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Non-ribosomal peptide synthetase docking domains : structure, function and engineering strategies
(2021)
Non-ribosomal peptide synthetases (NRPSs) are known for their capability to produce a wide range of natural compounds and some of them possess interesting bioactivities relevant for clinical application like antibiotics, anticancer, and immunosuppressive drugs. The diverse bioactivity of non-ribosomal peptides (NRPs) originates from their structural diversity, which results not only from the incorporation of non-proteinogenic amino acids into the growing peptide chain, but also the formation of heterocycles or further peptide modifications like methylation, hydroxylation and acetylation.
The biosynthesis of NRPs is achieved via the orchestrated interplay of distinct catalytic domains, which are grouped to modules that are located on one or more polypeptide chains. Each cycle starts with the selection and activation of a specific amino acid by the adenylation (A) domain, which catalyzes the aminoacyl adenylate formation under ATP consumption. This activated amino acid is then bound via a thioester bond to the 4’-phosphopantetheine cofactor (PPant-arm) of the following thiolation (T) domain. Before substrate loading, the PPant-arm is post-translationally added to the T domain by a phosphopantetheinyl transferase (PPTase), which converts the inactive apo-T domain in its active holo-form. In the last step of the catalytic cycle, two T domain bound peptide building blocks are connected by the condensation (C) domain, resulting in peptide bond formation and transfer of the nascent peptide chain to the following module. Each catalytic cycle is performed by a C-A-T elongation module until the termination module with a C-terminal thioesterase (TE) domain is reached. Here, the peptide product is released by hydrolysis or intramolecular cyclisation.
In comparison to single-protein NRPSs, where all modules are encoded on a single polypeptide chain, multi-protein NRPS systems must also maintain a specific module order during the peptide biosynthesis. Therefore, small C-terminal and N-terminal communication-mediating (COM) domains/docking domains (DD) were identified in the C- and N-terminal regions of multi-protein NRPSs. It was shown that these domains mediate specific and selective non-covalent protein-protein interaction, even though DD interactions are generally characterized by low affinities.
The first publication of this work focuses on the Peptide-Antimicrobial-Xenorhabdus peptide-producing NRPS called PaxS, which consists of the three proteins PaxA, PaxB and PaxC. Here, in particular the trans DD interface between the C-terminal attached DD of PaxB and N-terminal attached DD of PaxC was structurally investigated and thermodynamically characterized by isothermal titration calorimetry (ITC), yielding a dissociation constant (KD) of ~25 µM, which is a DD typical affinity known from further characterized DD pairs. The artificial linking of the PaxB/C C/NDD pair via a glycine-serine (GS) linker facilitated the structure determination of the DD complex by solution nuclear magnetic resonance (NMR) spectroscopy. In comparison to known docking domain structures, this DD complex assembles in a completely new fold which is characterized by a central α-helix of PaxC NDD wrapped in two V-shaped α-helices of PaxB CDD.
The first manuscript of this work focuses on the application of synthetic zippers (SZ) to mimic natural docking domains, enabling the easy assembly of NRPS building blocks encoded on different plasmids in a functional way. Here, the high-affinity interaction of SZs unambiguously defines the order of the synthetases derived from single-protein NRPSs in the engineered NRPS system and allows the recombination in a plug-and-play manner. Notably, the SZ engineering strategy even facilitates the functional assembly of NRPSs derived from Gram-positive and Gram-negative bacteria. Furthermore, the functional incorporation of SZs into NRPS modules is not limited to a specific linker region, so we could introduce them within all native NRPS linker regions (A-T, T-C, C-A).
The second publication and the second manuscript of this thesis again focus on the multi-protein PaxS, in particular on the trans interface between the proteins PaxA and PaxB on a molecular level by solution NMR. Therefore, the PaxA CDD adjacent T domain was included into the structural investigation besides the native interaction partner PaxB NDD. Before a three-dimensional structure could be obtained from NMR data, the NH groups located in the peptide bonds had to be assigned to the respective amino acids of the proteins (backbone assignment). Based on these backbone assignments, the secondary structure of PaxA T1-CDD and PaxB NDD in the absence and presence of the respective interaction partner were predicted.
The structural and functional characterization of the PaxA T1-CDD:PaxB NDD complex is summarized in manuscript two. The thermodynamic analysis of this complex by ITC determined a KD value of ~250 nM, whereas the discrete DDs did not interact at all. The high-affinity interaction allowed to determine the solution NMR structure of the PaxA T1-CDD:PaxB NDD complex without the covalent linkage of the interaction partners and an extended docking domain interface could be determined. This interface comprises on the one hand α-helix 4 of the PaxA T1 domain together with the α-helical CDD, and on the other hand the PaxB NDD, which is composed of two α-helices separated by a sharp bend.
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Most cellular processes are regulated by RNA-binding proteins (RBPs). These RBPs usually use defined binding sites to recognize and directly interact with their target RNA molecule. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments are an important tool to de- scribe such interactions in cell cultures in-vivo. This experimental protocol yields millions of individual sequencing reads from which the binding spec- trum of the RBP under study can be deduced. In this PhD thesis I studied how RNA processing is driven from RBP binding by analyzing iCLIP-derived sequencing datasets.
First, I described a complete data analysis pipeline to detect RBP binding sites from iCLIP sequencing reads. This workflow covers all essential process- ing steps, from the first quality control to the final annotation of binding sites. I described the accurate integration of biological iCLIP replicates to boost the initial peak calling step while ensuring high specificity through replicate re- producibility analysis. Further I proposed a routine to level binding site width to streamline downstream analysis processes. This was exemplified in the re- analysis of the binding spectrum of the U2 small nuclear RNA auxiliary factor 2 (U2AF2, U2AF65). I recaptured the known dominance of U2AF65 to bind to intronic sequences of protein-coding genes, where it likely recognizes the polypyrimidine tract as part of the core spliceosome machinery.
In the second part of my thesis, I analyzed the binding spectrum of the serine and arginine rich splicing factor 6 (SRSF6) in the context of diabetes. In pancreatic beta-cells, the expression of SRSF6 is regulated by the transcription factor GLIS3, which encodes for a diabetes susceptibility gene. It is known that SRSF6 promotes beta-cell death through the splicing dysregulation of genes essential to beta-cell function and survival. However, the exact mechanism of how these RNAs are targeted by SRSF6 remains poorly understood. Here, I applied the defined iCLIP processing pipeline to describe the binding landscape of the splicing factor SRSF6 in the human pancreatic beta-cell line EndoC-H1. The initial binding sites definition revealed a predominant binding to coding sequences (CDS) of protein-coding genes. This was followed up by extensive motif analysis which revealed a so far, in human, unknown purine-rich binding motif. SRSF6 seemed to specifically recognize repetitions of the triplet GAA. I also showed that the number of contiguous triplets correlated with increasing binding site strength. I further integrated RNA-sequencing data from the same cell type, with SRSF6 in KD and in basal conditions, to analyze SRSF6- related splicing changes. I showed that the exact positioning of SRSF6 on alternatively spliced exons regulates the produced transcript isoforms. This mechanism seemed to control exons in several known susceptibility genes for diabetes.
In summary, in my PhD thesis, I presented a comprehensive workflow for the processing of iCLIP-derived sequencing data. I applied this pipeline on a dataset from pancreatic beta-cells to unveil the impact of SRSF6-mediated splicing changes. Thus, my analysis provides novel insights into the regulation of diabetes susceptibility genes.
In times of global climate change and the fear of dwindling resources, we are facing different considerable challenges such as the replacement of fossil fuel–based energy carriers with the coincident maintenance of the increasing energy supply of our growing world population. Therefore, CO2 capturing and H2 storing solutions are urgently needed. In this study, we demonstrate the production of a functional and biotechnological interesting enzyme complex from acetogenic bacteria, the hydrogen-dependent CO2 reductase (HDCR), in the well-known model organism Escherichia coli. We identified the metabolic bottlenecks of the host organisms for the production of the HDCR enzyme complex. Here we show that the recombinant expression of a heterologous enzyme complex transforms E. coli into a whole-cell biocatalyst for hydrogen-driven CO2 reduction to formate without the need of any external co-factors or endogenous enzymes in the reaction process. This shifts the industrial platform organism E. coli more and more into the focus as biocatalyst for CO2-capturing and H2-storage. Key points: A functional HDCR enzyme complex was heterologously produced in E. coli; The metabolic bottlenecks for HDCR production were identified; HDCR enabled E. coli cell to capture and store H2 and CO2 in the form of formate.
Macrophages not only represent an integral part of innate immunity but also critically contribute to tissue and organ homeostasis. Moreover, disease progression is accompanied by macrophage accumulation in many cancer types and is often associated with poor prognosis and therapy resistance. Given their critical role in modulating tumor immunity in primary and metastatic brain cancers, macrophages are emerging as promising therapeutic targets. Different types of macrophages infiltrate brain cancers, including (i) CNS resident macrophages that comprise microglia (TAM-MG) as well as border-associated macrophages and (ii) monocyte-derived macrophages (TAM-MDM) that are recruited from the periphery. Controversy remained about their disease-associated functions since classical approaches did not reliably distinguish between macrophage subpopulations. Recent conceptual and technological advances, such as large-scale omic approaches, provided new insight into molecular profiles of TAMs based on their cellular origin. In this review, we summarize insight from recent studies highlighting similarities and differences of TAM-MG and TAM-MDM at the molecular level. We will focus on data obtained from RNA sequencing and mass cytometry approaches. Together, this knowledge significantly contributes to our understanding of transcriptional and translational programs that define disease-associated TAM functions. Cross-species meta-analyses will further help to evaluate the translational significance of preclinical findings as part of the effort to identify candidates for macrophage-targeted therapy against brain metastasis.
