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Neuroinflammatory and neurodegenerative diseases such as Parkinson's (PD) and multiple sclerosis (MS) often result in a severe impairment of the patient´s quality of life. Effective therapies for the treatment are currently not available, which results in a high socio-economic burden. Due to the heterogeneity of the disease subtypes, stratification is particularly difficult in the early phase of the disease and is mainly based on clinical parameters such as neurophysiological tests and central nervous imaging. Due to good accessibility and stability, blood and cerebrospinal fluid metabolite markers could serve as surrogates for neurodegenerative processes. This can lead to an improved mechanistic understanding of these diseases and further be used as "treatment response" biomarkers in preclinical and clinical development programs. Therefore, plasma and CSF metabolite profiles will be identified that allow differentiation of PD from healthy controls, association of PD with dementia (PDD) and differentiation of PD subtypes such as akinetic rigid and tremor dominant PD patients. In addition, plasma metabolites for the diagnosis of primary progressive MS (PPMS) should be investigated and tested for their specificity to relapsing-remitting MS (RRMS) and their development during PPMS progression.
By applying untargeted high-resolution metabolomics of PD patient samples and in using random forest and partial least square machine learning algorithms, this study identified 20 plasma metabolites and 14 CSF metabolite biomarkers. These differentiate against healthy individuals with an AUC of 0.8 and 0.9 in PD, respectively. We also identify ten PDD specific serum metabolites, which differentiate against healthy individuals and PD patients without dementia with an AUC of 1.0, respectively. Furthermore, 23 akinetic-rigid specific plasma markers were identified, which differentiate against tremor-dominant PD patients with an AUC of 0.94 and against healthy individuals with an AUC of 0.98. These findings also suggest more severe disease pathology in the akinetic-rigid PD than in tremor dominant PD. In the analysis of MS patient samples a partial least square analysis yielded predictive models for the classification of PPMS and resulted in 20 PPMS specific metabolites. In another MS study unknown changes in human metabolism were identified after administration of the multiple sclerosis drug dimethylfumarate, which is used for the treatment of RRMS. These results allow to describe and understand the hitherto completely unknown mechanism of action of this new drug and to use these findings for the further development of new drugs and targets against RRMS.
In conclusion, these results have the potential for improved diagnosis of these diseases and improvement of mechanistic understandings, as multiple deregulated pathways were identified. Moreover, novel Dimethylfumarate targets can be used to aid drug development and treatment efficiency. Overall, metabolite profiling in combination with machine learning identified as a promising approach for biomarker discovery and mode of action elucidation.
Characterization of altered inflorescence architecture in Arabidopsis thaliana BG-5 x Kro-0 hybrid
(2018)
A reciprocal cross between two A. thaliana accessions, Kro-0 (Krotzenburg, Germany) and BG-5 (Seattle, USA), displays purple rosette leaves and dwarf bushy phenotype in F1 hybrids when grown at 17 °C and a parental-like phenotype when grown at 21 °C. This F1 temperature-dependent-dwarf-bushy phenotype is characterized by reduced growth of the primary stem together with an increased number of branches. The reduced stem growth was the strongest at the first internode. In addition, we found that a temperature switch from 21 °C to 17 °C induced the phenotype only before the formation of the first internode of the stem. Similarly, the F1 dwarf-bushy phenotype could not be reversed when plants were shifted from 17 °C to 21 °C after the first internode was formed. Metabolic analysis showed that the F1 phenotype was associated with a significant upregulation of anthocyanin(s), kaempferol(s), salicylic acid, jasmonic acid and abscisic acid. As it has been previously shown that the dwarf-bushy phenotype is linked to two loci, one on chromosome 2 from Kro-0 and one on chromosome 3 from BG-5, an artificial micro-RNA approach was used to investigate the necessary genes on these intervals. From the results obtained, it was found that two genes, AT2G14120 that encodes for a DYNAMIN RELATED PROTEIN3B and AT2G14100 that encodes a member of the Cytochrome P450 family protein CYP705A13, were necessary for the appearance of the F1 phenotype on chromosome 2. It was also discovered that AT3G61035 that encodes for another cytochrome P450 family protein CYP705A13 and AT3G60840 that encodes for a MICROTUBULE-ASSOCIATED PROTEIN65-4 on chromosome 3 were both necessary for the induction of the F1 phenotype. To prove the causality of these genes, genomic constructs of the Kro-0 candidate genes on chromosome 2 were transferred to BG-5 and genomic constructs of the chromosome 3 candidate genes from BG-5 were transferred to Kro-0. The T1 lines showed that these genes are not sufficient alone to induce the phenotype. In addition to the F1 phenotype, more severe phenotypes were observed in the F2 generations that were grouped into five different phenotypic classes. Whilst seed yield was comparable between F1 hybrids and parental lines, three phenotypic classes in the F2 generation exhibited hybrid breakdown in the form of reproductive failure. This F2 hybrid breakdown was less sensitive to temperature and showed a dose-dependent effect of the loci involved in F1 phenotype. The severest class of hybrid breakdown phenotypes was observed only in the population of backcross with the parent Kro-0, which indicates a stronger contribution of the BG-5 allele when compared to the Kro-0 allele on the hybrid breakdown phenotypes. Overall, the findings of my thesis provide a further understanding of the genetic and metabolic factors underlying altered shoot architecture in hybrid dysfunction.
