570 Biowissenschaften; Biologie
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The importance of cryptic diversity in rotifers is well understood regarding its ecological consequences, but there remains an in depth comprehension of the underlying molecular mechanisms and forces driving speciation. Temperature has been found several times to affect species spatio-temporal distribution and organisms’ performance, but we lack information on the mechanisms that provide thermal tolerance to rotifers. High cryptic diversity was found recently in the freshwater rotifer “Brachionus calyciflorus”, showing that the complex comprises at least four species: B. calyciflorus sensu stricto (s.s.), B. fernandoi, B. dorcas, and B. elevatus. The temporal succession among species which have been observed in sympatry led to the idea that temperature might play a crucial role in species differentiation.
The central aim of this study was to unravel differences in thermal tolerance between species of the former B. calyciflorus species complex by comparing phenotypic and gene expression responses. More specifically, I used the critical maximum temperature as a proxy for inter-species differences in heat-tolerance; this was modeled as a bi-dimensional phenotypic trait taking into consideration the intention and the duration of heat stress. Significant differences on heat-tolerance between species were detected, with B. calyciflorus s.s. being able to tolerate higher temperatures than B. fernandoi.
Based on evidence of within species neutral genetic variation, I further examined adaptive genetic variability within two different mtDNA lineages of the heat tolerant B. calyciflorus s.s. to identify SNPs and genes under selection that might reflect their adaptive history. These analyses did not reveal adaptive genetic variation related to heat, however, they show putatively adaptive genetic variation which may reflect local adaptation. Functional enrichment of putatively positively selected genes revealed signals of adaptation in genes related to “lipid metabolism”, “xenobiotics biodegradation and metabolism” and “sensory system”, comprising candidate genes which can be utilized in studies on local adaptation. An absence of genetically-based differences in thermal adaptation between the two mtDNA lineages, together with our knowledge that B. calyciflorus s.s. can withstand a broad range of temperatures, led to the idea to further investigate shared transcriptomic responses to long-term exposure to high and low temperatures regimes. With this, I identified candidate genes that are involved in the response to temperature imposed stress. Lastly, I used comparative transcriptomics to examine responses to imposed heat-stress in heat-tolerant and heat-sensitive Brachionus species. I found considerably different patterns of gene expression in the two species. Most striking are patterns of expression regarding the heat shock proteins (hsps) between the two species. In the heat-tolerant, B. calyciflorus s.s., significant up-regulation of hsps at low temperatures was indicative of a stress response at the cooler end of the temperature regimes tested here. In contrast, in the heat-sensitive B. fernandoi, hsps generally exhibited up-regulation of these genes along with rising temperatures. Overall, identification of differences in expression of genes suggests suppression of protein biosynthesis to be a mechanism to increase thermal tolerance. Observed patterns in population growth are correlated with the hsp gene expression differences, indicating that this physiological stress response is indeed related to phenotypic life history performance.
Analysis of supramolecular assemblies of NE81, the first lamin protein in a non-metazoan organism
(2019)
Nuclear lamins are nucleus-specific intermediate filaments forming a network located at the inner nuclear membrane of the nuclear envelope. They form the nuclear lamina together with proteins of the inner nuclear membrane regulating nuclear shape and gene expression, among others. The amoebozoan Dictyostelium NE81 protein is a suitable candidate for an evolutionary conserved lamin protein in this non-metazoan organism. It shares the domain organization of metazoan lamins and is fulfilling major lamin functions in Dictyostelium. Moreover, field-emission scanning electron microscopy (feSEM) images of NE81 expressed on Xenopus oocytes nuclei revealed filamentous structures with an overall appearance highly reminiscent to that of metazoan Xenopus lamin B2. For the classification as a lamin-like or a bona fide lamin protein, a better understanding of the supramolecular NE81 structure was necessary. Yet, NE81 carrying a large N-terminal GFP-tag turned out as unsuitable source for protein isolation and characterization; GFP-NE81 expressed in Dictyostelium NE81 knock-out cells exhibited an abnormal distribution, which is an indicator for an inaccurate assembly of GFP-tagged