TY - GEN A1 - Liaimer, Anton A1 - Jensen, John B. A1 - Dittmann-Thünemann, Elke T1 - A genetic and chemical perspective on symbiotic recruitment of cyanobacteria of the genus Nostoc into the host plant Blasia pusilla L. T2 - Frontiers in microbiology N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 434 KW - cyanobacteria KW - secondary metabolites KW - symbiosis KW - Blasia KW - Nostoc KW - allelopathy Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-407179 ER - TY - GEN A1 - Kehr, Jan-Christoph A1 - Dittmann-Thünemann, Elke T1 - Biosynthesis and function of extracellular glycans in cyanobacteria N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 346 KW - cyanobacteria KW - exopolysaccharides KW - lipopolysaccharides KW - colony formation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-400121 ER - TY - JOUR A1 - Dittmann-Thünemann, Elke A1 - Fewer, David P. A1 - Neilan, Brett A. T1 - Cyanobacterial toxins biosynthetic routes and evolutionary roots JF - FEMS microbiology reviews N2 - 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. KW - microcystin KW - cylindrospermopsin KW - anatoxin KW - saxitoxin KW - cyanobacteria Y1 - 2013 U6 - https://doi.org/10.1111/j.1574-6976.2012.12000.x SN - 0168-6445 SN - 1574-6976 VL - 37 IS - 1 SP - 23 EP - 43 PB - Wiley-Blackwell CY - Hoboken ER - TY - THES A1 - Reyna González, Emmanuel T1 - Engineering of the microviridin post-translational modification enzymes for the production of synthetic protease inhibitors T1 - Manipulation der posttranslationalen Modifikationsenzyme von Microviridin zur Herstellung synthetischer Proteaseinhibitoren N2 - Natural products and their derivatives have always been a source of drug leads. In particular, bacterial compounds have played an important role in drug development, for example in the field of antibiotics. A decrease in the discovery of novel leads from natural sources and the hope of finding new leads through the generation of large libraries of drug-like compounds by combinatorial chemistry aimed at specific molecular targets drove the pharmaceutical companies away from research on natural products. However, recent technological advances in genetics, bioinformatics and analytical chemistry have revived the interest in natural products. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of natural products generated by the action of post-translationally modifying enzymes on precursor peptides translated from mRNA by ribosomes. The great substrate promiscuity exhibited by many of the enzymes from RiPP biosynthetic pathways have led to the generation of hundreds of novel synthetic and semisynthetic variants, including variants carrying non-canonical amino acids (ncAAs). The microviridins are a family of RiPPs characterized by their atypical tricyclic structure composed of lactone and lactam rings, and their activity as serine protease inhibitors. The generalities of their biosynthetic pathway have already been described, however, the lack of information on details such as the protease responsible for cleaving off the leader peptide from the cyclic core peptide has impeded the fast and cheap production of novel microviridin variants. In the present work, knowledge on leader peptide activation of enzymes from other RiPP families has been extrapolated to the microviridin family, making it possible to bypass the need of a leader peptide. This feature allowed for the exploitation of the microviridin biosynthetic machinery for the production of novel variants through the establishment of an efficient one-pot in vitro platform. The relevance of this chemoenzymatic approach has been exemplified by the synthesis of novel potent serine protease inhibitors from both rationally-designed peptide libraries and bioinformatically predicted microviridins. Additionally, new structure-activity relationships (SARs) could be inferred by screening microviridin intermediates. The significance of this technique was further demonstrated by the simple incorporation of ncAAs into the microviridin scaffold. N2 - Naturstoffe und ihre Derivate waren schon immer eine Quelle von Leitstrukturen. Insbesondere haben bakterielle Verbindungen eine wichtige Rolle bei der Arzneimittelentwicklung gespielt, zum Beispiel im Bereich der Antibiotika. Die Abnahme von Entdeckungen neuer Leitstrukturen aus natürlichen Quellen und die Hoffnung, neue Leitstrukturen in großen Bibliotheken medikamentenähnlicher Verbindungen zu finden, welche auf spezifische molekulare Ziele gerichtet sind und mithilfe kombinatorischer Chemie erstellt wurden, trieben die Pharmaunternehmen weg von der Naturstoffforschung. Allerdings haben moderne technologische Fortschritte in der Genetik, der Bioinformatik und der analytischen Chemie das Interesse an Naturstoffen wiederbelebt. Die ribosomal synthetisierten und posttranslational modifizierten Peptide (RiPPs) sind eine Gruppe von Naturstoffen, die durch das Einwirken posttranslational modifizierender Enzymen auf Präkursorpeptide entstehen, welche ihrerseits aus mRNA durch Translation an den Ribosomen