Systems biology aims at investigating biological systems in its entirety by gathering and analyzing large-scale data sets about the underlying components. Computational systems biology approaches use these large-scale data sets to create models at different scales and cellular levels. In addition, it is concerned with generating and testing hypotheses about biological processes. However, such approaches are inevitably leading to computational challenges due to the high dimensionality of the data and the differences in the dimension of data from different cellular layers.
This thesis focuses on the investigation and development of computational approaches to analyze metabolite profiles in the context of cellular networks. This leads to determining what aspects of the network functionality are reflected in the metabolite levels. With these methods at hand, this thesis aims to answer three questions: (1) how observability of biological systems is manifested in metabolite profiles and if it can be used for phenotypical comparisons; (2) how to identify couplings of reaction rates from metabolic profiles alone; and (3) which regulatory mechanism that affect metabolite levels can be distinguished by integrating transcriptomics and metabolomics read-outs.
I showed that sensor metabolites, identified by an approach from observability theory, are more correlated to each other than non-sensors. The greater correlations between sensor metabolites were detected both with publicly available metabolite profiles and synthetic data simulated from a medium-scale kinetic model. I demonstrated through robustness analysis that correlation was due to the position of the sensor metabolites in the network and persisted irrespectively of the experimental conditions. Sensor metabolites are therefore potential candidates for phenotypical comparisons between conditions through targeted metabolic analysis.
Furthermore, I demonstrated that the coupling of metabolic reaction rates can be investigated from a purely data-driven perspective, assuming that metabolic reactions can be described by mass action kinetics. Employing metabolite profiles from domesticated and wild wheat and tomato species, I showed that the process of domestication is associated with a loss of regulatory control on the level of reaction rate coupling. I also found that the same metabolic pathways in Arabidopsis thaliana and Escherichia coli exhibit differences in the number of reaction rate couplings.
I designed a novel method for the identification and categorization of transcriptional effects on metabolism by combining data on gene expression and metabolite levels. The approach determines the partial correlation of metabolites with control by the principal components of the transcript levels. The principle components contain the majority of the transcriptomic information allowing to partial out the effect of the transcriptional layer from the metabolite profiles. Depending whether the correlation between metabolites persists upon controlling for the effect of the transcriptional layer, the approach allows us to group metabolite pairs into being associated due to post-transcriptional or transcriptional regulation, respectively. I showed that the classification of metabolite pairs into those that are associated due to transcriptional or post-transcriptional regulation are in agreement with existing literature and findings from a Bayesian inference approach.
The approaches developed, implemented, and investigated in this thesis open novel ways to jointly study metabolomics and transcriptomics data as well as to place metabolic profiles in the network context. The results from these approaches have the potential to provide further insights into the regulatory machinery in a biological system.
Microbial processing of organic matter (OM) in the freshwater biosphere is a key component of global biogeochemical cycles. Freshwaters receive and process valuable amounts of leaf OM from their terrestrial landscape. These terrestrial subsidies provide an essential source of energy and nutrients to the aquatic environment as a function of heterotrophic processing by fungi and bacteria. Particularly in freshwaters with low in-situ primary production from algae (microalgae, cyanobacteria), microbial turnover of leaf OM significantly contributes to the productivity and functioning of freshwater ecosystems and not least their contribution to global carbon cycling.