NE81. Hence, a shorter 8×HisMyc construct was the tag of choice to investi-gate formation and structure of NE81 assemblies. One strategy was the structural analysis of NE81 in situ at the outer nuclear membrane in Dictyostelium cells; NE81 without a func-tional nuclear localization signal (NLS) forms assemblies at the outer face of the nucleus. Ultrastructural feSEM pictures of NE81ΔNLS nuclei showed a few filaments of the expected size but no repetitive filamentous structures. The former strategy should also be established for metazoan lamins in order to facilitate their structural analysis. However, heterologously expressed Xenopus and C. elegans lamins showed no uniform localization at the outer nucle-ar envelope of Dictyostelium and hence, no further ultrastructural analysis was undertaken. For in vitro assembly experiments a Dictyostelium mutant was generated, expressing NE81 without the NLS and the membrane-anchoring isoprenylation site (HisMyc-NE81ΔNLSΔCLIM). The cytosolic NE81 clusters were soluble at high ionic strength and were purified from Dictyostelium extracts using Ni-NTA Agarose. Widefield immunofluorescence microscopy, super-resolution light microscopy and electron microscopy images of purified NE81 showed its capability to form filamentous structures at low ionic strength, as described previously for metazoan lamins. Introduction of a phosphomimetic point mutation (S122E) into the CDK1-consensus sequence of NE81 led to disassembled NE81 protein in vivo, which could be reversibly stimulated to form supramolecular assemblies by blue light exposure.
The results of this work reveal that NE81 has to be considered a bona fide lamin, since it is able to form filamentous assemblies. Furthermore, they highlight Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope containing all rele-vant protein components known in animal cells.
ATP-binding cassette (ABC) transporters are present in all kingdoms of life and enable active transport of various different molecules across biological membranes. They all share an overall architecture of two lipophilic transmembrane spanning domains (TMDs) traversing the membrane and two hydrophilic nucleotide binding domains (NBDs) usually lacking sequence identity. The multiplicity in transported molecules is accompanied by extreme diversity in TMDs. Human mitochondria harbor four ABC transporters, namely ABCB6, ABCB7, ABCB8 and ABCB10 with functional homologues in yeast and plants. Except the ones found in Rickettsiae and related bacteria mitochondrial ABC transporters are absent in bacteria. In addition to converting energy mitochondria are important platforms for biosynthesizing various cofactors as iron sulfur clusters, molybdenum cofactor (Moco) or heme. ABCB7 (Atm1 in yeast) has been shown to connect mitochondrial with cytosolic iron sulfur cluster assembly by exporting a yet unknown sulfur containing molecule. In addition, TMDs of Atm1 display a glutathione binding pocket accessible from the matrix which has been identified in all ABCB7-like transporters and also exists in a bacterial ABC transporter homologue of Atm1 in Novosphingobium aromaticivorans. In addition, ATM3, a plant mitochondrial homologous ABC transporter to human ABCB7, has been associated with biosynthesizing Moco.
In this study we used the α-proteobacterium Rhodobacter capsulatus as a model organism to characterize mitochondrial ABC transporter homologues. R. capsulatus contains two homologues to mitochondrial ABC transporters with the corresponding gene loci rcc03139 and rcc02305. They share 38 to 47 % sequence identities to human mitochondrial ABC transporters ABCB8/ABCB10 and ABCB7/ABCB6, respectively. We created interposon mutants lacking either rcc03139 or rcc02305, analyzed the physiological effects on R. capsulatus and compared the findings especially to eukaryotic deletion studies. A viable bacterial double mutant strain lacking both mitochondrial ABC transporters was constructed to investigate possible overlapping functions. Both R. capsulatus single mutants showed a severe accumulation of intracellular reactive oxygen species (ROS) in comparison to ∆nifDK which revealed to be additive in the double mutant. In the proteome of ∆rcc03139I abundancies of tetrapyrrole related proteins were significantly increased in comparison to the proteome of parental strain, which was further validated by reduced amounts of tetrapyrrole intermediates in ∆rcc03139. In contrast, in ∆rcc02305I total glutathione (GSH) was elevated when endogenous GSH biosynthesis was inhibited. In conjunction with proteomic studies we uncovered misbalanced sulfur distribution in ∆rcc02305I. Furthermore, strains lacking Rcc02305 accumulated cyclic pyranopterin monophosphate (cPMP), an intermediate of Moco biosynthesis, as it was already shown for the deletion strain of the eukaryotic counterpart ATM3 in plants. In contrast single mutant strain Δrcc03139I neither accumulated cPMP nor glutathione.