Ribosome assembly is an essential and carefully choreographed cellular process. In eukaryotes, several 100 proteins, distributed across the nucleolus, nucleus, and cytoplasm, co-ordinate the step-wise assembly of four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins (RPs) into the mature ribosomal subunits. Due to the inherent complexity of the assembly process, functional studies identifying ribosome biogenesis factors and, more importantly, their precise functions and interplay are confined to a few and very well-established model organisms. Although best characterized in yeast (Saccharomyces cerevisiae), emerging links to disease and the discovery of additional layers of regulation have recently encouraged deeper analysis of the pathway in human cells. In archaea, ribosome biogenesis is less well-understood. However, their simpler sub-cellular structure should allow a less elaborated assembly procedure, potentially providing insights into the functional essentials of ribosome biogenesis that evolved long before the diversification of archaea and eukaryotes. Here, we use a comprehensive phylogenetic profiling setup, integrating targeted ortholog searches with automated scoring of protein domain architecture similarities and an assessment of when search sensitivity becomes limiting, to trace 301 curated eukaryotic ribosome biogenesis factors across 982 taxa spanning the tree of life and including 727 archaea. We show that both factor loss and lineage-specific modifications of factor function modulate ribosome biogenesis, and we highlight that limited sensitivity of the ortholog search can confound evolutionary conclusions. Projecting into the archaeal domain, we find that only few factors are consistently present across the analyzed taxa, and lineage-specific loss is common. While members of the Asgard group are not special with respect to their inventory of ribosome biogenesis factors (RBFs), they unite the highest number of orthologs to eukaryotic RBFs in one taxon. Using large ribosomal subunit maturation as an example, we demonstrate that archaea pursue a simplified version of the corresponding steps in eukaryotes. Much of the complexity of this process evolved on the eukaryotic lineage by the duplication of ribosomal proteins and their subsequent functional diversification into ribosome biogenesis factors. This highlights that studying ribosome biogenesis in archaea provides fundamental information also for understanding the process in eukaryotes.
In recent years, the popularity of rock-climbing has grown tremendously, setting an increasing pressure on cliff habitats. Climbing may be particularly harmful in the Mediterranean biome due to its appropriate environmental conditions for climbing. A few studies have identified the effect of climbing on plant diversity at a small-scale (namely locally or even just in specific climbing areas). However, no studies exist assessing the potential risk of rock-climbing on a broad-scale (e.g., regional or national). The study aims to identify the priority locations and priority cliff plant species in Spain to focus future study efforts. Spain was selected because it is a plant biodiversity hotspot, with a great diversity of endemic and endangered species, and one of the most popular destinations for climbers. We used a geographic information system-based approach to model the spatial concurrence among Spanish climbing areas (and climbing intensity), natural protected areas (NPAs), and distribution of threatened cliff plants (and their IUCN threat category). We found that 53.5% of climbing areas in Spain are located within a NPA, most of them falling into NPAs of medium protection level. We mapped 151 threatened cliff plants, identifying four medium priority Mediterranean locations and eight priority species in which future research efforts should be focused. High-priority study locations are absent in Spain according to our spatial modeling. For the first time on a national scale, this study identifies areas in which climbing represents a potential threat for cliff habitats and threatened plants. These findings contribute to designing field studies on the effects of rock-climbing on Mediterranean cliffs, laying the groundwork for a sustainable, yet challenging, balance between the protection of these unique habitats and rock-climbing.
Octanoic acid (C8 FA) is a medium-chain fatty acid which, in nature, mainly occurs in palm kernel oil and coconuts. It is used in various products including cleaning agents, cosmetics, pesticides and herbicides as well as in foods for preservation or flavoring. Furthermore, it is investigated for medical treatments, for instance, of high cholesterol levels. The cultivation of palm oil plants has surged in the last years to satisfy an increasing market demand. However, concerns about extensive monocultures, which often come along with deforestation of rainforest, have driven the search for more environmentally friendly production methods. A biotechnological production with microbial organisms presents an attractive, more sustainable alternative.
Traditionally, the yeast Saccharomyces cerevisiae has been utilized by mankind in bread, wine, and beer making. Based on comprehensive knowledge about its metabolism and genetics, it can nowadays be metabolically engineered to produce a plethora of compounds of industrial interest. To produce octanoic acid, the cytosolic fatty acid synthase (FAS) of S. cerevisiae was utilized and engineered. Naturally, the yeast produces mostly long-chain fatty acids with chain lengths of C16 and C18, and only trace amounts of medium-chain fatty acids, i.e. C8-C14 fatty acids. To generate an S. cerevisiae strain that produces primarily octanoic acid, a mutated version of the FAS was generated (Gajewski et al., 2017) and the resulting S. cerevisiae FASR1834K strain was utilized in this work as a starting strain.
The goal of this thesis was to develop and implement strategies to improve the production level of this strain. The current mode of quantification of octanoic acid includes labor-intensive, low-throughput sample preparation and measurement – a main obstacle in generating and screening for improved strain variants. To this end, a main objective of this thesis was the development of a biosensor. The biosensor was based on the pPDR12 promotor, which is regulated by the transcription factor War1. Coupling pPDR12 to GFP as the reporter gene on a multicopy plasmid allowed in vivo detection via fluorescence intensity. The developed biosensor enabled rapid and facile quantification of the short- and medium-chain fatty acids C6, C7 and C8 fatty acids (Baumann et al., 2018). This is the first biosensor that can quantify externally supplied octanoic acid as well as octanoic acid present in the culture supernatant of producer strains with a high linear and dynamic range. Its reliability was validated by correlation of the biosensor signal to the octanoic acid concentrations extracted from culture supernatants as determined by gas chromatography. The biosensor’s ability to detect octanoic acid in a linear range of 0.01-0.75 mM (≈1-110 mg/L), which is within the production range of the starting strain, and a response of up to 10-fold increase in fluorescence after activation was demonstrated.
A high-throughput FACS (fluorescence-activated cell sorting) screening of an octanoic acid producer strain library was performed with the biosensor to detect improved strain variants (Baumann et al., 2020a). For this purpose, the biosensor was genomically integrated into an octanoic acid producer strain, resulting in drastically reduced single cell noise. The additional knockout of FAA2 successfully prevented medium-chain fatty acid degradation. A high-throughput screening protocol was designed to include iterative enrichment rounds which decreased false positives. The functionality of the biosensor on single cell level was validated by adding octanoic acid in the range of 0-80 mg/L and subsequent flow cytometric analysis. The biosensor-assisted FACS screening of a plasmid overexpression library of the yeast genome led to the detection of two genetic targets, FSH2 and KCS1, that in combined overexpression enhanced octanoic acid titers by 55 % compared to the parental strain. This was the first report of an effect of FSH2 and KCS1 on fatty acid titers. The presented method can also be utilized to screen other genetic libraries and is a means to facilitate future engineering efforts.
In growth tests, the previously reported toxicity of octanoic acid on S. cerevisiae was confirmed. Different strategies were harnessed to create more robust strains. An adaptive laboratory evolution (ALE) experiment was conducted and several rational targets including transporter- (PDR12, TPO1) and transcription factor-encoding genes (PDR1, PDR3, WAR1) as well as the mutated acetyl-CoA carboxylase encoding gene ACC1S1157A were overexpressed or knocked out in producer or non-producer strains, respectively. Despite contrary previous reports for other strain backgrounds, an enhanced robustness was not observable. Suspecting that the utilized laboratory strains have a natively low tolerance level, four industrial S. cerevisiae strains were evaluated in growth assays with octanoic acid and inherently more robust strains were detected, which are suitable future production hosts.
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The magnetic field of the Earth provides animals with various kinds of information. Its use as a compass was discovered in the mid-1960s in birds, when it was first met with considerable skepticism, because it initially proved difficult to obtain evidence for magnetic sensitivity by conditioning experiments. Meanwhile, a magnetic compass was found to be widespread. It has now been demonstrated in members of all vertebrate classes, in mollusks and several arthropod species, in crustaceans as well as in insects. The use of the geomagnetic field as a ‘map’ for determining position, although already considered in the nineteenth century, was demonstrated by magnetically simulating displacements only after 2000, namely when animals, tested in the magnetic field of a distant site, responded as if they were physically displaced to that site and compensated for the displacement. Another use of the magnetic field is that as a ‘sign post’ or trigger: specific magnetic conditions elicit spontaneous responses that are helpful when animals reach the regions where these magnetic characteristics occur. Altogether, the geomagnetic field is a widely used valuable source of navigational information for mobile animals.
The oleochemical and petrochemical industries provide diverse chemicals used in personal care products, food and pharmaceutical industries or as fuels, oils, polymers and others. However, fossil resources are dwindling and concerns about these conventional production methods have risen due to their strong negative impact on the environment and contribution to climate change.
Therefore, alternative, sustainable and environmentally friendly production methods for oleochemical compounds such as fatty acids, fatty alcohols, hydroxy fatty acids and dicarboxylic acids are desired. The biotechnological production by engineered microorganism could fulfill these requirements. The concept of metabolic engineering, which is the modification of metabolic pathways of a host organism for increased production of a target compound, is a widely used strategy in biotechnology to generate cell factories or chassis strains for robust, efficient and high production. In this work, the versatile model and industrial yeast Saccharomyces cerevisiae was manipulated by metabolic engineering strategies for increased production of the medium-chain fatty acid octanoic acid and de novo production the derived 8-hydroxyoctanoic acid.
Octanoic acid production was enabled by the fatty acid biosynthesis pathway by use of a mutated fatty acid synthase (FASRK) in a wild type FAS deficient strain. The yeast fatty acid synthase (FAS) consists of two polypeptides, α and β, which assemble to a α6β6 complex in a co-translational manner by interaction of the subunits. Because this step might be subject to cellular regulation, the α- and β- subunits of fatty acid synthase were fused to form a single-chain construct (fusFASRK), which displayed superior octanoic acid production compared with split FASRK. Thus, FASRK expression was identified as a limiting step of octanoic acid production. But the strains that produce octanoic acid have a severe growth defect that is undesirable for biotechnological applications and could lead to lower production titers. One reason is the strong
inhibitory effect of octanoic acid. Another possibility is that the mutant FAS no longer produces enough essential long-chain fatty acids. To compensate for this, the mutated split and fused FAS variants were co-expressed individually in a strain harboring genomic wild type FAS alleles. In
addition, mutant and wild type variants of fused and split FAS were co-expressed together in a FAS deficient strain. However, both cases resulted in decreased octanoic acid titers potentially by physical and/or metabolic crosstalk of the FAS variants.