Based on differences in their chemical composition, it is believed that leaf OM is less bioavailable to microbial heterotrophs than OM photosynthetically produced by algae. Especially particulate leaf OM, consisting predominantly of structurally complex and aromatic polymers, is assumed highly resistant to enzymatic breakdown by microbial heterotrophs. However, recent research has demonstrated that OM produced by algae promotes the heterotrophic breakdown of leaf OM in aquatic ecosystems, with profound consequences for the metabolism of leaf carbon (C) within microbial food webs. In my thesis, I aimed at investigating the underlying mechanisms of this so called priming effect of algal OM on the use of leaf C in natural microbial communities, focusing on fungi and bacteria.
The works of my thesis underline that algal OM provides highly bioavailable compounds to the microbial community that are quickly assimilated by bacteria (Paper II). The substrate composition of OM pools determines the proportion of fungi and bacteria within the microbial community (Paper I). Thereby, the fraction of algae OM in the aquatic OM pool stimulates the activity and hence contribution of bacterial communities to leaf C turnover by providing an essential energy and nutrient source for the assimilation of the structural complex leaf OM substrate. On the contrary, the assimilation of algal OM remains limited for fungal communities as a function of nutrient competition between fungi and bacteria (Paper I, II). In addition, results provide evidence that environmental conditions determine the strength of interactions between microalgae and heterotrophic bacteria during leaf OM decomposition (Paper I, III). However, the stimulatory effect of algal photoautotrophic activities on leaf C turnover remained significant even under highly dynamic environmental conditions, highlighting their functional role for ecosystem processes (Paper III).
The results of my thesis provide insights into the mechanisms by which algae affect the microbial turnover of leaf C in freshwaters. This in turn contributes to a better understanding of the function of algae in freshwater biogeochemical cycles, especially with regard to their interaction with the heterotrophic community.
Um das Immunsystem der Pflanze zu manipulieren translozieren gram-negative pathogene Bakterien Typ-III Effektorproteine (T3E) über ein Typ-III Sekretionssystem (T3SS) in die pflanzliche Wirtszelle. Dort lokalisieren T3Es in verschiedenen subzellulären Kompartimenten, wo sie Zielproteine modifizieren und so die Infektion begünstigen. HopZ1a, ein T3E des Pflanzenpathogens Pseudomonas syringae pv. syringae, ist eine Acetyltransferase und lokalisiert über ein Myristolierungsmotiv an der Plasmamembran der Wirtszelle. Obwohl gezeigt wurde, dass HopZ1a die frühe Signalweiterleitung an der Plasmamembran stört, wurde bisher kein mit der Plasmamembran assoziiertes Zielprotein für diesen T3E identifiziert. Um bisher unbekannte HopZ1a-Zieleproteine zu identifizieren wurde im Vorfeld dieser Arbeit eine Hefe-Zwei-Hybrid-Durchmusterung mit einer cDNA-Bibliothek aus Tabak durchgeführt, wobei ein nicht näher charakterisiertes Remorin als Interaktor gefunden wurde.
Bei dem Remorin handelt es sich um einen Vertreter der Gruppe 4 der Remorin-Familie, weshalb es in NbREM4 umbenannt wurde. Durch den Einsatz verschiedener Interaktionsstudien konnte demonstriert werden, dass HopZ1a mit NbREM4 in Hefe, in vitro und in planta wechselwirkt. Es wurde ferner deutlich, dass HopZ1a auf spezifische Weise mit dem konservierten C-Terminus von NbREM4 interagiert, das Remorin jedoch in vitro nicht acetyliert. Analysen mittels BiFC haben zudem ergeben, dass NbREM4 in Homodimeren an der Plasmamembran lokalisiert, wo auch die Interaktion mit HopZ1a stattfindet.
Eine funktionelle Charakterisierung von NbREM4 ergab, dass das Remorin eine spezifische Rolle im Immunsystem der Pflanze einnimmt. Die transiente Expression in N. benthamiana induziert die Expression von Abwehrgenen sowie einen veränderten Blattphänotyp. In A. thaliana wird HopZ1a über das Decoy ZED1 und das R-Protein ZAR1 erkannt, was zur Auslösung einer starken Hypersensitiven Antwort (HR von hypersensitive response) führt. Es konnte im Rahmen dieser Arbeit gezeigt werden, dass ZAR1 in N. benthamiana konserviert ist, NbREM4 jedoch nicht in der ETI als Decoy fungiert. Mit Hilfe einer Hefe-Zwei-Hybrid-Durchmusterung mit NbZAR1 als Köder konnten zwei Proteine, die Catalase CAT1 und der Protonenpumpeninteraktor PPI1, als Interaktoren von NbZAR1 identifiziert werden, welche möglicherweise in der Regulation der HR eine Rolle spielen.