Bioinformatic analysis of the amino acid sequence of Rcc02305 revealed a pyridoxal 5´phosphate (PLP) binding site which overlaps with Walker A within the NBDs of Rcc02305 and other ABCB7-like transporters. The PLP cofactor is well studied in C-DES (L-cysteine/cystine lyase from Synechocystis) for persulfide production and in L-cysteine desulfurases such as IscS and NFS1 for its role in formation of protein-bound persulfides. Based on our findings we are able to propose a new modality for the transport of the sulfur containing molecule: first of all, the transporter produces a highly reactive persulfide which is then subsequently trapped by glutathione polysulfide, already bound within the binding pocket in TMDs. Walker A becomes accessible for ATP and after hydrolysis the mixed polysulfide is released.
Based on our studies we are convinced that both mitochondrial ABC transporter homologues fulfil distinct roles in R. capsulatus: Rcc02305 is a representative of Atm1/ABCB7-like transporters and important for proper sulfur distribution by exporting persulfides. In contrast Rcc03139 is a representative of ABCB6/ABCB10 related transporters and involved in biosynthesizing tetrapyrroles.
Im Mittelpunkt dieser Arbeit standen Analysen zur Charakterisierung der periplasmatischen Aldehyd Oxidoreduktase aus E. coli. Kinetische Untersuchungen mit Ferricyanid als Elektronenakzeptor unter anaeroben Bedingungen zeigten für dieses Enzym eine höhere Aktivität als unter aeroben Bedingungen. Die getroffene Hypothese, dass PaoABC fähig ist Elektronen an molekularen Sauerstoff weiter zu geben, konnte bestätigt werden. Für den Umsatz aromatischer Aldehyde mit molekularem Sauerstoff wurde ein Optimum von pH 6,0 ermittelt. Dies steht im Gegensatz zur Reaktion mit Ferricyanid, mit welchem ein pH-Optimum von 4,0 gezeigt wurde. Die Reaktion von PaoABC mit molekularem Sauerstoff generiert zwar Wasserstoffperoxid, die Produktion von Superoxid konnte dagegen nicht beobachtet werden. Dass aerobe Bedingungen einen Einfluss auf das Auslösen der Expression von PaoABC haben, wurde in dieser Arbeit ebenfalls ermittelt.
Im Zusammenhang mit der Produktion von ROS durch PaoABC wurde die Funktion eines kürzlich in Elektronentransfer-Distanz zum FAD identifizierten [4Fe4S]-Clusters untersucht. Ein Austausch der für die Bindung des Clusters zuständigen Cysteine führte zur Instabilität der Proteinvarianten, weswegen für diese keine weiteren Untersuchungen erfolgten. Daher wird zumindest ein struktur-stabilisierender Einfluss des [4Fe4S]-Clusters angenommen. Zur weiteren Untersuchung der Funktion dieses Clusters, wurde ein zwischen FAD und [4Fe4S]-Cluster lokalisiertes Arginin gegen ein Alanin ausgetauscht. Diese Proteinvariante zeigte eine reduzierte Geschwindigkeit der Reaktion gegenüber dem Wildtyp. Die Bildung von Superoxid konnte auch hier nicht beobachtet werden. Die Vermutung, dass dieser Cluster einen elektronen-sammelnden Mechanismus unterstützt, welcher die Radikalbildung verhindert, kann trotz allem nicht ausgeschlossen werden. Da im Umkreis des Arginins weitere geladene und aromatische Aminosäuren lokalisiert sind, können diese den notwendigen Elektronentransfer übernehmen.