hervorgehen. Die durch viele der Enzyme aus RiPP Biosynthesewege gezeigte große Substrat- Promiskuität führte zur Erzeugung hunderter neuartiger synthetischer und halbsynthetischer Varianten, einschließlich Varianten mit nicht-kanonischen Aminosäuren. Die Microviridine sind eine Familie von RiPPs, die durch ihre atypische trizyklische Struktur aus Lacton- und Lactamringen und ihre Aktivität als Serin-Protease-Inhibitoren gekennzeichnet sind. Die Grundlagen ihres Biosyntheseweges sind bereits beschrieben worden, aber wesentliche Fragestellungen, zum Beispiel die für die Spaltung des Leader-Peptids vom zyklischen Core- Peptid verantwortliche Protease betreffend, sind weitgehend ungeklärt und erschweren die schnelle und kostengünstige Herstellung neuer Microviridinvarianten. In der vorliegenden Arbeit wurde das Wissen über die durch Leader-Peptid Aktivierung von Enzymen aus anderen RiPP-Familien auf die Microviridinfamilie extrapoliert, wodurch es möglich wurde, die Notwendigkeit eines Leader-Peptids zu umgehen. Diese Besonderheit erlaubt nunmehr, die Microviridin-Biosynthese-Enzyme für die Herstellung von neuartigen Varianten durch die Etablierung einer effizienten in vitro Synthese-Plattform auszunutzen. Die Relevanz dieses chemoenzymatischen Ansatzes wurde durch die Synthese von neuen potenten Serin-Protease- Inhibitoren aus sowohl rational gestalteten Peptidbibliotheken als auch bioinformatisch vorhergesagten Microviridinen veranschaulicht. Darüber hinaus wurden durch das Screenen von Microviridinzwischenprodukten neue Struktur-Funktionsbeziehungen abgeleitet. Die Bedeutung dieser Technik wurde durch den einfachen Einbau von nicht-kanonischen Aminosäuren in das Microviridin-Gerüst weiter demonstriert. KW - RiPP KW - microviridin KW - biosynthesis KW - natural products KW - peptide KW - protease inhibitor KW - Biosynthese KW - Naturstoffe KW - cyanobacteria KW - Cyanobakterien KW - Microviridin KW - Protease-Inhibitoren Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-406979 ER - TY - JOUR A1 - Coloma, Sebastian A1 - Gaedke, Ursula A1 - Sivonen, Kaarina A1 - Hiltunen, Teppo T1 - Frequency of virus-resistant hosts determines experimental community dynamics JF - Ecology : a publication of the Ecological Society of America N2 - Parasites, such as bacterial viruses (phages), can have large effects on host populations both at the ecological and evolutionary levels. In the case of cyanobacteria, phages can reduce primary production and infected hosts release intracellular nutrients influencing planktonic food web structure, community dynamics, and biogeochemical cycles. Cyanophages may be of great importance in aquatic food webs during large cyanobacterial blooms unless the host population becomes resistant to phage infection. The consequences on plankton community dynamics of the evolution of phage resistance in bloom forming cyanobacterial populations are still poorly studied. Here, we examined the effect of different frequencies of a phage-resistant genotype within a filamentous nitrogen-fixing Nodularia spumigena population on an experimental plankton community. Three Nodularia populations with different initial frequencies (0%, 5%, and 50%) of phage-resistant genotypes were inoculated in separate treatments with the phage 2AV2, the green alga Chlorella vulgaris, and the rotifer Brachionus plicatilis, which formed the experimental plankton community subjected to either nitrogen-limited or nitrogen-rich conditions. We found that the frequency of the phage-resistant Nodularia genotype determined experimental community dynamics. Cyanobacterial populations with a high frequency (50%) of the phage-resistant genotype dominated the cultures despite the presence of phages, retaining most of the intracellular nitrogen in the plankton community. In contrast, populations with low frequencies (0% and 5%) of the phage-resistant genotype were lysed and reduced to extinction by the phage, transferring the intracellular nitrogen held by Nodularia to Chlorella and rotifers, and allowing Chlorella to dominate the communities and rotifers to survive. This study shows that even though phages represent minuscule biomass, they can have key effects on community composition and eco-evolutionary feedbacks in plankton communities. KW - cyanobacteria KW - eco-evolutionary feedbacks KW - experimental evolution KW - host-parasite interaction KW - phage resistance KW - predator-prey interaction Y1 - 2018 U6 - https://doi.org/10.1002/ecy.2554 SN - 0012-9658 SN - 1939-9170 VL - 100 IS - 1 PB - Wiley CY - Hoboken ER - TY - GEN A1 - Bolius, Sarah A1 - Morling, Karoline A1 - Wiedner, Claudia A1 - Weithoff, Guntram T1 - Genetic Identity and Herbivory Drive the Invasion of a Common Aquatic Microbial Invader T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Despite the increasing number of species invasions, the factors driving invasiveness are still under debate. This is particularly the case for “invisible” invasions by aquatic microbial species. Since in many cases only a few individuals or propagules enter a new