The fatty acid biosynthesis relies on cytosolic acetyl-CoA for initiation and derived malonyl-CoA for elongation and requires NADPH for reductive power. To increase production of octanoic acid, engineering strategies for increased acetyl-CoA and NADHP supply were investigated. First, the flux through the native cytosolic acetyl-CoA and NADPH providing pyruvate dehydrogenase bypass was enhanced by overexpression of the target genes ADH2, ALD6 and ACSL461P from Salmonella enterica in combination or individually. Next, the acety-CoA forming heterologous phosphoketolase/phosphotransacetylase pathway was expressed and NADPH formation was increased by redirecting the flux of glucose-6-phosphate into the NADPH producing oxidative branch of the pentose phosphate pathway. In particular, the flux through glycolysis and pyruvate dehydrogenase bypass was reduced by downregulating the expression of the phosphoglucose isomerase PGI1 and deleting the acetaldehyde dehydrogenase ALD6. Glucose-6-phosphate was guided into the pentose phosphate pathway by overexpressing the glucose-6-phosphate dehydrogenase ZWF1. The first approach did not influence octanoic acid production but the latter increased yields in the glucose consumption phase by 65 %. However,
combining the superior fusFASRK with acetyl-CoA and NADPH supply engineering strategies did not result in additive production effects, indicating that other limitations hinder high octanoic acid accumulation. Limitations could be caused in particular by the strong inhibitory effects of octanoic acid or by intrinsic limitations of the FASRK mutant. To enlarge the octanoic acid production platform towards other derived valuable oleochemical compounds the de novo production of 8-hydroxyoctanoic acid was targeted. Since short- and medium-chain fatty acids have a strong inhibitory effect on Saccharomyces cerevisiae, the inhibitory effect of hydroxy fatty acid and dicarboxylic with eight or ten carbon atoms were compared and revealed only little or no growth impairment. Subsequently, the formation of 8-hydroxyoctanoic acid was targeted by a terminal hydroxylation of externally supplied octanoic acid in a bioconversion. For that, three heterologous genes, encoding for cytochromes P450 enzymes and their cognate cytochrome P450 reductases were expressed and 8-hydroxyoctanoic acid production was compared. In addition, the use of different carbon sources was compared.
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Exploring the in vivo subthreshold membrane activity of phasic firing in midbrain dopamine neurons
(2021)
Dopamine is a key neurotransmitter that serves several essential functions in daily behaviors such as locomotion, motivation, stimulus coding, and learning. Disrupted dopamine circuits can result in altered functions of these behaviors which can lead to motor and psychiatric symptoms and diseases. In the central nervous system, dopamine is primarily released by dopamine neurons located in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) within the midbrain, where they signal behaviorally-relevant information to downstream structures by altering their firing patterns. Their “pacemaker” firing maintains baseline dopamine levels at projection sites, whereas phasic “burst” firing transiently elevates dopamine concentrations. Firing activity of dopamine neurons projecting to different brain regions controls the activation of distinct dopamine pathways and circuits. Therefore, characterization of how distinct firing patterns are generated in dopamine neuron populations will be necessary to further advance our understanding of dopamine circuits that encode environmental information and facilitate a behavior.
However, there is currently a large gap in the knowledge of biophysical mechanisms of phasic firing in dopamine neurons, as spontaneous burst firing is only observed in the intact brain, where access to intrinsic neuronal activity remains a challenge. So far, a series of highly-influential studies published in the 1980s by Grace and Bunney is the only available source of information on the intrinsic activity of midbrain dopamine neurons in vivo, in which sharp electrodes were used to penetrate dopamine neurons to record their intracellular activity. A novel approach is thus needed to fill in the gap. In vivo whole-cell patch-clamp method is a tool that enables access to a neuron’s intrinsic activity and subthreshold membrane potential dynamics in the intact brain. It has been used to record from neurons in superficial brain regions such as the cortex and hippocampus, and more recently in deeper regions such as the amygdala and brainstem, but has not yet been performed on midbrain dopamine neurons. Thus, the deep brain in vivo patch-clamp recording method was established in the lab in an attempt to investigate the subthreshold membrane potential dynamics of tonic and phasic firing in dopamine neurons in vivo.
The use of this method allowed the first in-depth examination of burst firing and its subthreshold membrane potential activity of in vivo midbrain dopamine neurons, which illuminated that firing activity and subthreshold membrane activity of dopamine neurons are very closely related. Furthermore, systematic characterization of subthreshold membrane patterns revealed that tonic and phasic firing patterns of in vivo dopamine neurons can be classified based on three distinct subthreshold membrane signatures: 1) tonic firing, characterized by stable, non-fluctuating subthreshold membrane potentials; 2) rebound bursting, characterized by prominent hyperpolarizations that initiate bursting; and 3) plateau bursting, characterized by transient, depolarized plateaus on which bursting terminates. The results thus demonstrated that different types of phasic firing are driven by distinct patterns of subthreshold membrane activity, which may potentially signal distinct types of information. Taken together, the deep brain in vivo patch-clamp technique can be used for the investigation of firing mechanisms of dopamine neurons in the intact brain and will help address open questions in the dopamine field, particularly regarding the biophysical mechanisms of burst firing in dopamine neurons that control behavior.
Plastics contain a complex mixture of chemicals including polymers, additives, starting substances and side-products of processing. These plastic chemicals are prone to leach into the packaged goods, in the case of food contact materials (FCMs), or into the natural environment, in the case of plastic debris. Thus, plastics represent an exposure source of chemicals for humans and wildlife alike. While it is widely known that individual plastic chemicals, such as bisphenol A and phthalates, are hazardous, little is known on the overall chemical composition and toxicity of plastics. When fragmented into smaller particles, referred to as microplastics (< 5 mm), the plastic itself can be ingested by many species. It is well established that microplastic ingestion can have negative consequences for a wide range of organisms including invertebrates, but the contribution of plastic chemicals to the toxicity of microplastics is unclear.
Given the above, the present thesis aimed at a comprehensive toxicological, ecotoxicological and chemical characterization of everyday plastics. For a comparative evaluation, 77 plastic products were selected covering 16 material types (e.g., polyethylene) made from petroleum or renewable feedstocks. These products included biodegradable products, FCMs and non-FCMs, as well as raw materials and final products, respectively. In the first two studies, the chemical mixtures contained in the 77 products were extracted with methanol and extracts were analyzed in a set of four in vitro bioassays and by non-target high-resolution gas or liquid chromatography mass spectrometry. Since an exposure only occurs if chemicals actually leach under realistic conditions, in a third study migration experiments with water were conducted for 24 out of the 77 products. The aqueous migrates were assessed in the same way as the methanolic extracts. In addition, the freshwater invertebrate Daphnia magna was exposed chronically to microplastics made of polyvinylchloride (PVC), polyurethane (PUR) and polylactic acid (PLA) to investigate the contribution of chemicals in microplastic toxicity, in a fourth study.
The experimental findings demonstrate that a wide variety of chemicals is present in plastics. A single plastic product can contain up to several thousand chemical features, most of which unique to that product and at the same time unknown. The results also indicate that the majority of these chemical mixtures are toxic in vitro. Accordingly, 65% of the plastic extracts induced baseline toxicity and 42% an oxidative stress response, while 25% had an antiandrogenic and 6% an estrogenic activity. This implies that chemicals causing unspecific toxicity are more prevalent in plastics than such with endocrine effects. These chemicals can also leach from plastics under realistic conditions. Between 17 and 8936 chemical features were detected in a single migrate sample and all 24 tested migrates induced in vitro toxicity. This means that humans and wildlife can actually be exposed to toxic plastic chemicals under realistic conditions. Generally, each product has its individual toxicological and chemical fingerprint. Thus, neither material type, feedstock, biodegradability nor the food contact suitability of a product can serve as a predictor for the toxicity, the chemical composition or complexity of a product. Likewise, this means that bio-based and biodegradable materials are not superior to their petroleum-based counterparts from a toxicological perspective despite being promoted as sustainable alternatives to conventional plastics.
Moreover, the present thesis demonstrates that plastic chemicals can be the main driver for microplastic toxicity. Irregular microplastics made of PVC, PUR and PLA adversely affected life-history traits of D. magna in a polymer type- and endpoint-dependent manner at concentrations between 100 and 500 mg L-1 and with a higher efficiency than natural kaolin particles. While the toxicity of PVC was triggered by the chemicals used in the material, the effects of PUR and PLA were induced by the physical properties of the particle.
In addition, in the fifth study, results and observations made during this thesis were integrated inter- and transdisciplinarily with the perspectives of a social scientist and a product manufacturer. This elucidated that knowledge on plastic ingredients is often concealed, is lacking or not applicable in practice. These intransparencies hinder the safety evaluation of plastic products as well as the choice and sale of the least toxic packaging material.
Overall, the present thesis highlights that the chemical safety of plastics and their bio-based and biodegradable alternatives is currently not ensured. Thus, chemicals require more consideration in the toxicity and risk assessment of plastics and microplastics. Product-specific and complex chemical compositions, including unknown compounds, pose a challenge here. Two essential steps towards non-toxic products are to increase transparency along the product life cycle and to reduce the chemical complexity of plastics by communication and regulation. The results of the present thesis indicate that products exist which do not contain toxic chemicals. These can serve to direct the design of safer plastics. Since toxicity and chemical complexity seem to increase with processing, the integration of toxicity testing during the production steps would further support the safe and sustainable production and use of plastic products.
Sympathetic influences on articular cartilage regeneration capacity and osteoarthritis manifestation
(2021)
The pathogenesis of osteoarthritis (OA) involves articular cartilage, synovial tissue and subchondral bone and is therefore a disease of the whole joint. OA is characterized by progressive degradation of cartilage, synovial inflammation, osteophyte formation and subchondral bone sclerosis. Cartilage-surrounding tissues are innervated by tyrosine hydroxylase (TH)-positive sympathetic nerve fibers with the most important sympathetic neurotransmitter norepinephrine (NE) detected in the synovial fluid of OA patients. Furthermore, adrenergic receptors are expressed in different knee joint tissues. Most in vitro studies indicate a potential role of the β2-adrenergic receptor, which has been not investigated during OA pathogenesis in vivo. The role of the sympathetic nervous system (SNS) in OA progression has not yet been studied. Therefore, the objective of this study was to analyze how the SNS and NE influence the MSC dependent cartilage regeneration in vitro and the OA pathogenesis and manifestation in vivo.