Neben der Ermittlung eines physiologischen Elektronenakzeptors und dessen Einfluss auf die Expression von PaoABC zeigt diese Arbeit auch, dass die Chaperone PaoD und MocA während der Reifung des MCD-Kofaktor eine gemeinsame Bindung an PaoABC realisieren. Es konnte im aktiven Zentrum von PaoABC ein Arginin beschrieben werden, welches auf Grund der engen Nachbarschaft zum MCD-Kofaktor und zum Glutamat (PaoABC-EC692) am Prozess der Substratbindung beteiligt ist. Im Zusammenhang mit dem Austausch dieses Arginins gegen ein Histidin oder ein Lysin wurden die Enzymspezifität und der Einfluss physiologischer Bedingungen, wie pH und Ionenstärke, auf die Reaktion des Enzyms untersucht. Gegenüber dem Wildtyp zeigten die Varianten mit molekularem Sauerstoff eine geringere Affinität zum Substrat aber auch eine höhere Geschwindigkeit der Reaktion. Vor allem für die Histidin-Variante konnte im gesamten pH-Bereich ein instabiles Verhalten bestimmt werden. Der Grund dafür wurde durch das Lösen der Struktur der Histidin-Variante beschreiben. Durch den Austausch der Aminosäuren entfällt die stabilisierende Wirkung der delokalisierten Elektronen des Arginins und es kommt zu einer Konformationsänderung im aktiven Zentrum.
Neben der Reaktion von PaoABC mit einer Vielzahl aromatischer Aldehyde konnte auch der Umsatz von Salicylaldehyd zu Salicylsäure durch PaoABC in einer Farbreaktion bestimmt werden. Durch Ausschluss von molekularem Sauerstoff als terminaler Elektronenakzeptor, in einer enzym-gekoppelten Reaktion, erfolgte ein Elektronentransport auf Ferrocencarboxylsäure. Die Kombination aus beiden Methoden ermöglichte eine Verwendung von Ferrocen-Derivaten zur Generierung einer enzym-gekoppelten Reaktion mit PaoABC.
Die Untersuchungen zu PaoABC zeigen, dass die Vielfalt der durch das Enzym katalysierten Rektionen weitere Möglichkeiten der enzymatischen Bestimmung biokatalytischer Prozesse bietet.
Force plays a fundamental role in the regulation of biological processes. Cells can sense the mechanical properties of the extracellular matrix (ECM) by applying forces and transmitting mechanical signals. They further use mechanical information for regulating a wide range of cellular functions, including adhesion, migration, proliferation, as well as differentiation and apoptosis. Even though it is well understood that mechanical signals play a crucial role in directing cell fate, surprisingly little is known about the range of forces that define cell-ECM interactions at the molecular level.
Recently, synthetic molecular force sensor (MFS) designs have been established for measuring the molecular forces acting at the cell-ECM interface. MFSs detect the traction forces generated by cells and convert this mechanical input into an optical readout. They are composed of calibrated mechanoresponsive building blocks and are usually equipped with a fluorescence reporter system. Up to date, many different MFS designs have been introduced and successfully used for measuring forces involved in the adhesion of mammalian cells. These MFSs utilize different molecular building blocks, such as double-stranded deoxyribonucleic acid (dsDNA) molecules, DNA hairpins and synthetic polymers like polyethylene glycol (PEG). These currently available MFS designs lack ECM mimicking properties.
In this work, I introduce a new MFS building block for cell biology applications, derived from the natural ECM. It combines mechanical tunability with the ability to mimic the native cellular microenvironment. Inspired by structural ECM proteins with load bearing function, this new MFS design utilizes coiled coil (CC)-forming peptides. CCs are involved in structural and mechanical tasks in the cellular microenvironment and many of the key protein components of the cytoskeleton and the ECM contain CC structures. The well-known folding motif of CC structures, an easy synthesis via solid phase methods and the many roles CCs play in biological processes have inspired studies to use CCs as tunable model systems for protein design and assembly. All these properties make CCs ideal candidates as building blocks for MFSs. In this work, a series of heterodimeric CCs were designed, characterized and further used as molecular building blocks for establishing a novel, next-generation MFS prototype.