habitat, their genetic variation is low and might limit their invasion success, known as the genetic bottleneck. Thus, a key question is, how genetic identity and diversity of invading species influences their invasion success and, subsequently, affect the resident community. We conducted invader-addition experiments using genetically different strains of the globally invasive, aquatic cyanobacterium Raphidiopsis raciborskii (formerly: Cylindrospermopsis raciborskii) to determine the role of invader identity and genetic diversity (strain richness) at four levels of herbivory. We tested the invasion success of solitary single strain invasions against the invader genetic diversity, which was experimentally increased up to ten strains (multi-strain populations). By using amplicon sequencing we determined the strain-specific invasion success in the multi-strain treatments and compared those with the success of these strains in the single-strain treatments. Furthermore, we tested for the invasion success under different herbivore pressures. We showed that high grazing pressure by a generalist herbivore prevented invasion, whereas a specialist herbivore enabled coexistence of consumer and invader. We found a weak effect of diversity on invasion success only under highly competitive conditions. When invasions were successful, the magnitude of this success was strain-specific and consistent among invasions performed with single-strain or multi-strain populations. A strain-specific effect was also observed on the resident phytoplankton community composition, highlighting the strong role of invader genetic identity. Our results point to a strong effect of the genetic identity on the invasion success under low predation pressure. The genetic diversity of the invader population, however, had little effect on invasion success in our study, in contrast to most previous findings. Instead, it is the interaction between the consumer abundance and type together with the strain identity of the invader that defined invasion success. This study underlines the importance of strain choice in invasion research and in ecological studies in general. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 971 KW - alien species KW - genotype KW - invasibility KW - cyanobacteria KW - consumptive resistance KW - phytoplankton KW - Raphidiopsis KW - genetic diversity Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-474333 SN - 1866-8372 IS - 971 ER - TY - JOUR A1 - Bolius, Sarah A1 - Karoline Morling, A1 - Wiedner, Claudia A1 - Weithoff, Guntram T1 - Genetic Identity and Herbivory Drive the Invasion of a Common Aquatic Microbial Invader JF - Frontiers in Microbiology N2 - Despite the increasing number of species invasions, the factors driving invasiveness are still under debate. This is particularly the case for “invisible” invasions by aquatic microbial species. Since in many cases only a few individuals or propagules enter a new habitat, their genetic variation is low and might limit their invasion success, known as the genetic bottleneck. Thus, a key question is, how genetic identity and diversity of invading species influences their invasion success and, subsequently, affect the resident community. We conducted invader-addition experiments using genetically different strains of the globally invasive, aquatic cyanobacterium Raphidiopsis raciborskii (formerly: Cylindrospermopsis raciborskii) to determine the role of invader identity and genetic diversity (strain richness) at four levels of herbivory. We tested the invasion success of solitary single strain invasions against the invader genetic diversity, which was experimentally increased up to ten strains (multi-strain populations). By using amplicon sequencing we determined the strain-specific invasion success in the multi-strain treatments and compared those with the success of these strains in the single-strain treatments. Furthermore, we tested for the invasion success under different herbivore pressures. We showed that high grazing pressure by a generalist herbivore prevented invasion, whereas a specialist herbivore enabled coexistence of consumer and invader. We found a weak effect of diversity on invasion success only under highly competitive conditions. When invasions were successful, the magnitude of this success was strain-specific and consistent among invasions performed with single-strain or multi-strain populations. A strain-specific effect was also observed on the resident phytoplankton community composition, highlighting the strong role of invader genetic identity. Our results point to a strong effect of the genetic identity on the invasion success under low predation pressure. The genetic diversity of the invader population, however, had little effect on invasion success in our study, in contrast to most previous findings. Instead, it is the interaction between the consumer abundance and type together with the strain identity of the invader that defined invasion success. This study underlines the importance of strain choice in invasion research and in