Aus Voruntersuchungen war bekannt, dass NbREM4 mit weiteren, nicht näher charakterisierten Proteinen aus Tabak interagieren könnte. Eine phylogenetische Einordnung hat gezeigt, dass es sich um die bekannte Immun-Kinase PBS1 sowie zwei E3-Ubiquitin-Ligasen, NbSINA1 und NbSINAL3, handelt. PBS1 interagiert mit NbREM4 an der Plasmamembran und phosphoryliert das Remorin innerhalb des intrinsisch ungeordneten N-Terminus. Mittels Massenspektrometrie konnten die Serine an Position 64 und 65 innerhalb der Aminosäuresequenz von NbREM4 als PBS1-abhängige Phosphorylierungsstellen identifiziert wurden.
NbSINA1 und NbSINAL3 besitzen in vitro Ubiquitinierungsaktivität, bilden Homo- und Heterodimere und interagieren ebenfalls mit dem N-terminalen Teil von NbREM4, wobei sie das Remorin in vitro nicht ubiquitinieren.
Aus den in dieser Arbeit gewonnenen Ergebnissen lässt sich ableiten, dass der bakterielle T3E HopZ1a gezielt mit dem Tabak-Remorin NbREM4 an der Plasmamembran interagiert und über einen noch unbekannten Mechanismus mit dem Immunsystem der Pflanze interferiert, wobei NbREM4 möglicherweise eine Rolle als Adapter- oder Ankerprotein zukommt, über welches HopZ1a mit weiteren Immunkomponenten interagiert. NbREM4 ist Teil eines größeren Immunnetzwerkes, zu welchem die bekannte Immun-Kinase PBS1 und zwei E3-Ubiquitin-Ligasen gehören. Mit NbREM4 konnte damit erstmalig ein membranständiges Protein mit einer Funktion im Immunsystem der Pflanze als Zielprotein von HopZ1a identifiziert werden.
Die Folgen einer lebensmittelbedingten Erkrankung sind zum Teil gravierend, insbesondere für Kinder und immunsupprimierte Menschen. Hierbei gehören Salmonella und Campylobacter zu den häufigsten Erregern, die verantwortlich für gastrointestinale Erkrankungen in Deutschland sind. Trotz umfassender Maßnahmen der EU zur Prävention und Bekämpfung von Salmonellen in Geflügelbeständen und der Lebensmittel-Industrie, wird von einem stagnierenden Trend von Infektionszahlen berichtet. Zoonose-Erreger wie Salmonellen können über Nutztiere in die Nahrungskette des Menschen gelangen, wodurch sich Infektionsherde schnell ausbreiten können. Dabei sind bestehende Präventionsstrategien für Geflügel vorhanden, die aber nicht auf den Menschen übertragbar sind. Folglich sind Diagnostik und Prävention in der Lebensmittelindustrie essentiell. Deshalb besteht ein hoher Bedarf für spezifische, sensitive und zuverlässige Nachweismethoden, die eine Point-of-care Diagnostik gewährleisten. Durch ein wachsendes Verständnis der wirtsspezifischen Faktoren von S. enterica Serovaren kann die Entwicklung sowohl neuartiger diagnostischer Methoden, als auch neuartiger Therapien und Impfstoffe maßgeblich vorangetrieben werden.