A mechanistic molecular understanding of their structural response to mechanical load is essential for revealing the sequence-structure-mechanics relationships of CCs. Here, synthetic heterodimeric CCs of different length were loaded in shear geometry and their mechanical response was investigated using a combination of atomic force microscope (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations. SMFS showed that the rupture forces of short heterodimeric CCs (3-5 heptads) lie in the range of 20-50 pN, depending on CC length, pulling geometry and the applied loading rate (dF/dt). Upon shearing, an initial rise in the force, followed by a force plateau and ultimately strand separation was observed in SMD simulations. A detailed structural analysis revealed that CC response to shear load depends on the loading rate and involves helix uncoiling, uncoiling-assisted sliding in the direction of the applied force and uncoiling-assisted dissociation perpendicular to the force axis.
The application potential of these mechanically characterized CCs as building blocks for MFSs has been tested in 2D cell culture applications with the goal of determining the threshold force for cell adhesion. Fully calibrated, 4- to 5-heptad long, CC motifs (CC-A4B4 and CC-A5B5) were used for functionalizing glass surfaces with MFSs. 3T3 fibroblasts and endothelial cells carrying mutations in a signaling pathway linked to cell adhesion and mechanotransduction processes were used as model systems for time-dependent adhesion experiments. A5B5-MFS efficiently supported cell attachment to the functionalized surfaces for both cell types, while A4B4-MFS failed to maintain attachment of 3T3 fibroblasts after the first 2 hours of initial cell adhesion. This difference in cell adhesion behavior demonstrates that the magnitude of cell-ECM forces varies depending on the cell type and further supports the application potential of CCs as mechanoresponsive and tunable molecular building blocks for the development of next-generation protein-based MFSs.This novel CC-based MFS design is expected to provide a powerful new tool for observing cellular mechanosensing processes at the molecular level and to deliver new insights into the mechanisms and forces involved. This MFS design, utilizing mechanically tunable CC building blocks, will not only allow for measuring the molecular forces acting at the cell-ECM interface, but also yield a new platform for the development of mechanically controlled materials for a large number of biological and medical applications.
The facilitation of species coexistence has been a central theme in ecological research for years, highlighting two key aspects: ecological niches and competition between species. According to the competitive exclusion principle, the overlap of species niches predicts the amount of shared resources and therefore competition between species, determining their ability to coexist. Only if niches of two species are sufficiently different, thus niche overlap is low, competition within species is higher than competition between species and stable coexistence is possible. Thereby, differences in species mean traits are focused on and conspecific individuals are assumed to be interchangeable. This approach might be outdated since behaviour, as a key aspect mediating niche differentiation between species, is individual based. Individuals from one species consistently differ across time and situations in their behavioural traits. Causes and consequences of consistent behavioural differences have been thoroughly investigated stimulating their recent incorporation into ecological interactions and niche theory. Spatial components have so far been largely overlooked, although animal movement is strongly connected to several aspects of ecological niches and interactions between individuals. Furthermore, numerous movement aspects haven been proven to be crucially influenced by consistent individual differences. Considering spatial parameters could therefore crucially broaden our understanding of how individual niches are formed and ecological interactions are shaped. Furthermore, extending established concepts on species interactions by an individual component could provide new insights into how species coexistence is facilitated and local biodiversity is maintained.
The main aim of this thesis was to test whether consistent inter-individual differences can facilitate the coexistence of ecological similar species. Therefore, the effects of consistent inter-individual differences on the spatial behaviour of two rodent species, the bank vole (Myodes glareolus) and the striped field mouse (Apodemus agrarius), were investigated and put in the context of: (i) individual spatial niches, (ii) interactions between species, and (iii) the importance of different levels of behavioural variation within species for their interactions. Consistent differences of study animals in boldness and exploration were quantified with the same tests in all presented studies and always combined with observations of movement and space use via automated VHF radio telemetry. Consequently, results are comparable throughout the thesis and the methods provide a common denominator for all chapters. The first two chapters are based on observations of free-ranging rodents in natural populations, while chapter III represents an experimental approach under semi-natural conditions.