In the first part of this study, the effect of NE on the chondrogenesis of sASC, which are known to play an important role in cartilage regeneration was analyzed in vitro. In the second part of this study, the role of the SNS was studied in vivo in mice that were sympathectomized chemically followed by surgically induced OA. The specific focus was on the β2-adrenergic receptor effects on OA pathogenesis, which were analyzed in β2-adrenergic receptor-deficient mice.
The in vitro experiments have shown that NE reduced the chondrogenic potential of sASCs by decreasing the expression of type II collagen and sGAG. NE mediated these effects mainly by the α2-AR signalling. Furthermore, NE treatment led to activation of the ERK1/2 signal pathway. These findings suggested that the sympathetic neurotransmitter NE might suppress the chondrogenic capacity of MSC and their dependent cartilage regeneration and may also play a role in OA progression and manifestation.
The in vivo study has shown that sympathectomy reduced synovial TH-positive nerve fibers in the synovium and the NE concentration in the spleen significantly. In WT mice, DMM leads to increased NE concentrations in the spleen compared to sham mice indicating an increased SNS activity after mechanical stress or inflammation due to DMM. Sympathectomy leads to less pronounced cartilage degeneration (OARSI score) after DMM compared to DMM in WT mice. Furthermore, the release of the type II collagen degradation fragment CTX-II was abolished in Syx DMM mice compared to WT DMM mice, suggesting that less SNS activity due to sympathectomy reduced the cartilage degeneration during OA pathogenesis. Similarly, sympathectomy decreased the synovitis score significantly after DMM compared to DMM in
WT mice. Synovitis in WT mice was accompanied by increased MMP-13 expression in the synovium after DMM, compared to Syx mice. Cartilage degeneration seemed to be driven mainly by the increased synovial inflammation accompanied by an increased MMP13 expression in synoviocytes and not in chondrocytes. The pathological changes in synovium and cartilage might also be linked to each other, as indicated by the moderate correlation between the synovial inflammation (synovitis score) and cartilage degeneration (OARSI score). Subchondral bone volume as well the thickness of the subchondral bone plate (SCBP) and calcified cartilage (CC) were increased in Syx mice compared to WT after DMM. The data on DMM induction in β2-AR deficient mice revealed that the β2-AR signaling is involved in cartilage degeneration and the aggravated subchondral bone changes as these mice had less pronounced cartilage degeneration compared to WT mice. While the cartilage degeneration was similar, the subchondral bone changes were more pronounced in β2-AR deficient mice compared to the Syx mice.
Overall, the SNS had differential effects in cartilage, synovium and subchondral bone. A reduced SNS activity by sympathectomy attenuated cartilage degeneration and synovitis but aggravated the OA specific subchondral bone changes. These findings provide new insights into the development of novel therapeutic strategies for OA by targeting the SNS in a tissue- specific manner.
The biotrophic pathogen Ustilago maydis causes smut disease on maize (Zea mays) and induces the formation of tumours on all aerial parts of the plant. Unlike in other biotrophic interactions, no gene-for-gene interactions have been identified in the maize–U. maydis pathosystem. Thus, maize resistance to U. maydis is considered a polygenic, quantitative trait. Here, we study the molecular mechanisms of quantitative disease resistance (QDR) in maize, and how U. maydis interferes with its components. Based on quantitative scoring of disease symptoms in 26 maize lines, we performed an RNA sequencing (RNA-Seq) analysis of six U. maydis-infected maize lines of highly distinct resistance levels. The different maize lines showed specific responses of diverse cellular processes to U. maydis infection. For U. maydis, our analysis identified 406 genes being differentially expressed between maize lines, of which 102 encode predicted effector proteins. Based on this analysis, we generated U. maydis CRISPR/Cas9 knock-out mutants for selected candidate effector sets. After infections of different maize lines with the fungal mutants, RNA-Seq analysis identified effectors with quantitative, maize line-specific virulence functions, and revealed auxin-related processes as a possible target for one of them. Thus, we show that both transcriptional activity and virulence function of fungal effector genes are modified according to the infected maize line, providing insights into the molecular mechanisms underlying QDR in the maize–U. maydis interaction.
1. During the last century, the practice of fur farming in Europe led to the introduction of two mammal species from opposite ends of the world. With their subsequent unintentional escape from captivity or intentional releases, the process of slow expansion and establishment in Europe began. The raccoon Procyon lotor and the raccoon dog Nyctereutes procyonoides are included on the European Union’s list of invasive alien species.
2. We characterised the current climatic niches of the two species in their native ranges in North America and Asia, and compared them with their non-native-range niches in Europe, where we also projected climatic suitability. The aim was to locate suitable habitats beyond their current ranges and assess where a range expansion can be expected.
3. Niche comparison and the projection of climatic suitability in Europe were based on eight bioclimatic variables and presence records from the Global Biodiversity Information Facility database. For niche modelling, we applied the maximum entropy approach (Maxent) and used the native-range data for training.
4. Minimum temperature of the coldest month (bio06) was identified as the most important bioclimatic variable in the habitat suitability models for both species. Different tolerance levels regarding this variable might explain small differences between the species’ projected ranges, especially in the north and east of Europe. The high niche unfilling for both species in Europe suggests a potential for expansion beyond their present ranges.
5. With only little understanding of their ecological impacts in their new ranges, including the potential risk of Nyctereutes procyonoides as SARS-CoV-2 reservoir hosts, further research and management is required at various spatial scales in Europe.
Locating a vocalizing animal can be useful in many fields of bioacoustics and behavioral research, and is often done in the wild, covering large areas. In zoos, however, the application of this method becomes particularly difficult, because, on the one hand, the animals are in a relatively small area and, on the other hand, reverberant environments and background noise complicate the analysis. Nevertheless, by localizing and analyzing animal sounds, valuable information on physiological state, sex, subspecies, reproductive state, social status, and animal welfare can be gathered. Therefore, we developed a sound localization software that is able to estimate the position of a vocalizing animal precisely, making it possible to assign the vocalization to the corresponding individual, even under difficult conditions. In this study, the accuracy and reliability of the software is tested under various conditions. Different vocalizations were played back through a loudspeaker and recorded with several microphones to verify the accuracy. In addition, tests were carried out under real conditions using the example of the giant otter enclosure at Dortmund Zoo, Germany. The results show that the software can estimate the correct position of a sound source with a high accuracy (median of the deviation 0.234 m). Consequently, this software could make an important contribution to basic research via position determination and the associated differentiation of individuals, and could be relevant in a long-term application for monitoring animal welfare in zoos.
Responses of southern ocean seafloor habitats and communities to global and local drivers of change
(2021)
Knowledge of life on the Southern Ocean seafloor has substantially grown since the beginning of this century with increasing ship-based surveys and regular monitoring sites, new technologies and greatly enhanced data sharing. However, seafloor habitats and their communities exhibit high spatial variability and heterogeneity that challenges the way in which we assess the state of the Southern Ocean benthos on larger scales. The Antarctic shelf is rich in diversity compared with deeper water areas, important for storing carbon (“blue carbon”) and provides habitat for commercial fish species. In this paper, we focus on the seafloor habitats of the Antarctic shelf, which are vulnerable to drivers of change including increasing ocean temperatures, iceberg scour, sea ice melt, ocean acidification, fishing pressures, pollution and non-indigenous species. Some of the most vulnerable areas include the West Antarctic Peninsula, which is experiencing rapid regional warming and increased iceberg-scouring, subantarctic islands and tourist destinations where human activities and environmental conditions increase the potential for the establishment of non-indigenous species and active fishing areas around South Georgia, Heard and MacDonald Islands. Vulnerable species include those in areas of regional warming with low thermal tolerance, calcifying species susceptible to increasing ocean acidity as well as slow-growing habitat-forming species that can be damaged by fishing gears e.g., sponges, bryozoan, and coral species. Management regimes can protect seafloor habitats and key species from fishing activities; some areas will need more protection than others, accounting for specific traits that make species vulnerable, slow growing and long-lived species, restricted locations with optimum physiological conditions and available food, and restricted distributions of rare species. Ecosystem-based management practices and long-term, highly protected areas may be the most effective tools in the preservation of vulnerable seafloor habitats. Here, we focus on outlining seafloor responses to drivers of change observed to date and projections for the future. We discuss the need for action to preserve seafloor habitats under climate change, fishing pressures and other anthropogenic impacts.
Land use change has led to large-scale insect decline, threatening ecosystem resilience through reduced functional diversity. Even in nature reserves, losses in insect diversity have been detected. Hereby, changes in local habitat quality and landscape-scale habitat quantity can play a role driving functional diversity toward erosion. Our aim was to analyze how local and landscape-scale factors simultaneously affect functional insect diversity. Therefore, we sampled moths in two Italian coastal forest reserves at 60 sites. Our focus was on functional richness, redundancy and niche occupation, being important for ecosystem resilience, following the insurance framework. Ecological information about 387 species and 14 traits was used to analyze functional diversity. Twenty-five functional groups were recognized and used to estimate niche occupation and redundancy. Fourteen local and 12 landscape-scale factors were measured and condensed by using Principal Components Analysis. The resulting PC-axes served as predictors in linear mixed effects models. Functional richness, redundancy and niche occupation of moths were lower at sites with low habitat quality and quantity, indicating reduced ecosystem resilience. Especially landscape diversity and habitat structure, viz. a humidity-nutrient gradient, but also plant diversity, were promoting functional richness. Landscape fragmentation, indicating increased impermeability for insects, reduced local functional richness, redundancy and niche occupation. Local habitat quality and landscape-wide habitat quantity are both important for maintaining functional insect diversity inside reserves. Therefore, small and isolated nature reserves might fail in preserving biodiversity and ecosystem functions through adverse effects acting from the surrounding landscape structure and configuration.
