@phdthesis{Bolius2018,
  author    = {Bolius, Sarah},
  title     = {Microbial invasions in aquatic systems - strain identity, genetic diversity and timing},
  school      = {Universit{\"a}t Potsdam},
  pages     = {154},
  year      = {2018},
  abstract  = {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 <i>Cylindrospermopsis raciborskii</i> 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 <i>C. raciborskii</i> 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 <i>C. raciborskii</i>, 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 <i>C. raciborskii</i> is not found in all lakes of a region.},
  language  = {en}
}
@phdthesis{Meissner2014,
  author    = {Meissner, Sven},
  title     = {Implications of Microcystin Production in Microcystis aeruginosa PCC 7806},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-75199},
  school      = {Universit{\"a}t Potsdam},
  pages     = {VII, 141},
  year      = {2014},
  abstract  = {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.},
  language  = {en}
}
@phdthesis{Krumbholz2021,
  author    = {Krumbholz, Julia},
  title     = {Identification of chemical mediators that regulate the specialized metabolism in Nostoc punctiforme},
  doi       = {10.25932/publishup-54024},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-540240},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xxiii, 187},
  year      = {2021},
  abstract  = {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.},
  language  = {en}
}
@phdthesis{ReynaGonzalez2017,
  author    = {Reyna Gonz{\´a}lez, Emmanuel},
  title     = {Engineering of the microviridin post-translational modification enzymes for the production of synthetic protease inhibitors},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-406979},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XI, 91, CI},
  year      = {2017},
  abstract  = {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.},
  language  = {en}
}