Chapter I focusses on the effect of consistent differences in boldness and exploration on movement and space use of bank voles and their contribution to individual spatial niche separation. Results show boldness to be the dominating predictor for spatial parameters in bank voles. Irrespective of sex, bolder individuals had larger home ranges, moved longer distances, had less spatial interactions with conspecifics and occupied different microhabitats compared to shy individuals. The same boldness-dependent spatial patterns could be observed in striped field mice which is reported in chapter II. Therefore, both study species showed individual spatial niche occupation.
Chapter II builds on findings from the first chapter, investigating the effect of boldness driven individual spatial niche occupation on the interactions between species. Irrespective of species and sex, bolder individuals had more interspecific spatial interactions, but less intraspecific interactions, compared to shy individuals. Due to individual niches occupation the competitive environment individuals experience is not random. Interactions are restricted to individuals of similar behavioural type with presumably similar competitive ability, which could balance differences on the species level and support coexistence.
In chapter III the experimental populations were either comprised of only shy or only bold bank voles, while striped field mice varied, creating either a shy- or bold-biased competitive community. Irrespective of behavioural type, striped field mice had more intraspecific interactions in bold-biased competitive communities. Only in a shy-biased competitive community, bolder striped field mice had less interspecific interactions compared to shy individuals. Bank voles showed no difference in intra- or interspecific interactions between populations. Chapter III highlights, that not only consistent inter-individual differences per se are important for interactions within and between species, but also the amount of behavioural variation within coexisting species.
Overall, this thesis highlights the importance of considering consistent inter-individual differences in a spatial context and their connection to individual spatial niche occupation, as well as the resulting effects on interactions within and between species. Individual differences are discussed in the context of similarity of individuals, individual and species niche width, and individual and species niche overlap. Thereby, this thesis makes one step further from the existing research on individual niches towards integrating consistent inter-individual differences into the larger framework of species coexistence.
Since half a century, cytometry has been a major scientific discipline in the field of cytomics - the study of system’s biology at single cell level. It enables the investigation of physiological processes, functional characteristics and rare events with proteins by analysing multiple parameters on an individual cell basis. In the last decade, mass cytometry has been established which increased the parallel measurement to up to 50 proteins. This has shifted the analysis strategy from conventional consecutive manual gates towards multi-dimensional data processing. Novel algorithms have been developed to tackle these high-dimensional protein combinations in the data. They are mainly based on clustering or non-linear dimension reduction techniques, or both, often combined with an upstream downsampling procedure. However, these tools have obstacles either in comprehensible interpretability, reproducibility, computational complexity or in comparability between samples and groups.
To address this bottleneck, a reproducible, semi-automated cytometric data mining workflow PRI (pattern recognition of immune cells) is proposed which combines three main steps: i) data preparation and storage; ii) bin-based combinatorial variable engineering of three protein markers, the so called triploTs, and subsequent sectioning of these triploTs in four parts; and iii) deployment of a data-driven supervised learning algorithm, the cross-validated elastic-net regularized logistic regression, with these triploT sections as input variables. As a result, the selected variables from the models are ranked by their prevalence, which potentially have discriminative value. The purpose is to significantly facilitate the identification of meaningful subpopulations, which are most distinguish between two groups. The proposed workflow PRI is exemplified by a recently published public mass cytometry data set. The authors found a T cell subpopulation which is discriminative between effective and ineffective treatment of breast carcinomas in mice. With PRI, that subpopulation was not only validated, but was further narrowed down as a particular Th1 cell population. Moreover, additional insights of combinatorial protein expressions are revealed in a traceable manner. An essential element in the workflow is the reproducible variable engineering. These variables serve as basis for a clearly interpretable visualization, for a structured variable exploration and as input layers in neural network constructs.
PRI facilitates the determination of marker levels in a semi-continuous manner. Jointly with the combinatorial display, it allows a straightforward observation of correlating patterns, and thus, the dominant expressed markers and cell hierarchies. Furthermore, it enables the identification and complex characterization of discriminating subpopulations due to its reproducible and pseudo-multi-parametric pattern presentation. This endorses its applicability as a tool for unbiased investigations on cell subsets within multi-dimensional cytometric data sets.