Learning and animal movement
(2021)
Integrating diverse concepts from animal behavior, movement ecology, and machine learning, we develop an overview of the ecology of learning and animal movement. Learning-based movement is clearly relevant to ecological problems, but the subject is rooted firmly in psychology, including a distinct terminology. We contrast this psychological origin of learning with the task-oriented perspective on learning that has emerged from the field of machine learning. We review conceptual frameworks that characterize the role of learning in movement, discuss emerging trends, and summarize recent developments in the analysis of movement data. We also discuss the relative advantages of different modeling approaches for exploring the learning-movement interface. We explore in depth how individual and social modalities of learning can matter to the ecology of animal movement, and highlight how diverse kinds of field studies, ranging from translocation efforts to manipulative experiments, can provide critical insight into the learning process in animal movement.
The pyruvate:ferredoxin oxidoreductase of the thermophilic acetogen, Thermoanaerobacter kivui
(2021)
Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low-potential ferredoxin (Fd2−) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg−1) and ferredoxin (Fd; 27.2 U·mg−1) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein−1, consistent with the presence of three predicted [4Fe–4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His-tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron-bifurcating enzymes, to keep a constant level of reduced Fd.
The alpha-proteobacterium Zymomonas mobilis is a promising biofuel producer, based on its native metabolism that efficiently converts sugars to ethanol. Therefore, it has a high potential for industrial-scale biofuel production. Two previous studies suggested that Z. mobilis strain Zm4 might not be monoploid. However, a systematic analysis of the genome copy number is still missing, in spite of the high potential importance of Z. mobilis. To get a deep insight into the ploidy level of Z. mobilis and its regulation, the genome copy numbers of three strains were quantified. The analyses revealed that, during anaerobic growth, the lab strain Zm6, the Zm6 type strain obtained from DSMZ (German Collection of Microorganisms), and the lab strain Zm4, have copy numbers of 18.9, 22.3 and 16.2, respectively, of an origin-adjacent region. The copy numbers of a terminus-adjacent region were somewhat lower with 9.3, 15.8, and 12.9, respectively. The values were similar throughout the growth curves, and they were only slightly downregulated in late stationary phase. During aerobic growth, the copy numbers of the lab strain Zm6 were much higher with around 40 origin-adjacent copies and 17 terminus-adjacent copies. However, the cells were larger during aerobic growth, and the copy numbers per μm3 cell volume were rather similar. Taken together, this first systematic analysis revealed that Z. mobilis is polyploid under regular laboratory growth conditions. The copy number is constant during growth, in contrast to many other polyploid bacteria. This knowledge should be considered in further engineering of the strain for industrial applications.
Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source
(2021)
Eubacterium limosum KIST612 is one of the few acetogenic bacteria that has the genes encoding for butyrate synthesis from acetyl-CoA, and indeed, E. limosum KIST612 is known to produce butyrate from CO but not from H2 + CO2. Butyrate production from CO was only seen in bioreactors with cell recycling or in batch cultures with addition of acetate. Here, we present detailed study on growth of E. limosum KIST612 on different carbon and energy sources with the goal, to find other substrates that lead to butyrate formation. Batch fermentations in serum bottles revealed that acetate was the major product under all conditions investigated. Butyrate formation from the C1 compounds carbon dioxide and hydrogen, carbon monoxide or formate was not observed. However, growth on glucose led to butyrate formation, but only in the stationary growth phase. A maximum of 4.3 mM butyrate was observed, corresponding to a butyrate:glucose ratio of 0.21:1 and a butyrate:acetate ratio of 0.14:1. Interestingly, growth on the C1 substrate methanol also led to butyrate formation in the stationary growth phase with a butyrate:methanol ratio of 0.17:1 and a butyrate:acetate ratio of 0.33:1. Since methanol can be produced chemically from carbon dioxide, this offers the possibility for a combined chemical-biochemical production of butyrate from H2 + CO2 using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.
The abyssal seafloor is a mosaic of highly diverse habitats that represent the least known marine ecosystems on Earth. Some regions enriched in natural resources, such as polymetallic nodules in the Clarion-Clipperton Zone (CCZ), attract much interest because of their huge commercial potential. Since nodule mining will be destructive, baseline data are necessary to measure its impact on benthic communities. Hence, we conducted an environmental DNA and RNA metabarcoding survey of CCZ biodiversity targeting microbial and meiofaunal eukaryotes that are the least known component of the deep-sea benthos. We analyzed two 18S rRNA gene regions targeting eukaryotes with a focus on Foraminifera (37F) and metazoans (V1V2), sequenced from 310 surface-sediment samples from the CCZ and other abyssal regions. Our results confirm huge unknown deep-sea biodiversity. Over 60% of benthic foraminiferal and almost a third of eukaryotic operational taxonomic units (OTUs) could not be assigned to a known taxon. Benthic Foraminifera are more common in CCZ samples than metazoans and dominated by clades that are only known from environmental surveys. The most striking results are the uniqueness of CCZ areas, both datasets being characterized by a high number of OTUs exclusive to the CCZ, as well as greater beta diversity compared to other abyssal regions. The alpha diversity in the CCZ is high and correlated with water depth and terrain complexity. Topography was important at a local scale, with communities at CCZ stations located in depressions more diverse and heterogeneous than those located on slopes. This could result from eDNA accumulation, justifying the interim use of eRNA for more accurate biomonitoring surveys. Our descriptions not only support previous findings and consolidate our general understanding of deep-sea ecosystems, but also provide a data resource inviting further taxon-specific and large-scale modeling studies. We foresee that metabarcoding will be useful for deep-sea biomonitoring efforts to consider the diversity of small taxa, but it must be validated based on ground truthing data or experimental studies.
Similar to chloroplast loci, mitochondrial markers are frequently used for genotyping, phylogenetic studies, and population genetics, as they are easily amplified due to their multiple copies per cell. In a recent study, it was revealed that the chloroplast offers little variation for this purpose in central European populations of beech. Thus, it was the aim of this study to elucidate, if mitochondrial sequences might offer an alternative, or whether they are similarly conserved in central Europe. For this purpose, a circular mitochondrial genome sequence from the more than 300-year-old beech reference individual Bhaga from the German National Park Kellerwald-Edersee was assembled using long and short reads and compared to an individual from the Jamy Nature Reserve in Poland and a recently published mitochondrial genome from eastern Germany. The mitochondrial genome of Bhaga was 504,730 bp, while the mitochondrial genomes of the other two individuals were 15 bases shorter, due to seven indel locations, with four having more bases in Bhaga and three locations having one base less in Bhaga. In addition, 19 SNP locations were found, none of which were inside genes. In these SNP locations, 17 bases were different in Bhaga, as compared to the other two genomes, while 2 SNP locations had the same base in Bhaga and the Polish individual. While these figures are slightly higher than for the chloroplast genome, the comparison confirms the low degree of genetic divergence in organelle DNA of beech in central Europe, suggesting the colonisation from a common gene pool after the Weichsel Glaciation. The mitochondrial genome might have limited use for population studies in central Europe, but once mitochondrial genomes from glacial refugia become available, it might be suitable to pinpoint the origin of migration for the re-colonising beech population.
In der vorliegenden Arbeit wurde das Zinkfinger-µ-Protein HVO_2753 des halophilen Archaeons Haloferax volcanii hinsichtlich seiner biologischen Funktion und seiner Struktur charakterisiert.
Zinkfinger-µ-Proteine wurden bisher nur sehr wenig untersucht, während ihnen jedoch in den letzten Jahren steigendes Interesse entgegengebracht wird. Im Genom von H. volcanii sind mehr als 40 solcher Zinkfinger-µ-Proteine codiert. Von diesen besitzt mit HVO_2753 lediglich eines nicht nur zwei, sondern vier der charakteristischen C(P)XCG-Muster, was für die Anwesenheit von zwei Zinkfinger-Motiven spricht. Während Homologe von HVO_2753 in vielen Euryachaeota vorkommen und manche davon als Zink-Ribbon RNA-Bindeproteine annotiert sind, ist über ihre Funktion jedoch nichts bekannt. Zur Charakterisierung des Proteins wurde zunächst eine in frame-Deletionsmutante seines Gens erstellt und diese einer phänotypischen Charakterisierung unterzogen. Die Mutante wies, verglichen mit dem Wildtyp, keine Unterschiede im Wachstum in Komplexmedium oder in synthetischem Medium mit Glukose als Kohlenstoffquelle auf. Ein schweres Defizit konnte jedoch sowohl bei der Adhäsion und Biofilmbildung als auch der Schwärmfähigkeit der Deletionsmutante festgestellt werden. Während die Schwärmfähigkeit des Wildtyps durch plasmidische Expression von HVO_2753 in der Deletionsmutante teilweise wiederhergestellt werden konnte, war eine solche Komplementation bei der Biofilmbildung nicht möglich. Die Analyse der Relevanz ausgewählter Aminosäuren, wie beispielsweise das jeweils erste Cystein in jedem C(P)XCG-Muster zeigte, dass die Substitution jeder einzelnen der getesteten Aminosäuren einen Funktionsverlust des Proteins nach sich zieht. Die Untersuchung des HVO_2753-Transkripts mittels Northern Blot-Analyse bestätigte erste Hinweise aus vorangegangenen dRNA- und RNA-Seq-Studien, die eine Co-Transkription von HVO_2753 mit dem Nachbargen HVO_2752, das für den Translations-Elongationsfaktor aEF-1 beta codiert, aufzeigten. Daraufhin erfolgte eine Untersuchung des Ribosomenprofils, bei der keine Unterschiede zwischen der Deletionsmutante und der Überexpressionsmutante von HVO_2753 festgestellt werden konnten.
Eine Variante von HVO_2753 mit N-terminalem Hexahistidin-Tag wurde homolog überproduziert und aufgereinigt. Die Überproduktion und Aufreinigung wurden im Zuge dieser Arbeit weiter, speziell für HVO_2753, optimiert. So konnten große Mengen von HVO_2753n überproduziert und bei nativen Salzbedingungen mittels Nickel-Affinitätschromatographie und anschließender Größenausschlusschromatographie aufgereinigt werden. Eine massenspektrometrische Analyse bestätigte sowohl das Molekulargewicht als auch die Abwesenheit posttranslationaler Modifikationen. Die Untersuchung der Menge an gebundenem Zink im Protein erfolgte beim Zink-Assay mit Hilfe des hochsensitiven und hochspezifischen Fluorophors ZnAF-2F. Dabei konnte gezeigt werden, dass überraschenderweise lediglich ein Zink-Ion in HVO_2753 gebunden vorliegt.
