, Keishi and Mikkat, Stefan and Hagemann, Martin and Kaplan, Aaron and B{\"o}rner, Thomas and Dittmann-Th{\"u}nemann, Elke}, title = {The cyanobacterial hepatotoxin microcystin binds to proteins and increases the fitness of microcystis under oxidative stress conditions}, series = {PLoS one}, volume = {6}, journal = {PLoS one}, number = {3}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0017615}, pages = {11}, year = {2011}, abstract = {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 Delta mcyB 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.}, language = {en} } @misc{DittmannThuenemannGuggerSivonenetal.2015, author = {Dittmann-Th{\"u}nemann, Elke and Gugger, Muriel and Sivonen, Kaarina and Fewer, David P.}, title = {Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria}, series = {Trends in microbiology}, volume = {23}, journal = {Trends in microbiology}, number = {10}, publisher = {Elsevier}, address = {Oxford}, issn = {0966-842X}, doi = {10.1016/j.tim.2015.07.008}, pages = {642 -- 652}, year = {2015}, abstract = {Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with intricate chemical structures and potent biological activities. The bulk of these natural products are known from just a handful of genera. Recent efforts have elucidated the mechanisms underpinning the biosynthesis of a diverse array of natural products from cyanobacteria. Many of the biosynthetic mechanisms are unique to cyanobacteria or rarely described from other organisms. Advances in genome sequence technology have precipitated a deluge of genome sequences for cyanobacteria. This makes it possible to link known natural products to biosynthetic gene clusters but also accelerates the discovery of new natural products through genome mining. These studies demonstrate that cyanobacteria encode a huge variety of cryptic gene clusters for the production of natural products, and the known chemical diversity is likely to be just a fraction of the true biosynthetic capabilities of this fascinating and ancient group of organisms.}, language = {en} } @unpublished{KehrDittmannThuenemann2015, author = {Kehr, Jan-Christoph and Dittmann-Th{\"u}nemann, Elke}, title = {Protective tunicate endosymbiont with extreme genome reduction}, series = {Environmental microbiology}, volume = {17}, journal = {Environmental microbiology}, number = {10}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1462-2912}, doi = {10.1111/1462-2920.12941}, pages = {3430 -- 3432}, year = {2015}, language = {en} } @misc{DittmannThuenemannFewerNeilan2013, author = {Dittmann-Th{\"u}nemann, Elke and Fewer, David P. and Neilan, Brett A.}, title = {Cyanobacterial toxins biosynthetic routes and evolutionary roots}, series = {FEMS microbiology reviews}, volume = {37}, journal = {FEMS microbiology reviews}, number = {1}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0168-6445}, doi = {10.1111/j.1574-6976.2012.12000.x}, pages = {23 -- 43}, year = {2013}, abstract = {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.}, language = {en} } @misc{ArnisonBibbBierbaumetal.2013, author = {Arnison, Paul G. and Bibb, Mervyn J. and Bierbaum, Gabriele and Bowers, Albert A. and Bugni, Tim S. and Bulaj, Grzegorz and Camarero, Julio A. and Campopiano, Dominic J. and Challis, Gregory L. and Clardy, Jon and Cotter, Paul D. and Craik, David J. and Dawson, Michael and Dittmann-Th{\"u}nemann, Elke and Donadio, Stefano and Dorrestein, Pieter C. and Entian, Karl-Dieter and Fischbach, Michael A. and Garavelli, John S. and Goeransson, Ulf and Gruber, Christian W. and Haft, Daniel H. and Hemscheidt, Thomas K. and Hertweck, Christian and Hill, Colin and Horswill, Alexander R. and Jaspars, Marcel and Kelly, Wendy L. and Klinman, Judith P. and Kuipers, Oscar P. and Link, A. James and Liu, Wen and Marahiel, Mohamed A. and Mitchell, Douglas A. and Moll, Gert N. and Moore, Bradley S. and Mueller, Rolf and Nair, Satish K. and Nes, Ingolf F. and Norris, Gillian E. and Olivera, Baldomero M. and Onaka, Hiroyasu and Patchett, Mark L. and Piel, J{\"o}rn and Reaney, Martin J. T. and Rebuffat, Sylvie and Ross, R. Paul and Sahl, Hans-Georg and Schmidt, Eric W. and Selsted, Michael E. and Severinov, Konstantin and Shen, Ben and Sivonen, Kaarina and Smith, Leif and Stein, Torsten and Suessmuth, Roderich D. and Tagg, John R. and Tang, Gong-Li and Truman, Andrew W. and Vederas, John C. and Walsh, Christopher T. and Walton, Jonathan D. and Wenzel, Silke C. and Willey, Joanne M. and van der Donk, Wilfred A.