Determining the relationship between genotype and phenotype is the key to understand the plasticity and robustness of phenotypes in nature. While the directly observable plant phenotypes (e.g. agronomic, yield and stress resistance traits) have been well-investigated, there is still a lack in our knowledge about the genetic basis of intermediate phenotypes, such as metabolic phenotypes. Dissecting the links between genotype and phenotype depends on suitable statistical models. The state-of-the-art models are developed for directly observable phenotypes, regardless the characteristics of intermediate phenotypes. This thesis aims to fill the gaps in understanding genetic architecture of intermediate phenotypes, and how they tie to composite traits, namely plant growth. The metabolite levels and reaction fluxes, as two aspects of metabolic phenotypes, are shaped by the interrelated chemical reactions formed in genome-scale metabolic network. Here, I attempt to answer the question: Can the knowledge of underlying genome-scale metabolic network improve the model performance for prediction of metabolic phenotypes and associated plant growth? To this end, two projects are investigated in this thesis. Firstly, we propose an approach that couples genomic selection with genome-scale metabolic network and metabolic profiles in Arabidopsis thaliana to predict growth. This project is the first integration of genomic data with fluxes predicted based on constraint-based modeling framework and data on biomass composition. We demonstrate that our approach leads to a considerable increase of prediction accuracy in comparison to the state-of-the-art methods in both within and across environment predictions. Therefore, our work paves the way for combining knowledge on metabolic mechanisms in the statistical approach underlying genomic selection to increase the efficiency of future plant breeding approaches. Secondly, we investigate how reliable is genomic selection for metabolite levels, and which single nucleotide polymorphisms (SNPs), obtained from different neighborhoods of a given metabolic network, contribute most to the accuracy of prediction. The results show that the local structure of first and second neighborhoods are not sufficient for predicting the genetic basis of metabolite levels in Zea mays. Furthermore, we find that the enzymatic SNPs can capture most the genetic variance and the contribution of non-enzymatic SNPs is in fact small. To comprehensively understand the genetic architecture of metabolic phenotypes, I extend my study to a local Arabidopsis thaliana population and their hybrids. We analyze the genetic architecture in primary and secondary metabolism as well as in growth. In comparison to primary metabolites, compounds from secondary metabolism were more variable and show more non-additive inheritance patterns which could be attributed to epistasis. Therefore, our study demonstrates that heterozygosity in local Arabidopsis thaliana population generates metabolic variation and may impact several tasks directly linked to metabolism. The studies in this thesis improve the knowledge of genetic architecture of metabolic phenotypes in both inbreed and hybrid population. The approaches I proposed to integrate genome-scale metabolic network with genomic data provide the opportunity to obtain mechanistic insights about the determinants of agronomically important polygenic traits.
A contemporary challenge in Ecology and Evolutionary Biology is to anticipate the fate of populations of organisms in the context of a changing world. Climate change and landscape changes due to anthropic activities have been of major concern in the contemporary history. Organisms facing these threats are expected to respond by local adaptation (i.e., genetic changes or phenotypic plasticity) or by shifting their distributional range (migration). However, there are limits to their responses. For example, isolated populations will have more difficulties in developing adaptive innovations by means of genetic changes than interconnected metapopulations. Similarly, the topography of the environment can limit dispersal opportunities for crawling organisms as compared to those that rely on wind. Thus, populations of species with different life history strategy may differ in their ability to cope with changing environmental conditions. However, depending on the taxon, empirical studies investigating organisms’ responses to environmental change may become too complex, long and expensive; plus, complications arising from dealing with endangered species. In consequence, eco-evolutionary modeling offers an opportunity to overcome these limitations and complement empirical studies, understand the action and limitations of underlying mechanisms, and project into possible future scenarios. In this work I take a modeling approach and investigate the effect and relative importance of evolutionary mechanisms (including phenotypic plasticity) on the ability for local adaptation of populations with different life strategy experiencing climate change scenarios. For this, I performed a review on the state of the art of eco-evolutionary Individual-Based Models (IBMs) and identify gaps for future research. Then, I used the results from the review to develop an eco-evolutionary individual-based modeling tool to study the role of genetic and plastic mechanisms in promoting local adaption of populations of organisms with different life strategies experiencing scenarios of climate change and environmental stochasticity. The environment was simulated through a climate variable (e.g., temperature) defining a phenotypic optimum moving at a given rate of change. The rate of change was changed to simulate different scenarios of climate change (no change, slow, medium, rapid climate change). Several scenarios of stochastic noise color resembling different climatic conditions were explored. Results show that populations of sexual species will rely mainly on standing genetic variation and phenotypic plasticity for local adaptation. Population of species with relatively slow growth rate (e.g., large mammals) – especially those of small size – are the most vulnerable, particularly if their plasticity is limited (i.e., specialist species). In addition, whenever organisms from these populations are capable of adaptive plasticity, they can buffer fitness losses in reddish climatic conditions. Likewise, whenever they can adjust their plastic response (e.g., bed-hedging strategy) they will cope with bluish environmental conditions as well. In contrast, life strategies of high fecundity can rely on non-adaptive plasticity for their local adaptation to novel environmental conditions, unless the rate of change is too rapid. A recommended management measure is to guarantee interconnection of isolated populations into metapopulations, such that the supply of useful genetic variation can be increased, and, at the same time, provide them with movement opportunities to follow their preferred niche, when local adaptation becomes problematic. This is particularly important for bluish and reddish climatic conditions, when the rate of change is slow, or for any climatic condition when the level of stress (rate of change) is relatively high.