ecological studies in general. KW - alien species KW - genotype KW - invasibility KW - cyanobacteria KW - consumptive resistance KW - phytoplankton KW - Raphidiopsis KW - genetic diversity Y1 - 2019 U6 - https://doi.org/10.3389/fmicb.2020.01598 SN - 1664-302X VL - 11 PB - Frontiers Media CY - Lausanne ER - TY - JOUR A1 - Weiz, Annika R. A1 - Ishida, Keishi A1 - Quitterer, Felix A1 - Meyer, Sabine A1 - Kehr, Jan-Christoph A1 - Mueller, Kristian M. A1 - Groll, Michael A1 - Hertweck, Christian A1 - Dittmann-Thünemann, Elke T1 - Harnessing the evolvability of tricyclic microviridins to dissect protease-inhibitor interactions JF - Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition N2 - 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. KW - cyanobacteria KW - peptide engineering KW - protease inhibitors KW - RiPPs KW - structure elucidation Y1 - 2014 U6 - https://doi.org/10.1002/anie.201309721 SN - 1433-7851 SN - 1521-3773 VL - 53 IS - 14 SP - 3735 EP - 3738 PB - Wiley-VCH CY - Weinheim ER - TY - THES A1 - Krumbholz, Julia T1 - Identification of chemical mediators that regulate the specialized metabolism in Nostoc punctiforme T1 - Identifizierung chemischer Mediatoren, die den spezialisierten Metabolismus in Nostoc punctiforme regulieren N2 - Specialized metabolites, so-called natural products, are produced by a variety of different organisms, including bacteria and fungi. Due to their wide range of different biological activities, including pharmaceutical relevant properties, microbial natural products are an important source for drug development. They are encoded by biosynthetic gene clusters (BGCs), which are a group of locally clustered genes. By screening genomic data for genes encoding typical core biosynthetic enzymes, modern bioinformatical approaches are able to predict a wide range of BGCs. To date, only a small fraction of the predicted BGCs have their associated products identified. The phylum of the cyanobacteria has been shown to be a prolific, but largely untapped source for natural products. Especially multicellular cyanobacterial genera, like Nostoc, harbor a high amount of BGCs in their genomes. A main goal of this study was to develop new concepts for the discovery of natural products in cyanobacteria. Due to its diverse setup of orphan BGCs and its amenability to genetic manipulation, Nostoc punctiforme PCC 73102 (N. punctiforme) appeared to be a promising candidate to be established as a model organism for natural product discovery in cyanobacteria. By utilizing a combination of genome-mining, bioactivity-screening, variations of culture conditions, as well as metabolic engineering, not only two new polyketides were discovered, but also first-time insights into the regulation of the specialized metabolism in N. punctiforme were gained during this study. The cultivation of N. punctiforme to very high densities by utilizing increasing light intensities and CO2 levels, led to an enhanced metabolite production, causing rather complex metabolite extracts. By utilizing a library of CFP reporter mutant strains, each strain reporting for one of the predicted BGCs, it was shown that eight out of 15 BGCs were upregulated under high density (HD) cultivation conditions. Furthermore, it could be demonstrated that the supernatant of an HD culture can increase the expression of four of the influenced BGCs, even under conventional cultivation conditions. This led to the hypothesis that a chemical mediator encoded by one of the affected BGCs is accumulating in the HD supernatant and is able to increase the expression of other BGCs as part of a cell-density dependent regulatory circuit. To identify which of the BGCs could be a main trigger of the presumed regulatory circuit, it was tried to activate four BGCs (pks1, pks2, ripp3, ripp4) selectively by overexpression of putative pathway-specific regulatory genes that were found inside the gene clusters. Transcriptional analysis of the mutants revealed that only the mutant strain targeting the pks1 BGC, called AraC_PKS1, was able to upregulate the expression of its associated BGC. From an RNA sequencing study of the AraC_PKS1 mutant strain, it was discovered that beside pks1, the orphan BGCs ripp3 and ripp4 were also upregulated in the mutant strain. Furthermore, it was observed that secondary metabolite production in the AraC_PKS1 mutant strain is further enhanced under high-light and high-CO2 cultivation conditions. The increased production of the pks1 regulator NvlA also had an impact on other regulatory factors, including sigma factors and the RNA chaperone Hfq. Analysis of the AraC_PKS1 cell and supernatant extracts led to the discovery of two novel polyketides, nostoclide and nostovalerolactone, both encoded by the pks1 BGC. Addition of the polyketides to N. punctiforme WT demonstrated that the pks1-derived compounds are able to partly reproduce the effects on secondary metabolite production found in the AraC_PKS1 mutant strain. This indicates that both compounds are acting as extracellular signaling factors as part of a