Zur weiteren Funktionsaufklärung erfolgte eine Interaktionspartnersuche. Hierfür wurde HVO_2753 überproduziert, ein in vivo-Crosslink und anschließend eine native Aufreinung durchgeführt. Die massenspektrometrische Analyse ausgewählter Fraktionen nach der Größenausschlusschromatographie ergaben eine Vielzahl an möglichen Bindepartnern. Besonders häufig wurde hier die GalE family Epimerase/Dehydratase gefunden. Eine weitere Methode zur Suche nach Interaktionspartnern richtete sich auf RNAs. Hier konnten mittels eines eigens entwickelten Protokolls neben RNAs des Translationsapparates auch mehrfach die tRNA(Glu) gefunden werden.
Zusätzlich sollte die Transkriptomanalyse mittels RNA-Sequenzierung Unterschiede zwischen Wildtyp, Deletionsmutante und Komplementationsmutante aufzeigen. Hier wurden weitreichende Auswirkungen der Deletion von HVO_2753 gefunden. Zahlreiche Gene in mehreren Operons zur Motilität und Chemotaxis lagen in der Deletionsmutante stark herunterreguliert vor, während die Gene einiger Metallionen-Transporter und der Eisen(III)-Siderophor-Biosynthese hochreguliert vorlagen. In der Komplementationsmutante konnten nur von den letzteren Genen Transkriptlevel vergleichbar mit denen des Wildtyps wiedergefunden werden.
In dieser Arbeit konnte gezeigt werden, dass das kleine Zinkfinger-Protein HVO_2753 eine essenzielle Rolle in der positiven Regulation der Motilität, Chemotaxis und der Adhäsion bzw. Biofilmbildung spielt. Gleichzeitig übt HVO_2753 eine negative Regulation auf den Metallionen-Transport und die Biosynthese des Eisen(III)-Siderophors aus.
Climate change imposes severe stress on European forests, with forest degradation already visible in several parts of Europe. Thus adaptation of forestry applications in Mediterranean areas and central Europe is necessary. Proactive forestry management may include the planting of Mediter- ranean oak species in oak-bearing Central European regions. Five replicate common gardens of Greek and Italian provenances of Quercus ilex, Q. pubescens and Q. frainetto seedlings (210 each per plantation) were established in Central Italy, NE Greece (two) and Southern Germany (two, including Q. robur) to assess their performance under different climate conditions. Climate and soil data of the plantation sites are given and seedling establishment was monitored for survival and morphological parameters. After 3 years (2019) survival rates were satisfactory in the German and Italian sites, whereas the Greek sites exerted extremely harsh conditions for the seedlings, including extreme frost and drought events. In Germany, seedlings suffered extreme heat and drought periods in 2018 and 2019 but responded well. Provenances were ranked for each country for their performance after plan- tation. In Greece and Italy, Q. pubescens was the best performing species. In Germany, Q. pubescens and Q. robur performed best. We suggest that Greek or Italian provenances of Q. pubescens may be effectively used for future forestation purposes in Central Europe. For the establishment of Quercus plantations in Northern Greece, irrigation appears to be a crucial factor in seedling establishment.
Microplastics (MPs) are ubiquitous and persistent pollutants, and have been detected in a wide variety of media, from soils to aquatic systems. MPs, consisting primarily of polyethylene, polypropylene, and polyacrylamide polymers, have recently been found in 12% of samples of honey collected in Ecuador. Recently, MPs have also been identified in honey bees collected from apiaries in Copenhagen, Denmark, as well as nearby semiurban and rural areas. Given these documented exposures, assessment of their effects is critical for understanding the risks of MP exposure to honey bees. Exposure to polystyrene (PS)-MPs decreased diversity of the honey bee gut microbiota, followed by changes in gene expression related to oxidative damage, detoxification, and immunity. As a result, the aim of this perspective was to investigate whether wide-spread prevalence of MPs might have unintended negative effects on health and fitness of honey bees, as well as to draw the scientific community’s attention to the possible risks of MPs to the fitness of honey bees. Several research questions must be answered before MPs can be considered a potential threat to bees.
Photorhabdus and Xenorhabdus are Gram-negative, entomopathogenic bacteria, living in endosymbiosis with the soil-dwelling nematode of the genera Steinernema and Heterorhabditis. The life cycle of these nematodes consists of non-feeding infective juvenile (IJ) stage, which actively searches for insects in the soil. After penetrating the insect prey, Photorhabdus and Xenorhabdus bacteria are released from the nematode gut. The bacteria proliferate and produce toxins to kill the insect. Photorhabdus and Xenorhabdus support nematode development throughout the life cycle and to get rid of food competitors by providing a wide variety of specialized metabolites (SMs). However, little is known about which SMs function as so called “food signals” to trigger the development process.
The IJs develop into adult, self-fertilizing hermaphrodites in a process called recovery, while feeding on cadaver and bacterial biomass. Heterorhabditis and Steinernema proceed to breed until nutrients are exhausted. Next generation IJs (NG-IJs) develop and leave the cadaver to search for another insect prey.
Photorhabdus and Xenorhabdus can be cultivated in defined medium under laboratory conditions. By placing IJs on a plate containing their respective bacterial symbiont, the complete life cycle of the nematodes can be observed in vitro. The in vitro nematode bioassay was used as a tool to investigate the development of the nematode.
The aim of this study was to find the food signals responsible for nematode development. Different Photorhabdus deletion strains unable to produce one or several SMs were co-cultivated with nematodes in the nematode bioassay. Subsequently, two aspects of the life cycle were investigated: recovery and NG-IJ development.
As isopropyl stilbene (IPS) is postulated to function as a food signal to support nematode recovery, it was used as a starting point for investigations. This study was focused on the biosynthetic pathway of IPS, including intermediates, side products and derivatives to investigate which one is in fact responsible for supporting nematode development.
The biosynthesis of IPS requires two precursors, phenylalanine and leucine (Figure 5). The first topic was focused on the phenylalanine derived pathway. Photorhabdus laumondii deletion mutants, defective in intermediate steps of this pathway, were created. The deletion of the genes coding for the phenylalanine ammonium lyase (stlA), converting phenylalanine into cinnamic acid (CA), the coenzyme A (CoA) ligase (stlB) and the operon coding for a ketosynthase and aromatase (stlCDE), were used. These strains were used for nematode bioassay including complementation of mutant phenotypes by feeding experiments. Recovery of nematodes grown on the deletion strains was always lower than recovery of nematodes grown on wild type bacteria. Feeding IPS to a deletion strain did not restore wild type level nematode recovery, thus IPS cannot be the food signal. Instead, the food signal must be another compound derived from this part of biosynthetic pathway. Lumiquinone and 2,5-dihydrostilbene are suggested to function as food signals and need to be investigated in future work.
The second part of this study was focused on the leucine derived pathway, which involved the Bkd complex forming the iso-branched part of IPS. A deletion of bkd was created and phenotypically analysed, subsequently performed with the nematode bioassay. Not only IPS but also other branched SMs, like photopyrones and phurealipids are synthetised by the Bkd complex. Deletions strains defective in producing photopyrones and phurealipids were also performed in nematode bioassays to investigate effects of these SMs individually. Branched SMs did not have an impact on nematode development, but nematodes grown on the ΔbkdABC strain showed a reduced nematode recovery and almost diminished NG-IJs development. As the Bkd complex also produces branched chain fatty acids (BCFAs), feeding experiments were performed with lipid extracts of wild type and mutant strain. All lipid extracts improved recovery, but only wild type lipids could complement NG-IJ development. This strongly indicates that BCFAs play an important role in NG-IJ development, which needs to be proven with purified BCFA feeding. This is an interesting finding, which could improve nematode production for biocontrol agent usage.
The role of IPS derived to epoxy stilbene (EPS) for nematode development, was another focus in the nematode life cycle. Recently it was demonstrated that EPS does not support nematode development. However, EPS forms adducts with amino acids. In my thesis, novel adducts containing the amino acid phenylalanine or a tetrapeptide were characterized. Another adduct, most likely being an EPS dimer, was also characterized. The biological role of such adducts was discussed to be potentially important for insect weakening and the structure of the novel compounds need to be structure elucidated and tested for bioactivity.
Climatic niches describe the climatic conditions in which species can persist. Shifts in climatic niches have been observed to coincide with major climatic change, suggesting that species adapt to new conditions. We test the relationship between rates of climatic niche evolution and paleoclimatic conditions through time for 65 Old-World flycatcher species (Aves: Muscicapidae). We combine niche quantification for all species with dated phylogenies to infer past changes in the rates of niche evolution for temperature and precipitation niches. Paleoclimatic conditions were inferred independently using two datasets: a paleoelevation reconstruction and the mammal fossil record. We find changes in climatic niches through time, but no or weak support for a relationship between niche evolution rates and rates of paleoclimatic change for both temperature and precipitation niche and for both reconstruction methods. In contrast, the inferred relationship between climatic conditions and niche evolution rates depends on paleoclimatic reconstruction method: rates of temperature niche evolution are significantly negatively related to absolute temperatures inferred using the paleoelevation model but not those reconstructed from the fossil record. We suggest that paleoclimatic change might be a weak driver of climatic niche evolution in birds and highlight the need for greater integration of different paleoclimate reconstructions.
The factors that vary the aroma of Tuber magnatum fruiting bodies are poorly understood. The study determined the headspace aroma composition, sensory aroma profiles, maturity and bacterial communities from T. magnatum originating from Italy, Croatia, Hungary, and Serbia, and tested if truffle aroma is dependent on provenance and if fruiting body volatiles are explained by maturity and/or bacterial communities.
Headspace volatile profiles were determined using gas chromatography–mass spectrometry–olfactometry (GC-MS-O) and aroma of fruiting body extracts were sensorially assessed. Fruiting body maturity was estimated through spore melanisation. Bacterial community was determined using 16S rRNA amplicon sequencing.
Main odour active compounds were present in all truffles but varied in concentration. Aroma of truffle extracts were sensorially discriminated by sites. However, volatile profiles of individual fruiting bodies varied more within sites than across geographic area, while maturity level did not play a role. Bacterial communities varied highly and were partially explained by provenance. A few rare bacterial operational taxonomical units associated with a select few nonodour active volatile compounds.