Infolgedessen wurde in dieser Arbeit ein infektionsähnliches in vitro Modell für S. Enteritidis etabliert und darauf basierend eine umfassende Untersuchung zur Identifizierung neuer Zielstrukturen für den Erreger durchgeführt. Während einer Salmonellen-Infektion ist die erste zelluläre Barriere im Wirt die Epithelschicht. Dementsprechend wurde eine humane Zelllinie (CaCo 2, Darmepithel) für die Pathogen-Wirt-Studie ausgewählt. Das Salmonellen-Transkriptom und morphologische Eigenschaften der Epithelzellen wurden in verschiedenen Phasen der Salmonellen-Infektion untersucht und mit bereits gut beschriebenen Virulenzfaktoren und Beobachtungen in Bezug gesetzt. Durch dieses Infektionsmodell konnte ein spezifischer Phänotyp für die intrazellulären Salmonellen in den Epithelzellen nachgewiesen werden. Zudem wurde aufgezeigt, dass bereits die Kultivierung in Flüssigmedium einen invasionsaktiven Zustand der Salmonellen erzeugt. Allerdings wurde durch die Kokultivierung mit Epithelzellen eine zusätzliche Expression relevanter Gene induziert, um eine effiziente Adhäsion und Transmembran-Transport zu gewährleisten. Letzterer ist charakteristisch für die intrazelluläre Limitierung von Nährstoffen und prägt den infektionsrelevanten Status. Unter Berücksichtigung dieser Faktoren ergab sich ein Phänotyp, der eindeutig Mechanismen zur Wirtsadaptation und möglicherweise auch Pathogenese aufzeigt. Die intrazellulären Bakterien müssen vom Wirt separiert werden, was ein wesentlicher Schritt für Pathogen-bestimmende Analysen ist. Hierbei wurde mithilfe einer Detergenz-basierten Lyse der eukaryotischen Zellmembran und differentieller Zentrifugation, der eukaryotische Eintrag minimal gehalten. Unter Verwendung der Virulenz-adaptierten Salmonellen wurden Untersuchungen in Hinblick auf die Identifizierung neuer Zielstrukturen für S. Enteritidis durchgeführt. Mithilfe eines immunologischen Screenings wurden neue potentielle Antigene entdeckt. Zu diesem Zweck wurden bakterielle cDNA-basierte Expressionsbibliotheken hergestellt, die durch eine vereinfachte Microarray-Anwendung ein Hochdurchsatzscreening von Proteinen als potentielle Binder ermöglichen. Folglich konnten neue unbeschriebene Proteine identifiziert werden, die sich durch eine Salmonella-Spezifität oder Membranständigkeit auszeichnen. Ebenso wurde ein Vergleich der im Screening identifizierten Proteine mit der Regulation der kodierenden Gene im infektionsähnlichen Modell durchgeführt. Dabei wurde deutlich, dass die Häufigkeit von Transkripten einen Einfluss auf die Verfügbarkeit in der cDNA-Bibliothek und folglich auch auf die Expressionsbibliothek nimmt. Angesichts eines Ungleichgewichts zwischen der Gesamtzahl protein-kodierender Gene in S. Enteritidis zu möglichen Klonen, die während des Microarray-Screenings untersucht werden können, besteht der Bedarf einer Anreicherung von Proteinen in der Expressionsbibliothek. Das infektionsähnliche Modell zeigte, dass nicht nur Virulenz-assoziierte, sondern auch Stress- und Metabolismus-relevante Gene hochreguliert werden. Durch die Konstruktion dieser spezifischen cDNA-Bibliotheken ist die Erkennung von charakteristischen molekularen Markern gegeben.
Weiterhin wurden anhand der Transkriptomanalyse spezifisch hochregulierte Gene identifiziert, die relevant für das intrazelluläre Überleben von S. Enteritidis in humanen Epithelzellen sind. Hiervon wurden drei Gene näher untersucht, indem ihr Einfluss im infektionsähnlichen Modell mittels entsprechender Gen-Knockout-Stämme analysiert wurde. Dabei wurde für eine dieser Mutanten ein reduziertes Wachstum in der späten intrazellulären Phase nachgewiesen. Weiterführende in vitro Analysen sind für die Charakterisierung des Knockout-Stamms notwendig, um den Einsatz als potenzielles Therapeutikum zu verifizieren.
Zusammenfassend wurde ein in vitro Infektionsmodell für S. Enteritidis etabliert, wodurch neue Zielstrukturen des Erregers identifiziert wurden. Diese sind für diagnostische oder therapeutische Anwendungen interessant. Das Modell lässt sich ebenso für andere intrazelluläre Pathogene übertragen und gewährleistet eine zuverlässige Identifizierung von potentiellen Antigenen.