regulatory network. Since not all transcriptional effects that were found in the AraC_PKS1 mutant strain could be reproduced by the pks1 products, it can be assumed that the regulator NvlA has a global effect and is not exclusively specific to the pks1 pathway. This study was the first to use a putative pathway specific regulator for the specific activation of BGC expression in cyanobacteria. This strategy did not only lead to the detection of two novel polyketides, it also gave first-time insights into the regulatory mechanism of the specialized metabolism in N. punctiforme. This study illustrates that understanding regulatory pathways can aid in the discovery of novel natural products. The findings of this study can guide the design of new screening strategies for bioactive compounds in cyanobacteria and help to develop high-titer production platforms for cyanobacterial natural products. N2 - Sekundärmetabolite, auch Naturstoffe genannt, werden von einer Vielzahl an Organismen, darunter Bakterien und Pilzen, hergestellt. Aufgrund ihrer Vielzahl an verschiedenen Bioaktivitäten, einschließlich pharmakologisch relevanter Wirkungen, sind mikrobielle Naturstoffe eine wichtige Grundlage für die Arzneimittelentwicklung. Naturstoffe werden durch eine Ansammlung lokal gruppierter Gene, sogenannten Biosynthese-Genclustern (BGC), im Genom kodiert. Moderne bioinformatische Methoden durchsuchen Genom-Daten nach Genen, die typische biosynthetische Enzyme kodieren. Auf Grundlage dessen können verschiedenste BGCs vorhergesagt werden. Bislang konnte allerdings nur für einen kleinen Teil der vorhergesagten BGCs das dazugehörige Produkt identifiziert und charakterisiert werden. Cyanobakterien sind nachweislich eine reichhaltige, aber weitestgehend unerschlossene Quelle für Naturstoffe. Insbesondere mehrzellige Gattungen, wie Nostoc, tragen eine Vielzahl an BGCs in ihren Genomen. Ein Hauptziel dieser Studie war es, neue Konzepte für die Entdeckungen von Naturstoffen in Cyanobakterien zu entwickeln. Nostoc punctiforme PCC 73102 (N. punctiforme) erwies sich als besonders geeigneter Stamm für diese Aufgabe, da er eine Vielzahl weitestgehend ununtersuchter Gencluster besitzt und zugänglich für genetische Modifikationen ist. Eine Kombination aus Genome Mining, Bioaktivitäts-Screening, verschiedenen Kultivierungsbedingungen und Metabolic Engineering führte zur Entdeckung zweier neuer Polyketide und gewährte im Verlauf der Studie erstmals Einblicke in den spezialisierten Metabolismus von N. punctiforme. Die Kultivierung von N. punctiforme in sehr hohen Zelldichten, ermöglicht durch sehr hohe Lichtintensitäten und erhöhte CO2-Verfügbarkeit, führte zu einer verstärkten Metabolitproduktion und komplexen Metabolitextrakten. Unter Verwendung einer Bibliothek von CFP-Reportermutanten, bei der jede Mutante eines der vorhergesagten BGCs repräsentiert, konnte gezeigt werden, dass 8 von 15 BGCs unter Hochzelldichte-Kultivierungsbedingungen hochreguliert wurden. Zudem zeigte sich, dass der Überstand einer dichten Kultur, auch unter konventionellen Kultivierungsbedingungen, vier der regulierten BGCs beeinflussen kann. Dies lässt vermuten, dass sich unter Hochzelldichte-Kultivierungsbedingungen ein chemischer Mediator, welcher von einem der beeinflussten BGCs produziert wird, im Überstand anhäuft und die Expression anderer BGCs als Teil eines zelldichte-abhängigen Regelkreises kontrollieren kann. Um herauszufinden, welches der BGCs ein Hauptauslöser des vermuteten Regelkreises sein könnte, wurde versucht die Expression von vier BGCs (pks1, pks2, ripp3, ripp4) mittels Überexpression von potentiell biosynthese-spezifischen regulatorischen Genen zu aktivieren. Eine transkriptionelle Analyse der Mutanten ergab, dass nur der Stamm, welcher das pks1 BGC aktivieren sollte (AraC_PKS1), einen positiven Effekt auf die Expression des zu erwartenden BGCs hatte. Eine RNA-Sequenzierungsstudie ergab, dass in der AraC_PKS1 Mutante neben dem pks1 BGC auch die kryptischen BGCs ripp3 und ripp4 eine erhöhte Transkription aufwiesen. Zudem wurde beobachtet, dass sich die Sekundärmetabolitproduktion in der Mutante durch Kultivierung unter erhöhten Licht-Intensitäten und CO2-Leveln erweitern lässt. Unabhängig von den Kultivierungsbedingungen, hat die erhöhte Produktion des pks1 Regulators NvlA in der Mutante einen Einfluss auf andere regulatorische Faktoren, wie Sigma-Faktoren und das RNA-Chaperon Hfq. Die Analyse des Zell- und Überstandsextrakts der AraC_PKS1 Mutante führte zur Entdeckung zweier neuer Polyketide, Nostoclid und Nostovalerolacton, welche beide vom pks1 BGC codiert werden. Die Zugabe dieser Polyketide zum N. punctiforme Wildtyp zeigte, dass diese in der Lage sind einen Teil der Sekundärmetabolit-Effekte der AraC_PKS1 Mutante zu reproduzieren. Dies lässt darauf schließen, dass beide Polyketide als Signalstoffe innerhalb eines regulatorischen Netzwerks agieren. Da nicht alle transkriptionellen Effekte der AraC_PKS1 Mutante durch die Zugabe der pks1 Produkte reproduziert werden konnten, ist anzunehmen, dass der Regulator NvlA einen