Specificities of the aroma of T. magnatum truffles are more likely to be linked to individual properties than provenance. Some constituents of bacteria may provide biomarkers of provenance and be linked to nonodour active volatiles.
Although macroecology is a well-established field, much remains to be learned about the large-scale variation of fungal traits. We conducted a global analysis of mean fruit body size of 59 geographical regions worldwide, comprising 5340 fungal species exploring the response of fruit body size to latitude, resource availability and temperature. The results showed a hump-shaped relationship between mean fruit body size and distance to the equator. Areas with large fruit bodies were characterised by a high seasonality and an intermediate mean temperature. The responses of mutualistic species and saprotrophs were similar. These findings support the resource availability hypothesis, predicting large fruit bodies due to a seasonal resource surplus, and the thermoregulation hypothesis, according to which small fruit bodies offer a strategy to avoid heat and cold stress and therefore occur at temperature extremes. Fruit body size may thus be an adaptive trait driving the large-scale distribution of fungal species.
Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt.
Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.
Eukaryotic ribosome assembly starts in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into the 35S pre-ribosomal RNA (pre-rRNA). More than two-hundred ribosome biogenesis factors (RBFs) and more than two-hundred small nucleolar RNAs (snoRNA) catalyze the processing, folding and modification of the rRNA in Arabidopsis thaliana. The initial pre-ribosomal 90S complex is formed already during transcription by association of ribosomal proteins (RPs) and RBFs. In addition, small nucleolar ribonucleoprotein particles (snoRNPs) composed of snoRNAs and RBFs catalyze the two major rRNA modification types, 2′-O-ribose-methylation and pseudouridylation. Besides these two modifications, rRNAs can also undergo base methylations and acetylation. However, the latter two modifications have not yet been systematically explored in plants. The snoRNAs of these snoRNPs serve as targeting factors to direct modifications to specific rRNA regions by antisense elements. Today, hundreds of different sites of modifications in the rRNA have been described for eukaryotic ribosomes in general. While our understanding of the general process of ribosome biogenesis has advanced rapidly, the diversities appearing during plant ribosome biogenesis is beginning to emerge. Today, more than two-hundred RBFs were identified by bioinformatics or biochemical approaches, including several plant specific factors. Similarly, more than two hundred snoRNA were predicted based on RNA sequencing experiments. Here, we discuss the predicted and verified rRNA modification sites and the corresponding identified snoRNAs on the example of the model plant Arabidopsis thaliana. Our summary uncovers the plant modification sites in comparison to the human and yeast modification sites.
Biological and environmental factors as sources of variation in nocturnal behavior of giraffe
(2021)
Upon a drastic decline of the giraffe population in the wild, conservation efforts and therefore the role of zoos have become more important than ever. With their unique opportunities, zoos provide excellent conditions to study animal behavior, expanding the knowledge about the giraffe's behavior repertoire and their ability to adapt. This study therefore examined the nocturnal behavior of 63 giraffe living in 13 different EAZA zoos across Germany and the Netherlands. Giraffe were observed and videos recorded via infrared sensitive cameras during the winter seasons 2015–2018. The observation period spanned nightly from 17:00 to 7:00. Thus, 198 nights, with a total of 2772 h were recorded and analyzed. Linear mixed models were then used to assess potential biological and environmental factors influencing behavior during the dark phase. Results show that individual variables such as age, subspecies and motherhood determined nocturnal activity and sleep behavior most. Among the variables studied, husbandry conditions and environmental factors complying with EAZA standards had no influence on the giraffe's nocturnal behavior. By combining nocturnal activity analyses and an assessment of potential influencing factors, our findings present a holistic approach to a better understanding of captive giraffe behavior and allow for management implications.
Cercosporoid fungi (Mycosphaerellaceae, Mycosphaerellales, Ascomycota) are one of the largest and most diverse groups of hyphomycetes causing a wide range of diseases of economically important plants as well as of plants in the wild. Although more than 6000 species are known for this group, the documentation of this fungal group is far from complete. Especially in the tropics, the diversity of cercosporoid fungi is poorly known. The present study aims to identify and characterise cercosporoid fungi collected on host plants belonging to Fabaceae in Benin, West Africa. Information on their morphology, host species and DNA sequence data (18S rDNA, 28S rDNA, ITS and tef1) is provided. DNA sequence data were obtained by a simple and non-culture-based method for DNA isolation which has been applied for cercosporoid fungi for the first time in the context of the present study. Among the loci used for the phylogenetic analysis, tef1 provided the best resolution together with the multigene dataset. Species delimitation in many cases, however, was only possible by combining molecular sequence data with morphological characteristics. Based on forty specimens recently collected in Benin, 18 species are presented with morphological descriptions, illustrations and sequence data. Among these, six species in the genus Cercospora and two species in Pseudocercospora are proposed as species new to science. The newly described species are Cercospora (C.) beninensis on Crotalaria macrocalyx, C. parakouensis on Desmodium tortuosum, C. rhynchophora on Vigna unguiculata, C. vignae-subterraneae on Vigna subterranea, C. tentaculifera on Vigna unguiculata, C. zorniicola on Zornia glochidiata, Pseudocercospora sennicola on Senna occidentalis and Pseudocercospora tabei on Vigna unguiculata. Eight species of cercosporoid fungi are reported for Benin for the first time, three of them, namely C. cf. canscorina, C. cf. fagopyri and C. phaseoli-lunati are new for West Africa. The presence of two species of cercosporoid fungi on Fabaceae previously reported from Benin, namely Nothopassalora personata and Passalora arachidicola, is confirmed.
Measuring connection to nature - a illustrated extension of the inclusion of nature in self scale
(2021)
The human-nature connection is an important factor that is frequently the subject of environmental education research and environmental psychology. Therefore, over the years, numerous measuring instruments have been established to quantitatively record a person’s connection to nature. However, there is no instrument specifically for children with cognitive limitations. For this reason, in this study, an established scale for connection to nature, the inclusion of nature in self scale (INS), was modified especially for the needs of this group. Study 1 investigated what students understand by the term “nature” in order to create an illustrated version of the INS. In study 2, the new instrument was tested on university students and compared with the original INS and the connectedness to nature scale (CNS). No significant differences between the original INS and the new developed scale were found (p = 0.247), from which it can be concluded that the illustrated INS (IINS) measures the connection to nature with similar accuracy as the original INS. In study 3, the instrument was tested together with other established nature connection instruments on the actual target group, students with disabilities. The correlation between the IINS, the CNS, and nature connectedness scale (NR) were in accordance with the expected literature values (rIINS-CNS = 0.570 & rIINS-NR = 0.605). The results of this study also prove effectiveness of the developed illustrated scale. This research thus provides a suitable measuring instrument for people with learning difficulties and can make a contribution to the investigation of human-nature connections and conservation education.
The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis
(2021)
The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T-cell-directed therapies fail to elicit effective anti-tumor immune responses. Here, we seek to evaluate the applicability of radio- immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapy- induced immune modulation resulted in an increase in cytotoxic T-cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experi- mental breast-to-brain metastasis. However, long-term efficacy was not observed. Recurrent brain metastases showed accumula- tion of blood-borne PD-L1+ myeloid cells after radio-immunother- apy indicating the establishment of an immune suppressive environment to counteract re-activated T-cell responses. This finding was further supported by transcriptional analyses indicat- ing a crucial role for monocyte-derived macrophages in mediating immune suppression and regulating T-cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases.
The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881–1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
Studium der Lebewesen : im Masterprogramm »Ökologie und Evolution« wird Diversität großgeschrieben
(2021)
Viele Gruppen der Lebewesen, insbesondere Insekten breiten sich durch steigende Temperaturen zunehmend in Gebieten aus, in denen sie ursprünglich nicht vorkommen(Novikov und Vaulin 2014; Bebber 2015). Hierbei ist die steigende Temperatur in
verschiedenen Gebieten der Hauptfaktor für Expansionen dieser Arten in Richtung des nördlichen Polarkreises. Einige dieser Arten sind sehr tolerant für verschiedene Variablen und können damit ihr Verbreitungsgebiet deutlich nach Norden hin ausdehnen. Aufgrund steigender Temperaturen werden jedoch andere Arten in ihrem Verbreitungsgebiet eingeschränkt oder ihre Verbreitung verschiebt sich in nördliche Richtung (Ogden und Lindsay 2016; Lawler et al. 2009). Auch für die Verbreitung von Krankheiten spielen Temperaturen, Ausbreitungen oder Verbreitungsverschiebungen eine wichtige Rolle (Mordecai et al. 2019).
So können, durch die Etablierung der passenden Vektoren, bisher nur in wärmeren Gebieten auftretende Krankheiten zukünftig auch in unseren Breitengraden eingeschleppt und
verbreitet werden. Bremsen, invasive Stechmücken aber auch einheimische Mücken tragen alle ein Potential,verschiedenste Krankheitserreger zu verbreiten, auch wenn die Eignung als
Vektor für jede Art unterschiedlich groß ausfällt und manche Arten daher kaum beobachtet und untersucht werden. Mit dem Augenmerk auf sich ändernde Verbreitungsgebiete hinsichtlich zukünftigen klimatischen Veränderungen und sich wandelnden anthropogenen Einflüssen sollten jedoch auch Arten mit bisher geringem Vektorpotential mit in Beobachtungsprogramme aufgenommen werden.