Investigating the role of fluorinated amino acids on protein structure and function using simulation
(2018)
The continuously increasing pollution of aquatic environments with microplastics (plastic particles < 5 mm) is a global problem with potential implications for organisms of all trophic levels. For microorganisms, trillions of these floating microplastics particles represent a huge surface area for colonization. Due to the very low biodegradability, microplastics remain years to centuries in the environment and can be transported over thousands of kilometers together with the attached organisms. Since also pathogenic, invasive, or otherwise harmful species could be spread this way, it is essential to study microplastics-associated communities.
For this doctoral thesis, eukaryotic communities were analyzed for the first time on microplastics in brackish environments and compared to communities in the surrounding water and on the natural substrate wood. With Illumina MiSeq high-throughput sequencing, more than 500 different eukaryotic taxa were detected on the microplastics samples. Among them were various green algae, dinoflagellates, ciliates, fungi, fungal-like protists and small metazoans such as nematodes and rotifers. The most abundant organisms was a dinoflagellate of the genus Pfiesteria, which could include fish pathogenic and bloom forming toxigenic species. Network analyses revealed that there were numerous interaction possibilities among prokaryotes and eukaryotes in microplastics biofilms. Eukaryotic community compositions on microplastics differed significantly from those on wood and in water, and compositions were additionally distinct among the sampling locations. Furthermore, the biodiversity was clearly lower on microplastics in comparison to the diversity on wood or in the surrounding water.
In another experiment, a situation was simulated in which treated wastewater containing microplastics was introduced into a freshwater lake. With increasing microplastics concentrations, the resulting bacterial communities became more similar to those from the treated wastewater. Moreover, the abundance of integrase I increased together with rising concentrations of microplastics. Integrase I is often used as a marker for anthropogenic environmental pollution and is further linked to genes conferring, e.g., antibiotic resistance.
This dissertation gives detailed insights into the complexity of prokaryotic and eukaryotic communities on microplastics in brackish and freshwater systems. Even though microplastics provide novel microhabitats for various microbes, they might also transport toxigenic, pathogenic, antibiotic-resistant or parasitic organisms; meaning their colonization can pose potential threats to humans and the environment. Finally, this thesis explains the urgent need for more research as well as for strategies to minimize the global microplastic pollution.
Plastic pollution is ubiquitous on the planet since several millions of tons of plastic waste enter aquatic ecosystems each year. Furthermore, the amount of plastic produced is expected to increase exponentially shortly. The heterogeneity of materials, additives and physical characteristics of plastics are typical of these emerging contaminants and affect their environmental fate in marine and freshwaters. Consequently, plastics can be found in the water column, sediments or littoral habitats of all aquatic ecosystems. Most of this plastic debris will fragment as a product of physical, chemical and biological forces, producing particles of small size. These particles (< 5mm) are known as “microplastics” (MP). Given their high surface-to-volume ratio, MP stimulate biofouling and the formation of biofilms in aquatic systems.
As a result of their unique structure and composition, the microbial communities in MP biofilms are referred to as the “Plastisphere.” While there is increasing data regarding the distinctive composition and structure of the microbial communities that form part of the plastisphere, scarce information exists regarding the activity of microorganisms in MP biofilms. This surface-attached lifestyle is often associated with the increase in horizontal gene transfer (HGT) among bacteria. Therefore, this type of microbial activity represents a relevant function worth to be analyzed in MP biofilms. The horizontal exchange of mobile genetic elements (MGEs) is an essential feature of bacteria. It accounts for the rapid evolution of these prokaryotes and their adaptation to a wide variety of environments. The process of HGT is also crucial for spreading antibiotic resistance and for the evolution of pathogens, as many MGEs are known to contain antibiotic resistance genes (ARGs) and genetic determinants of pathogenicity.
In general, the research presented in this Ph.D. thesis focuses on the analysis of HGT and heterotrophic activity in MP biofilms in aquatic ecosystems. The primary objective was to analyze the potential of gene exchange between MP bacterial communities vs. that of the surrounding water, including bacteria from natural aggregates. Moreover, the thesis addressed the potential of MP biofilms for the proliferation of biohazardous bacteria and MGEs from wastewater treatment plants (WWTPs) and associated with antibiotic resistance. Finally, it seeks to prove if the physiological profile of MP biofilms under different limnological conditions is divergent from that of the water communities. Accordingly, the thesis is composed of three independent studies published in peer-reviewed journals. The two laboratory studies were performed using both model and environmental microbial communities. In the field experiment, natural communities from freshwater ecosystems were examined.