globalen Effekt hat und nicht ausschließlich die pks1 Biosynthese reguliert. Diese Studie war die erste, welche einen potentiell biosynthese-spezifischen Regulator für die gezielte Aktivierung von BGC-Expression in Cyanobakterien verwendet hat. Diese Strategie führte neben der Entdeckung zweier neuer Polyketide, zu ersten Einblicken in den regulatorischen Mechanismus, der den spezialisierten Metabolismus in N. punctiforme kontrolliert. Diese Studie veranschaulicht, dass das Verstehen regulatorischer Mechanismen für die Entdeckung neuer Naturstoffe hilfreich sein kann. Die Studien-Ergebnisse können die Entwicklung neuer Screening-Strategien für bioaktive Metabolite in Cyanobakterien anregen und können dabei helfen Hochtiter-Produktionsplattformen für cyanobakterielle Naturstoffe zu entwickeln. KW - cyanobacteria KW - natural products KW - specialized metabolites KW - gene cluster activation KW - Nostoc punctiforme KW - Cyanobakterien KW - Sekundärmetabolite KW - Naturstoffe KW - Gencluster-Aktivierung Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-540240 ER - TY - THES A1 - Meissner, Sven T1 - Implications of Microcystin Production in Microcystis aeruginosa PCC 7806 T1 - Effekte der Produktion von Microcystin in Microcystis aeruginosa PCC 7806 N2 - 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. N2 - Cyanobakterien produzieren etwa 40 Prozent der primären Biomasse auf der Welt aber auch giftige Peptide wie das leberschädigende Microcystin. Massenvorkommen, so genannte Blaualgenblüten, gefährden vielerorts regelmäßig die Trinkwasserversorgung. Diese Arbeit hatte zum Ziel, den Einfluss der Microcystinproduktion auf physiologische Abläufe in dem weit verbreiteten blütenbildenden Cyanobakterium Microcystis aeruginosa zu charakterisieren. Zum einen, wurde hierfür der Einfluss der Beleuchtungsintensität auf die Produktion von Microcystin und dessen Bindung an zelluläre Proteine ermittelt. Hierzu wurden etablierte Quantifizierungstechniken mit biochemischen Methoden kombiniert. RubisCO, das Schlüsselenzym zur primären Kohlenstofffixierung, war ein Hauptinteraktionspartner von Microcystin. Hohe Beleuchtungsintensität erhöhte die Menge von an Proteine gebundenem Microcystin. Bis zu 60 Prozent des gesamten zellulären Microcystins lag an Proteine gebunden vor, d.h. es wurde durch Standardquantifizierungsmethoden nicht erfasst. Die Notwendigkeit, zur Quantifizierung des gesamten Microcystins die Proteinfraktion mit einzubeziehen, wurde auch in Freilandproben demonstriert. Die Entwicklung einer immunofluoreszenzbasierten Methode erlaubte die Unterscheidung von toxischen und nichttoxischen Microcystis-Kolonien in Freilandproben. Die starklichtinduzierte Interaktion von Microcystin mit Proteinen deutete auf einen möglichen Einfluss des Sekundärmetabolits auf den Primärstoffwechsel von Microcystis hin. Um dieser Frage nachzugehen, wurde ein umfassender GC/MS-basierter Versuch durchgeführt, um die Akkumulation von Metaboliten im Microcystin produzierenden Stamm Microcystis aeruginosa PCC 7806 und dessen microcystinfreier ΔmcyB-mutierten Variante vergleichen zu können. Es zeigte sich, dass Microcystin einen Einfluss auf die Akkumulation von 85 (17 Prozent) aller 501 detektierten Metaboliten unter erhöhter Beleuchtungsstärke hatte. Besonders die vermehrte Synthese osmotisch aktiver Substanzen in der ΔmcyB Mutante, verstanden als generelle Reaktion auf allgemeinen Stress, deutete auf eine Beteiligung von Microcystin in der metabolischen Justierung von Microcystis hin. Die Parallelanalyse des Modellstamms Synechocystis PCC 6803 offenbarte grundsätzliche metabolische Unterschiede zwischen verschiedenen Cyanobakterienspezies. Demnach produzierte Microcystis vor allem Kohlehydrate und Synechocystis eher Aminosäuren. Die GC/MS-basierten Ergebnisse wurden durch elektronmikroskopische Aufnahmen und die Quantifizierung von Speichermetaboliten gestützt. Innerhalb drei Stunden bewirkte Starklicht die Akkumulation von Glykogen in Microcystis auf ca. 8.5 Prozent des Frischgewichts, wohingegen Synechocystis mehr Cyanophycin produzierte. Die Ergebnisse zeigten, dass im Hinblick auf die biotechnologische Nutzung von Cyanobakterien, die Charakterisierung speziesspezifischer metabolischer Eigenschaften mehr Beachtung finden sollte. KW - microcystin KW - Microcystin KW - cyanobacteria KW - Cyanobakterien Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-75199 ER - TY - GEN A1 - Van Donk, Ellen A1 - Ianora, Adrianna A1 - Vos, Matthijs T1 - Induced defences in marine and freshwater phytoplankton BT - a review T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - Many organisms have developed defences to avoid predation by species at higher trophic levels. The capability of primary producers to defend themselves against herbivores affects their own survival, can modulate the strength of trophic cascades and changes rates of competitive exclusion in aquatic communities. Algal species are highly flexible in their morphology, growth form, biochemical composition and production of toxic and deterrent compounds. Several