}, title = {Ribosomally synthesized and post-translationally modified peptide natural products overview and recommendations for a universal nomenclature}, series = {Natural product reports : a journal of current developments in bio-organic chemistry}, volume = {30}, journal = {Natural product reports : a journal of current developments in bio-organic chemistry}, number = {1}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {0265-0568}, doi = {10.1039/c2np20085f}, pages = {108 -- 160}, year = {2013}, abstract = {This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.}, language = {en} } @misc{WelkerDittmannThuenemannvonDoehren2012, author = {Welker, Martin and Dittmann-Th{\"u}nemann, Elke and von Doehren, Hans}, title = {Cyanobacteria as a source of natural products}, series = {Methods in enzymology}, volume = {517}, journal = {Methods in enzymology}, number = {1}, editor = {Hopwood, DA}, publisher = {Elsevier}, address = {San Diego}, isbn = {978-0-12-404634-4}, issn = {0076-6879}, doi = {10.1016/B978-0-12-404634-4.00002-4}, pages = {23 -- 46}, year = {2012}, abstract = {Cyanobacteria or blue-green algae from various environments have been recognized as sources of a variety of bioactive metabolites. Strategies of strain isolation from aquatic habitats, and cultivation and harvesting for metabolite production are described. Strategies for screening of compounds are discussed, including their direct MALDI-TOF mass spectrometric detection in whole cells. Genetic approaches including genomic mining, mutagenesis including transcriptional activation, heterologous expression, and in vitro. reconstitution of pathways are presented.}, language = {en} } @article{GuljamowDelissenBaumannetal.2012, author = {Guljamow, Arthur and Delissen, Friedmar and Baumann, Otto and Thuenemann, Andreas F. and Dittmann-Th{\"u}nemann, Elke}, title = {Unique properties of eukaryote-type actin and profilin horizontally transferred to cyanobacteria}, series = {PLoS one}, volume = {7}, journal = {PLoS one}, number = {1}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0029926}, pages = {221 -- 231}, year = {2012}, abstract = {A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 mu m in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 510 lam and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity.}, language = {en} } @article{LiaimerJenkeKodamaIshidaetal.2011, author = {Liaimer, Anton and Jenke-Kodama, Holger and Ishida, Keishi and Hinrichs, Katrin and Stangeland, Janne and Hertweck, Christian and Dittmann-Th{\"u}nemann, Elke}, title = {A polyketide interferes with cellular differentiation in the symbiotic cyanobacterium Nostoc punctiforme}, series = {Environmental microbiology reports}, volume = {3}, journal = {Environmental microbiology reports}, number = {5}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {1758-2229}, doi = {10.1111/j.1758-2229.2011.00258.x}, pages = {550 -- 558}, year = {2011}, abstract = {Nostoc punctiforme is a filamentous cyanobacterium capable of forming symbiotic associations with a wide range of plants. The strain exhibits extensive phenotypic characteristics and can differentiate three mutually exclusive cell types: nitrogen-fixing heterocysts, motile hormogonia and spore-like akinetes. Here, we provide evidence for a crucial role of an extracellular metabolite in balancing cellular differentiation. Insertional mutagenesis of a gene of the polyketide synthase gene cluster pks2 led to the accumulation of short filaments carrying mostly terminal heterocysts under diazotrophic conditions. The mutant has a strong tendency to form biofilms on solid surfaces as well as in liquid culture. The pks2-strain keeps forming hormogonia over the entire growth curve and shows an early onset of akinete formation. We could isolate two fractions of the wildtype supernatant that could restore the capability to form long filaments with intercalary heterocysts. Growth of the mutant cells in the neighbourhood of wild-type cells on plates led to a reciprocal influence and a partial reconstruction of wild-type and mutant phenotype respectively. We postulate that extracellular metabolites of Nostoc punctiforme act as life cycle governing factors (LCGFs) and that the ratio between distinct factors may guide the differentiation into different life stages.