In Chapter I, the inflow of treated wastewater into a temperate lake was simulated with a concentration gradient of MP particles. The effects of MP on the microbial community structure and the occurrence of integrase 1 (int 1) were followed. The int 1 is a marker associated with mobile genetic elements and known as a proxy for anthropogenic effects on the spread of antimicrobial resistance genes. During the experiment, the abundance of int1 increased in the plastisphere with increasing MP particle concentration, but not in the surrounding water. In addition, the microbial community on MP was more similar to the original wastewater community with increasing microplastic concentrations. Our results show that microplastic particles indeed promote persistence of standard indicators of microbial anthropogenic pollution in natural waters.
In Chapter II, the experiments aimed to compare the permissiveness of aquatic bacteria towards model antibiotic resistance plasmid pKJK5, between communities that form biofilms on MP vs. those that are free-living. The frequency of plasmid transfer in bacteria associated with MP was higher when compared to bacteria that are free-living or in natural aggregates. Moreover, comparison increased gene exchange occurred in a broad range of phylogenetically-diverse bacteria. The results indicate a different activity of HGT in MP biofilms, which could affect the ecology of aquatic microbial communities on a global scale and the spread of antibiotic resistance.
Finally, in Chapter III, physiological measurements were performed to assess whether microorganisms on MP had a different functional diversity from those in water. General heterotrophic activity such as oxygen consumption was compared in microcosm assays with and without MP, while diversity and richness of heterotrophic activities were calculated by using Biolog® EcoPlates. Three lakes with different nutrient statuses presented differences in MP-associated biomass build up. Functional diversity profiles of MP biofilms in all lakes differed from those of the communities in the surrounding water, but only in the oligo-mesotrophic lake MP biofilms had a higher functional richness compared to the ambient water. The results support that MP surfaces act as new niches for aquatic microorganisms and can affect global carbon dynamics of pelagic environments.
Overall, the experimental works presented in Chapters I and II support a scenario where MP pollution affects HGT dynamics among aquatic bacteria. Among the consequences of this alteration is an increase in the mobilization and transfer efficiency of ARGs. Moreover, it supposes that changes in HGT can affect the evolution of bacteria and the processing of organic matter, leading to different catabolic profiles such as demonstrated in Chapter III. The results are discussed in the context of the fate and magnitude of plastic pollution and the importance of HGT for bacterial evolution and the microbial loop, i.e., at the base of aquatic food webs. The thesis supports a relevant role of MP biofilm communities for the changes observed in the aquatic microbiome as a product of intense human intervention.
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.
East Africa is a natural laboratory: Studying its unique geological and biological history can help us better inform our theories and models. Studying its present and future can help us protect its globally important biodiversity and ecosystem services. East African vegetation plays a central role in all these aspects, and this dissertation aims to quantify its dynamics through computer simulations.
Computer models help us recreate past settings, forecast into the future or conduct simulation experiments that we cannot otherwise perform in the field. But before all that, one needs to test their performance. The outputs that the model produced using the present day-inputs, agreed well with present-day observations of East African vegetation. Next, I simulated past vegetation for which we have fossil pollen data to compare. With computer models, we can fill the gaps of knowledge between sites where we have fossil pollen data from, and create a more complete picture of the past. Good level of agreement between model and pollen data where they overlapped in space further validated our model performance.
Once the model was tested and validated for the region, it became possible to probe one of the long standing questions regarding East African vegetation: How did East Africa lose its tropical forests? The present-day vegetation in the tropics is mainly characterized by continuous forests worldwide except in tropical East Africa, where forests only occur as patches. In a series of simulation experiments, I was able to show under which conditions these forest patches could have been connected and fragmented in the past. This study showed the sensitivity of East African vegetation to climate change and variability such as those expected under future climate change.
El Niño Southern Oscillation (ENSO) events that result from the fluctuations in temperature between the ocean and atmosphere, bring further variability to East African climate and are predicted to increase in intensity in the future. But climate models are still not good at capturing the pattens of these events. In a study where I quantified the influence of ENSO events on East African vegetation, I showed how different the future vegetation could be from what we currently predict with these climate models that lack accurate ENSO contribution. Consideration of these discrepancies is important for our future global carbon budget calculations and management decisions.