of these variable traits in phytoplankton have been interpreted as defence mechanisms against grazing. Zooplankton feed with differing success on various phytoplankton species, depending primarily on size, shape, cell wall structure and the production of toxins and deterrents. Chemical cues associated with (i) mechanical damage, (ii) herbivore presence and (iii) grazing are the main factors triggering induced defences in both marine and freshwater phytoplankton, but most studies have failed to disentangle the exact mechanism(s) governing defence induction in any particular species. Induced defences in phytoplankton include changes in morphology (e.g. the formation of spines, colonies and thicker cell walls), biochemistry (such as production of toxins, repellents) and in life history characteristics (formation of cysts, reduced recruitment rate). Our categorization of inducible defences in terms of the responsible induction mechanism provides guidance for future work, as hardly any of the available studies on marine or freshwater plankton have performed all the treatments that are required to pinpoint the actual cue(s) for induction. We discuss the ecology of inducible defences in marine and freshwater phytoplankton with a special focus on the mechanisms of induction, the types of defences, their costs and benefits, and their consequences at the community level. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 881 KW - defenses KW - algae KW - review KW - plankton community KW - cyanobacteria KW - toxins Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-435130 SN - 1866-8372 IS - 881 ER - TY - JOUR A1 - Reyna-González, Emmanuel A1 - Schmid, Bianca A1 - Petras, Daniel A1 - Süssmuth, Roderich D. A1 - Dittmann, Elke T1 - Leader Peptide-Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease-Targeted Libraries JF - Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition N2 - 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. KW - biosynthesis KW - cyanobacteria KW - microviridins KW - natural products KW - peptides Y1 - 2016 U6 - https://doi.org/10.1002/anie.201604345 SN - 1433-7851 SN - 1521-3773 VL - 55 SP - 9398 EP - 9401 PB - Wiley-VCH CY - Weinheim ER - TY - THES A1 - Bolius, Sarah T1 - Microbial invasions in aquatic systems – strain identity, genetic diversity and timing N2 - Biological invasions are the dispersal and following establishment of species outside their native habitat. Due to globalisation, connectivity of regions and climate changes the number of invasive species and their successful establishment is rising. The impact of these species is mostly negative, can induce community and habitat alterations, and is one main cause for biodiversity loss. This impact is particularly high and less researched in aquatic systems and microbial organisms and despite the high impact, the knowledge about overall mechanisms and specific factors affecting invasions are not fully understood. In general, the characteristics of the habitat, native community and invader determine the invasiveness. In this thesis, I aimed to provide a better understanding of aquatic invasions focusing on the invader and its traits and identity. This thesis used a set of 12 strains of the invasive cyanobacterium Cylindrospermopsis raciborskii to examine the effect and impact of the invaders’ identity and genetic diversity. Further, the effect of timing on the invasion potential and success was determined, because aquatic systems in particular undergo seasonal fluctuations. Most studies revealed a higher invasion success with increasing genetic diversity. Here, the increase of the genetic diversity, by either strain richness or phylogenetic dissimilarity, is not firstly driving the invasion, but the strain-identity. The high variability among the strains in traits important for invasions led to the highly varying strain-specific invasion success. This success was most dependent on nitrogen uptake and efficient resource use. The lower invasion success into communities comprising further N-fixing species indicates C. raciborskii can use this advantage only without the presence of competitive species. The relief of grazing pressure, which is suggested to be more important in aquatic invasions, was only promoting the invasion when unselective and larger consumers were present. High abundances of unselective consumers hampered the invasion success. This indicates a more complex and temporal interplay of competitive and consumptive resistance mechanisms during the invasion process. Further, the fluctuation abundance and presence of competitors (= primary producers) and consumers (= zooplankton) in lakes can open certain ‘invasion windows’. Remarkably, the composition of the resident community was also strain-specific affected and altered, independent of a high or low invasion success. Prior, this was only documented on the species level. Further, investigations on the population of invasive strains can reveal more about the invasion patterns and how multiple strain invasions change resident communities. The