}, language = {en} } @article{WeizIshidaQuittereretal.2014, author = {Weiz, Annika R. and Ishida, Keishi and Quitterer, Felix and Meyer, Sabine and Kehr, Jan-Christoph and Mueller, Kristian M. and Groll, Michael and Hertweck, Christian and Dittmann-Th{\"u}nemann, Elke}, title = {Harnessing the evolvability of tricyclic microviridins to dissect protease-inhibitor interactions}, series = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition}, volume = {53}, journal = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition}, number = {14}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1433-7851}, doi = {10.1002/anie.201309721}, pages = {3735 -- 3738}, year = {2014}, abstract = {Understanding and controlling proteolysis is an important goal in therapeutic chemistry. Among the natural products specifically inhibiting proteases microviridins are particularly noteworthy. Microviridins are ribosomally produced and posttranslationally modified peptides that are processed into a unique, cagelike architecture. Here, we report a combined rational and random mutagenesis approach that provides fundamental insights into selectivity-conferring moieties of microviridins. The potent variant microviridin J was co-crystallized with trypsin, and for the first time the three-dimensional structure of microviridins was determined and the mode of inhibition revealed.}, language = {en} } @article{HarelWeissDanieletal.2012, author = {Harel, Moshe and Weiss, Gad and Daniel, Einat and Wilenz, Avraham and Hadas, Ora and Sukenik, Assaf and Sedmak, Bojan and Dittmann-Th{\"u}nemann, Elke and Braun, Sergei and Kaplan, Aaron}, title = {Casting a net fibres produced by Microcystis sp in field and laboratory populations}, series = {Environmental microbiology reports}, volume = {4}, journal = {Environmental microbiology reports}, number = {3}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {1758-2229}, doi = {10.1111/j.1758-2229.2012.00339.x}, pages = {342 -- 349}, year = {2012}, abstract = {The reasons for the apparent dominance of the toxic cyanobacterium Microcystis sp., reflected by its massive blooms in many fresh water bodies, are poorly understood. We show that in addition to a large array of secondary metabolites, some of which are toxic to eukaryotes, Microcystis sp. secretes large amounts of fibrous exopolysaccharides that form extremely long fibres several millimetres in length. This phenomenon was detected in field and laboratory cultures of various Microcystis strains. In addition, we have identified and characterized three of the proteins associated with the fibres and the genes encoding them in Microcystis sp. PCC 7806 but were unable to completely delete them from its genome. Phylogenetic analysis of the most abundant one, designated IPF-469, showed its presence only in cyanobacteria. Its closest relatives were detected in Synechocystis sp. PCC 6803 and in Cyanothece sp. strains; in the latter the genomic organization of the IPF-469 was highly conserved. IPF-469 and the other two proteins identified here, a haloperoxidase and a haemolysin-type calcium-binding protein, may be part of the fibres secretion pathway. The biological role of the fibres in Microcystis sp. is discussed.}, language = {en} } @misc{KaplanHarelKaplanLevyetal.2012, author = {Kaplan, Aaron and Harel, Moshe and Kaplan-Levy, Ruth N. and Hadas, Ora and Sukenik, Assaf and Dittmann-Th{\"u}nemann, Elke}, title = {The languages spoken in the water body (or the biological role of cyanobacterial toxins)}, series = {Frontiers in microbiology}, volume = {3}, journal = {Frontiers in microbiology}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {1664-302X}, doi = {10.3389/fmicb.2012.00138}, pages = {11}, year = {2012}, abstract = {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.}, language = {en} } @misc{LiaimerJensenDittmannThuenemann2016, author = {Liaimer, Anton and Jensen, John B. and Dittmann-Th{\"u}nemann, Elke}, title = {A genetic and chemical perspective on symbiotic recruitment of cyanobacteria of the genus Nostoc into the host plant Blasia pusilla L.}, series = {Frontiers in microbiology}, journal = {Frontiers in microbiology}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-407179}, pages = {16}, year = {2016}, abstract = {Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin.}, language = {en} } @article{LiaimerJensenDittmann2016, author = {Liaimer, Anton and Jensen, John B. and Dittmann, Elke}, title = {A Genetic and Chemical Perspective on Symbiotic Recruitment of Cyanobacteria of the Genus Nostoc into the Host Plant Blasia pusilla L.}, series = {Frontiers in microbiology}, volume = {7}, journal = {Frontiers in microbiology}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {1664-302X}, doi = {10.3389/fmicb.2016.01693}, pages = {449 -- 474}, year = {2016}, abstract = {Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin.}, language = {en} } @article{ReynaGonzalezSchmidPetrasetal.2016, author = {Reyna-Gonz{\´a}lez, Emmanuel and Schmid, Bianca and Petras, Daniel and S{\"u}ssmuth, Roderich D. and Dittmann, Elke}, title = {Leader Peptide-Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease-Targeted Libraries}, series = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition}, volume = {55}, journal = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1433-7851}, doi = {10.1002/anie.201604345}, pages = {9398 -- 9401}, year = {2016}, abstract = {Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.}, language = {en} } @misc{PearsonDittmannMazmouzetal.2016, author = {Pearson, Leanne A. and Dittmann, Elke and Mazmouz, Rabia and Ongley, Sarah E. and Neilan, Brett A.}, title = {The genetics, biosynthesis and regulation of toxic specialized metabolites of cyanobacteria}, series = {Harmful algae}, volume = {54}, journal = {Harmful algae}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1568-9883}, doi = {10.1016/j.hal.2015.11.002}, pages = {98 -- 111}, year = {2016}, abstract = {The production of toxic metabolites by cyanobacterial blooms represents a significant threat to the health of humans and ecosystems worldwide. Here we summarize the current state of the knowledge regarding the genetics, biosynthesis and regulation of well-characterized cyanotoxins, including the microcystins, nodularin, cylindrospermopsin, saxitoxins and antitoxins, as well as the lesser-known marine toxins (e.g. lyngbyatoxin, aplysiatoxin, jamaicamides, barbamide, curacin, hectochlorin and apratoxins). (C) 2015 Elsevier B.V. All rights reserved.}, language = {en} } @article{MakowerSchuurmansGrothetal.2015, author = {Makower, A. Katharina and Schuurmans, J. Merijn and Groth, Detlef and Zilliges, Yvonne and Matthijs, Hans C. P. and Dittmann-Th{\"u}nemann, Elke}, title = {Transcriptomics-Aided dissection of the intracellular and extracellular roles of microcystin in microcystis aeruginosa PCC 7806}, series = {Applied and environmental microbiology}, volume = {81}, journal = {Applied and environmental microbiology}, number = {2}, publisher = {American Society for Microbiology}, address = {Washington}, issn = {0099-2240}, doi = {10.1128/AEM.02601-14}, pages = {544 -- 554}, year = {2015}, abstract = {Recent studies have provided evidence for both intracellular and extracellular roles of the potent hepatotoxin microcystin (MC) in the bloom-forming cyanobacterium Microcystis. Here, we surveyed transcriptomes of the wild-type strain M. aeruginosa PCC 7806 and the microcystin-deficient Delta mcyB mutant under low light conditions with and without the addition of external MC of the LR variant (MC-LR). Transcriptomic data acquired by microarray and quantitative PCR revealed substantial differences in the relative expression of genes of the central intermediary metabolism, photosynthesis, and energy metabolism. In particular, the data provide evidence for a lower photosystem I (PSI)-to-photosystem II (PSII) ratio and a more pronounced carbon limitation in the microcystin-deficient mutant. Interestingly, only 6\% of the transcriptional differences could be complemented by external microcystin-LR addition. This MC signaling effect was seen exclusively for genes of the secondary metabolism category. The orphan polyketide synthase gene cluster IPF38-51 was specifically downregulated in response to external MC-LR under low light. Our data suggest a hierarchical and light-dependent cross talk of secondary metabolites and support both an intracellular and an extracellular role of MC in Microcystis.}, language = {en} } @misc{KehrDittmannThuenemann2015, author = {Kehr, Jan-Christoph and Dittmann-Th{\"u}nemann, Elke}, title = {Biosynthesis and function of extracellular glycans in cyanobacteria}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-400121}, pages = {17}, year = {2015}, abstract = {The cell surface of cyanobacteria is covered with glycans that confer versatility and adaptability to a multitude of environmental factors. The complex carbohydrates act as barriers against different types of stress and play a role in intra- as well as inter-species interactions. In this review, we summarize the current knowledge of the chemical composition, biosynthesis and biological function of exo- and lipo-polysaccharides from cyanobacteria and give an overview of sugar-binding lectins characterized from cyanobacteria. We discuss similarities with well-studied enterobacterial systems and highlight the unique features of cyanobacteria. We pay special attention to colony formation and EPS biosynthesis in the bloom-forming cyanobacterium, Microcystis aeruginosa.}, language = {en} } @article{AhmedReynaGonzalezSchmidetal.2017, author = {Ahmed, Muhammad N. and Reyna-Gonzalez, Emmanuel and Schmid, Bianca and Wiebach, Vincent and Suessmuth, Roderich D. and Dittmann, Elke and Fewer, David P.