Electrosynthesis and characterization of molecularly imprinted polymers for peptides and proteins
(2019)
Increasing concerns regarding the environmental impact of our chemical production have shifted attention towards possibilities for sustainable biotechnology. One-carbon (C1) compounds, including methane, methanol, formate and CO, are promising feedstocks for future bioindustry. CO2 is another interesting feedstock, as it can also be transformed using renewable energy to other C1 feedstocks for use. While formaldehyde is not suitable as a feedstock due to its high toxicity, it is a central intermediate in the process of C1 assimilation. This thesis explores formaldehyde metabolism and aims to engineer formaldehyde assimilation in the model organism Escherichia coli for the future C1-based bioindustry.
The first chapter of the thesis aims to establish growth of E. coli on formaldehyde via the most efficient naturally occurring route, the ribulose monophosphate pathway. Linear variants of the pathway were constructed in multiple-gene knockouts strains, coupling E. coli growth to the activities of the key enzymes of the pathway. Formaldehyde-dependent growth was achieved in rationally designed strains. In the final strain, the synthetic pathway provides the cell with almost all biomass and energy requirements.
In the second chapter, taking advantage of the unique feature of its reactivity, formaldehyde assimilation via condensation with glycine and pyruvate by two promiscuous aldolases was explored. Facilitated by these two reactions, the newly designed homoserine cycle is expected to support higher yields of a wide array of products than its counterparts. By dividing the pathway into segments and coupling them to the growth of dedicated strains, all pathway reactions were demonstrated to be sufficiently active. The work paves a way for future implementation of a highly efficient route for C1 feedstocks into commodity chemicals.
In the third chapter, the in vivo rate of the spontaneous formaldehyde tetrahydrofolate condensation to methylene-tetrahydrofolate was assessed in order to evaluate its applicability as a biotechnological process. Tested within an E. coli strain deleted in essential genes for native methylene-tetrahydrofolate biosynthesis, the reaction was shown to support the production of this essential intermediate. However, only low growth rates were observed and only at high formaldehyde concentrations. Computational analysis dependent on in vivo evidence from this strain deduced the slow rate of this spontaneous reaction, thus ruling out its substantial contribution to growth on C1 feedstocks.
The reactivity of formaldehyde makes it highly toxic. In the last chapter, the formation of thioproline, the condensation product of cysteine and formaldehyde, was confirmed to contribute this toxicity effect. Xaa-Pro aminopeptidase (PepP), which genetically links with folate metabolism, was shown to hydrolyze thioproline-containing peptides. Deleting pepP increased strain sensitivity to formaldehyde, pointing towards the toxicity of thioproline-containing peptides and the importance of their removal. The characterization in this study could be useful in handling this toxic intermediate.
Overall, this thesis identified challenges related to formaldehyde metabolism and provided novel solutions towards a future bioindustry based on sustainable C1 feedstocks in which formaldehyde serves as a key intermediate.