present dissertation emphasises the importance of invader-addition experiments with a community context and the importance of the strain-level for microbial invasions and in general, e.g. for community assemblies and the outcome of experiments. The strain-specific community changes, also after days, may explain some sudden changes in communities, which have not been explained yet. This and further knowledge may also facilitate earlier and less cost-intensive management to step in, because these species are rarely tracked until they reach a high abundance or bloom, because of their small size. Concluded for C. raciborskii, it shows that this species is no ‘generalistic’ invader and its invasion success depends more on the competitor presence than grazing pressure. This may explain its, still unknown, invasion pattern, as C. raciborskii is not found in all lakes of a region. N2 - Biologische Invasionen beschreiben die Ausbreitung und Etablierung von Arten außerhalb ihres natürlichen Verbreitungsgebiets. Das Eindringen dieser invasiven Arten in ein neues Ökosystem hat meist negative Auswirkungen. Beispiele sind unter anderem veränderte Ökosystemprozesse, Lebensräume und Zusammensetzungen der einheimischen Arten, die zu einem Verlust der biologischen Vielfalt führen. Durch die fortschreitende Globalisierung und den Klimawandel steigt die Anzahl invasiver Arten weltweit. Um dies möglichst zu verhindern, müssen die zugrundeliegenden Mechanismen und Faktoren verstanden sein. Besonders bei aquatischen Mikroorganismen ist die Wissenslücke dabei groß und umso drängender, da diese Arten ein hohes Invasionspotential und potentiell stärkere negative Auswirkungen haben. Die vorliegende Dissertation untersuchte anhand der invasiven Cyanobakterie Cylindrospermopsis raciborskii, den anfänglichen Invasionsprozess, unter besonderer Berücksichtigung der Stamm-Identität, der genetischen Diversität und des Zeitpunkts der Invasion. Die meisten Studien zu Invasionen zeigen einen positiven Effekt der genetischen Diversität auf Invasionen. Diese Arbeit konnte zeigen, dass der Invasionserfolg auf bestimmte Stamm-spezifische Eigenschaften zurückzuführen ist. Für C. raciborskii war dies eine erhöhte Aufnahme von Stickstoff und eine effizientere Nutzung von Ressourcen. Wie einige andere aquatischen Arten hat C. raciborskii die Fähigkeit über differenzierte Zellen Stickstoff aus der Luft zu fixieren. Des Weiteren fördern bestimmte Umweltbedingungen, wie eine niedrige Nährstoffkonzentration, das Wachstum von C. raciborskii. Fraßdruck wirkte sich nur negativ aus, wenn unselektive Prädatoren anwesend waren. Zudem zeigten weitere Versuche, dass ihr Konkurrenzvorteil nur in Gemeinschaften ohne weitere Stickstoff-Fixierer und in Stickstoff-reduzierten Habitaten die Etablierung positiv beeinflusst. Diese Erkenntnisse lassen darauf schließen, dass diverse Eigenschaften und eine zeitliche Abfolge dieser, den Invasionserfolg beeinflussen. Dieser kann einerseits durch den Widerstand der heimischen Arten-Gemeinschaft und zum anderem durch die herrschenden abiotischen Bedingungen verhindert werden. Gerade aquatische Systeme unterlaufen saisonalen Schwankungen und diese erlauben somit bestimmte, temporäre Invasionsmöglichkeiten. Zusätzlich führte die Stamm-Identität zu Änderungen in der einheimischen Artenzusammensetzung, unabhängig vom Erfolg der Invasion - dies wurde bis jetzt nur Art-spezifisch gezeigt. Damit betont die vorliegende Arbeit die Bedeutung von Stamm-Identitäten auf Invasionen und deren Auswirkungen auf ökologische Prozesse und den Ausgang von Experimenten. Zusammengefasst für C. raciborskii zeigt die Arbeit, dass diese Art keine ‚generell‘ erfolgreiche invasive Art ist und dass der Invasionserfolg eher von den vorhanden konkurrierenden Arten abhängt. Dies könnte das noch unklare Ausbreitungs-Muster erklären. KW - invasion KW - cyanobacteria KW - population KW - Cylindrospermopsis raciborskii Y1 - 2018 ER - TY - JOUR A1 - Kehr, Jan-Christoph A1 - Picchi, Douglas Gatte A1 - Dittmann-Thünemann, Elke T1 - Natural product biosyntheses in cyanobacteria a treasure trove of unique enzymes JF - Beilstein journal of organic chemistry N2 - 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. KW - cyanobacteria KW - natural products KW - NRPS KW - PKS KW - ribosomal peptides Y1 - 2011 U6 - https://doi.org/10.3762/bjoc.7.191 SN - 1860-5397 VL - 7 IS - 2 SP - 1622 EP - 1635 PB - Beilstein-Institut zur Förderung der Chemischen Wissenschaften CY - Frankfurt, Main ER - TY - JOUR A1 - Meyer, Sabine A1 - Mainz, Andi A1 - Kehr, Jan-Christoph A1 - Suessmuth, Roderich A1 - Dittmann, Elke T1 - Prerequisites of Isopeptide Bond Formation in Microcystin Biosynthesis JF - ChemBioChem : a European journal of chemical biology N2 - 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. KW - amino acids KW - biosynthesis KW - cyanobacteria KW - nonribosomal peptide KW - substrate specificity Y1 - 2017 U6 - https://doi.org/10.1002/cbic.201700389 SN - 1439-4227 SN - 1439-7633 VL - 18 SP - 2376 EP - 2379 PB - Wiley-VCH CY - Weinheim ER -