}, title = {Phylogenomic Analysis of the Microviridin Biosynthetic Pathway Coupled with Targeted Chemo-Enzymatic Synthesis Yields Potent Protease Inhibitors}, series = {ACS chemical biology}, volume = {12}, journal = {ACS chemical biology}, publisher = {American Chemical Society}, address = {Washington}, issn = {1554-8929}, doi = {10.1021/acschembio.7b00124}, pages = {1538 -- 1546}, year = {2017}, abstract = {Natural products and their semisynthetic derivatives are an important source of drugs for the pharmaceutical industry. Bacteria are prolific producers of natural products and encode a vast diversity of natural product biosynthetic gene clusters. However, much of this diversity is inaccessible to natural product discovery. Here, we use a combination of phylogenomic analysis of the microviridin biosynthetic pathway and chemo-enzymatic synthesis of bioinformatically predicted microviridins to yield new protease inhibitors. Phylogenomic analysis demonstrated that microviridin biosynthetic gene clusters occur across the bacterial domain and encode three distinct subtypes of precursor peptides. Our analysis shed light on the evolution of microviridin biosynthesis and enabled prioritization of their chemo-enzymatic production. Targeted one-pot synthesis of four microviridins encoded by the cyanobacterium Cyanothece sp. PCC 7822 identified a set of novel and potent serine protease inhibitors, the most active of which had an IC50 value of 21.5 nM. This study advances the genome mining techniques available for natural product discovery and obviates the need to culture bacteria.}, language = {en} } @article{SoeriyadiOngleyKehretal.2021, author = {Soeriyadi, Angela H. and Ongley, Sarah E. and Kehr, Jan-Christoph and Pickford, Russel and Dittmann, Elke and Neilan, Brett A.}, title = {Tailoring enzyme stringency masks the multispecificity of a lyngbyatoxin (indolactam alkaloid) nonribosomal peptide synthetase}, series = {ChemBioChem}, volume = {23}, journal = {ChemBioChem}, number = {3}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1439-4227}, doi = {10.1002/cbic.202100574}, pages = {6}, year = {2021}, abstract = {Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.}, language = {en} } @article{desAulnoisReveillonRobertetal.2020, author = {des Aulnois, Maxime Georges and R{\´e}veillon, Damien and Robert, Elise and Caruana, Amandine and Briand, Enora and Guljamow, Arthur and Dittmann, Elke and Amzil, Zouher and Bormans, Myriam}, title = {Salt shock responses of Microcystis revealed through physiological, transcript, and metabolomic analyses}, series = {Toxins}, volume = {12}, journal = {Toxins}, number = {3}, publisher = {MDPI}, address = {Basel}, issn = {2072-6651}, doi = {10.3390/toxins12030192}, pages = {18}, year = {2020}, abstract = {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.}, language = {en} } @article{KrumbholzIshidaBaunachetal.2022, author = {Krumbholz, Julia and Ishida, Keishi and Baunach, Martin and Teikari, Jonna and Rose, Magdalena M. and Sasso, Severin and Hertweck, Christian and Dittmann, Elke}, title = {Deciphering chemical mediators regulating specialized metabolism in a symbiotic cyanobacterium}, series = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker. International edition}, journal = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker. International edition}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1433-7851}, doi = {10.1002/anie.202204545}, pages = {10}, year = {2022}, abstract = {Genomes of cyanobacteria feature a variety of cryptic biosynthetic pathways for complex natural products, but the peculiarities limiting the discovery and exploitation of the metabolic dark matter are not well understood. Here we describe the discovery of two cell density-dependent chemical mediators, nostoclide and nostovalerolactone, in the symbiotic model strain Nostoc punctiforme, and demonstrate their pronounced impact on the regulation of specialized metabolism. Through transcriptional, bioinformatic and labeling studies we assigned two adjacent biosynthetic gene clusters to the biosynthesis of the two polyketide mediators. Our findings provide insight into the orchestration of specialized metabolite production and give lessons for the genomic mining and high-titer production of cyanobacterial bioactive compounds.}, language = {en} } @article{NwosuRoeserYangetal.2021, author = {Nwosu, Ebuka Canisius and Roeser, Patricia Angelika and Yang, Sizhong and Ganzert, Lars and Dellwig, Olaf and Pinkerneil, Sylvia and Brauer, Achim and Dittmann, Elke and Wagner, Dirk and Liebner, Susanne}, title = {From water into sediment-tracing freshwater cyanobacteria via DNA analyses}, series = {Microorganisms : open access journal}, volume = {9}, journal = {Microorganisms : open access journal}, number = {8}, publisher = {MDPI}, address = {Basel}, issn = {2076-2607}, doi = {10.3390/microorganisms9081778}, pages = {20}, year = {2021}, abstract = {Sedimentary ancient DNA-based studies have been used to probe centuries of climate and environmental changes and how they affected cyanobacterial assemblages in temperate lakes. Due to cyanobacteria containing potential bloom-forming and toxin-producing taxa, their approximate reconstruction from sediments is crucial, especially in lakes lacking long-term monitoring data. To extend the resolution of sediment record interpretation, we used high-throughput sequencing, amplicon sequence variant (ASV) analysis, and quantitative PCR to compare pelagic cyanobacterial composition to that in sediment traps (collected monthly) and surface sediments in Lake Tiefer See. Cyanobacterial composition, species richness, and evenness was not significantly different among the pelagic depths, sediment traps and surface sediments (p > 0.05), indicating that the cyanobacteria in the sediments reflected the cyanobacterial assemblage in the water column. However, total cyanobacterial abundances (qPCR) decreased from the metalimnion down the water column. The aggregate-forming (Aphanizomenon) and colony-forming taxa (Snowella) showed pronounced sedimentation. In contrast, Planktothrix was only very poorly represented in sediment traps (meta- and hypolimnion) and surface sediments, despite its highest relative abundance at the thermocline (10 m water depth) during periods of lake stratification (May-October). We conclude that this skewed representation in taxonomic abundances reflects taphonomic processes, which should be considered in future DNA-based paleolimnological investigations.}, language = {en} } @article{NwosuRoeserYangetal.2021, author = {Nwosu, Ebuka Canisius and Roeser, Patricia Angelika and Yang, Sizhong and Pinkerneil, Sylvia and Ganzert, Lars and Dittmann, Elke and Brauer, Achim and Wagner, Dirk and Liebner, Susanne}, title = {Species-level spatio-temporal dynamics of cyanobacteria in a hard-water temperate lake in the Southern Baltics}, series = {Frontiers in microbiology}, volume = {12}, journal = {Frontiers in microbiology}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {1664-302X}, doi = {10.3389/fmicb.2021.761259}, pages = {17}, year = {2021}, abstract = {Cyanobacteria are important primary producers in temperate freshwater ecosystems. However, studies on the seasonal and spatial distribution of cyanobacteria in deep lakes based on high-throughput DNA sequencing are still rare. In this study, we combined monthly water sampling and monitoring in 2019, amplicon sequence variants analysis (ASVs; a proxy for different species) and quantitative PCR targeting overall cyanobacteria abundance to describe the seasonal and spatial dynamics of cyanobacteria in the deep hard-water oligo-mesotrophic Lake Tiefer See, NE Germany. We observed significant seasonal variation in the cyanobacterial community composition (p < 0.05) in the epi- and metalimnion layers, but not in the hypolimnion. In winter-when the water column is mixed-picocyanobacteria (Synechococcus and Cyanobium) were dominant. With the onset of stratification in late spring, we observed potential niche specialization and coexistence among the cyanobacteria taxa driven mainly by light and nutrient dynamics. Specifically, ASVs assigned to picocyanobacteria and the genus Planktothrix were the main contributors to the formation of deep chlorophyll maxima along a light gradient. While Synechococcus and different Cyanobium ASVs were abundant in the epilimnion up to the base of the euphotic zone from spring to fall, Planktothrix mainly occurred in the metalimnetic layer below the euphotic zone where also overall cyanobacteria abundance was highest in summer. Our data revealed two potentially psychrotolerant (cold-adapted) Cyanobium species that appear to cope well under conditions of lower hypolimnetic water temperature and light as well as increasing sediment-released phosphate in the deeper waters in summer. The potential cold-adapted Cyanobium species were also dominant throughout the water column in fall and winter. Furthermore, Snowella and Microcystis-related ASVs were abundant in the water column during the onset of fall turnover. Altogether, these findings suggest previously unascertained and considerable spatiotemporal changes in the community of cyanobacteria on the species level especially within the genus Cyanobium in deep hard-water temperate lakes.}, language = {en} }