Wir untersuchten in Projekt I auf kontinentaler Skala die Verbreitung von sechs verschiedenen Bremsenarten und konnten sowohl Rückschlüsse auf eine mangelhafte Beobachtung der
Arten ziehen als auch Artpräferenzen hinsichtlich der Landschaftsnutzung, Auswirkungen des Klimas auf die Verbreitung der Art und bisher unbekannte Toleranzen hinsichtlich tiefen Temperaturen und äußerst verkürzten Wärmeperioden aufdecken. Eine Größenordnung niedriger wurde in Projekt II, basierend auf aktuellen und Vergangenen Klimadaten, die zukünftige und aktuelle Verbreitung einer invasiven, sich zukünftig ausbreitenden Stechmückenart innerhalb Deutschlands modelliert. Durch bisherig im Untersuchungsgebiet nur begrenztes Auftreten konnten noch keine Rückschlüsse auf die unterschiedlichen Präferenzen für das Habitat gezogen werden, es können jedoch für zukünftige Berechnungen Habitatpräferenzen aus anderen Gebieten hinzugezogen werden um die Art und ihre fortschreitende Ausbreitung genauer zu beobachten. Auf der kleinsten untersuchten Ebene konnten in Projekt III innerhalb eines Mikrohabitates verschiedenste Rückschlüsse auf limitierende oder förderliche abiotische Faktoren, die teilweise bisherig nicht oder nur geringfügig beobachtet wurden, gezogen werden. Ebenfalls konnten Auswirkungen der umgebenden Landschaft auf die Abundanzen der Tiere beobachtet werden. Mithilfe von verschiedenen Modellen und in Abhängigkeit von Klimakarten, Landbedeckungsdaten und Landnutzung sowie Eigenschaften und Toleranzen der untersuchten Arten lassen sich in verschiedenen Größenordnungen geeignete Habitate von einheimischen sowie invasiven Arten identifizieren und zukünftige Verbreitungen effizient vorhersagen.
Insgesamt können, basierend auf all diesen Daten, dadurch für alle untersuchten Faktoren Modelle auf andere Gebiete übertragen werden um somit potentielle Verbreitungen dort
vorherzusagen. Auf unseren Daten basierend können so zum Beispiel Modellierungen für die potentielle Ausbreitung der untersuchten Tabaniden innerhalb anderer Kontinente berechnet werden und Monitoringprogramme können die Ergebnisse unserer Studie als Startpunkt aufgreifen, um durch Beprobung an modellierten Standorten die Korrektheit unserer Modelle zu überprüfen und sowohl Landschaftstypen als auch Artzusammensetzung aufzunehmen um das Modell zu bestätigen oder zu verbessern. Die Modellierung der invasiven Art Aedes albopictus bietet die Möglichkeit, diese Art in Zukunft innerhalb der möglichen Ausbreitungskorridore genauer zu beobachten um ihre fortschreitende Verbreitung zu
verifizieren oder eventuelle Änderungen des klimatischen Verlaufes mit einzubinden und das Modell anzupassen. Die Untersuchung des Mikrohabitats von Culex pipiens pipiens und Culex torrentium bietet, auch hinsichtlich anderer Arten in diesem Habitat, eine potente Methode, Vorhersagen für Artvorkommen innerhalb anderer Unterirdischen Objekte zu berechnen. Hier können, bei ausreichend großer Datenlage, eine Vielzahl von Faktoren in die Auswertung mit einfließen.
Die durchgeführten Studien bestätigen die Notwendigkeit für verbesserte Monitoringkonzepte für alle vektorkompetenten Tiergruppen hinsichtlich der sich ändernden klimatischen Bedingungen, des globalen Handels und die sich wandelnde Nutzung der Landschaften durch den Menschen und darin begründete Veränderungen der Artenzusammensetzung eines Habitates, zeigen Möglichkeiten, diese Konzepte mit bisher
ungenutzten Daten aufzubauen und zu verbessern und können gleichzeitig zu deren Verbesserung herangezogen werden.
Gene conversion is defined as the non-reciprocal transfer of genetic information from one site to a homologous, but not identical site of the genome. In prokaryotes, gene conversion can increase the variance of sequences, like in antigenic variation, but can also lead to a homogenization of sequences, like in the concerted evolution of multigene families. In contrast to these intramolecular mechanisms, the intermolecular gene conversion in polyploid prokaryotes, which leads to the equalization of the multiple genome copies, has hardly been studied. We have previously shown the intermolecular gene conversion in halophilic and methanogenic archaea is so efficient that it can be studied without selecting for conversion events. Here, we have established an approach to characterize unselected intermolecular gene conversion in Haloferax volcanii making use of two genes that encode enzymes involved in carotenoid biosynthesis. Heterozygous strains were generated by protoplast fusion, and gene conversion was quantified by phenotype analysis or/and PCR. It was verified that unselected gene conversion is extremely efficient and it was shown that gene conversion tracts are much longer than in antigenic variation or concerted evolution in bacteria. Two sites were nearly always co-converted when they were 600 bp apart, and more than 30% co-conversion even occurred when two sites were 5 kbp apart. The gene conversion frequency was independent from the extent of genome differences, and even a one nucleotide difference triggered conversion.
The geomagnetic field provides directional information for birds. The avian magnetic compass is an inclination compass that uses not the polarity of the magnetic field but the axial course of the field lines and their inclination in space. It works in a flexible functional window, and it requires short-wavelength light. These characteristics result from the underlying sensory mechanism based on radical pair processes in the eyes, with cryptochrome suggested as the receptor molecule. The chromophore of cryptochrome, flavin adenine dinucleotide (FAD), undergoes a photocycle, where radical pairs are formed during photo-reduction as well as during re-oxidation; behavioral data indicate that the latter is crucial for detecting magnetic directions. Five types of cryptochromes are found in the retina of birds: cryptochrome 1a (Cry1a), cryptochrome 1b, cryptochrome 2, cryptochrome 4a, and cryptochrome 4b. Because of its location in the outer segments of the ultraviolet cones with their clear oil droplets, Cry1a appears to be the most likely receptor molecule for magnetic compass information.
Microglia, the primary immune cells of the central nervous system, hold a multitude of tasks in order to ensure brain homeostasis and are one of the best predictors of biological age on a cellular level. We and others have shown that these long-lived cells undergo an aging process that impedes their ability to perform some of the most vital homeostatic functions such as immune surveillance, acute injury response, and clearance of debris. Microglia have been described as gradually transitioning from a homeostatic state to an activated state in response to various insults, as well as aging. However, microglia show diverse responses to presented stimuli in the form of acute injury or chronic disease. This complexity is potentially further compounded by the distinct alterations that globally occur in the aging process. In this review, we discuss factors that may contribute to microglial aging, as well as transcriptional microglia alterations that occur in old age. We then compare these distinct phenotypic changes with microglial phenotype in neurodegenerative disease.
Taxa under scrutiny in this thesis are Halophytophthora-like oomycetes. The genus Halophytophthora, proposed in 1990, is an assemblage of unrelated species grouped together on the basis habitat preference, i.e. the mangrove or saltmarsh biome, and morphological similarity to Phytophthora. The premise “Phytophthora-like species from the mangrove environment” became the genus concept for Halophytophthora and lasted for almost 2 decades which resulted to the addition of several species (i.e. H. elongata, H. exoprolifera, H. porrigovesica, H. kandeliae, H. masteri, and H. tartarea). At the onset of molecular phylogenetics, Halophytophthora was inferred as a highly polyphyletic taxon and the genus concept was found to be unsuitable. This thesis adds to this, since six Phytophthora spp. were isolated from the mangrove environment, two of which were found in the Philippines (Phytophthora elongata and Phytophthora insolita). After a thorough assessment of the morphologic and phylogenetic data of taxa included in this thesis, several taxonomic novelties were introduced – a new family (Salispinaceae), a new genus (Calycofera), new species (Calycofera cryptica, Phytopythium dogmae, Phytopythium leanoi, Salisapilia coffeyi, and Salispina hoi), and new combinations (Calycofera operculata, Salisapilia bahamensis, S. elongata, S. epistomia, S. masteri, S. mycoparasitica). In addition, Salisapiliaceae and Salisapilia were emended.
Research on Podospora anserina unraveled a network of molecular pathways affecting biological aging. In particular, a number of pathways active in the control of mitochondria were identified on different levels. A long-known key process active during aging of P. anserina is the age- related reorganization of the mitochondrial DNA (mtDNA). Mechanisms involved in the stabilization of the mtDNA lead to lifespan extension. Another critical issue is to balance mitochondrial levels of reactive oxygen species (ROS). This is important because ROS are essential signaling molecules, but at increased levels cause molecular damage. At a higher level of the network, mechanisms are active in the repair of damaged compounds. However, if damage passes critical limits, the corresponding pathways are overwhelmed and impaired molecules as well as those present in excess are degraded by specific enzymes or via different forms of autophagy. Subsequently, degraded units need to be replaced by novel functional ones. The corresponding processes are dependent on the availability of intact genetic information. Although a number of different pathways involved in the control of cellular homeostasis were uncovered in the past, certainly many more exist. In addition, the signaling pathways involved in the control and coordination of the underlying pathways are only initially understood. In some cases, like the induction of autophagy, ROS are active. Additionally, sensing and signaling the energetic status of the organism plays a key role. The precise mechanisms involved are elusive and remain to be elucidated.
Mitochondrial F1Fo-ATP-synthase dimers play a critical role in shaping and maintenance of mitochondrial ultrastructure. Previous studies have revealed that ablation of the F1Fo-ATP-synthase assembly factor PaATPE of the ascomycete Podospora anserina strongly affects cristae formation, increases hydrogen peroxide levels, impairs mitochondrial function and leads to premature cell death. In the present study, we investigated the underlying mechanistic basis. Compared to the wild type, we observed a slight increase in non-selective and a pronounced increase in mitophagy, the selective vacuolar degradation of mitochondria. This effect depends on the availability of functional cyclophilin D (PaCYPD), the regulator of the mitochondrial permeability transition pore (mPTP). Simultaneous deletion of PaAtpe and PaAtg1, encoding a key component of the autophagy machinery or of PaCypD, led to a reduction of mitophagy and a partial restoration of the wild-type specific lifespan. The same effect was observed in the PaAtpe deletion strain after inhibition of PaCYPD by its specific inhibitor, cyclosporin A. Overall, our data identify autophagy-dependent cell death (ADCD) as part of the cellular response to impaired F1Fo-ATP-synthase dimerization, and emphasize the crucial role of functional mitochondria in aging.
Nomadic movements are often a consequence of unpredictable resource dynamics. However, how nomadic ungulates select dynamic resources is still understudied. Here we examined resource selection of nomadic Mongolian gazelles (Procapra gutturosa) in the Eastern Steppe of Mongolia. We used daily GPS locations of 33 gazelles tracked up to 3.5 years. We examined selection for forage during the growing season using the Normalized Difference Vegetation Index (NDVI). In winter we examined selection for snow cover which mediates access to forage and drinking water. We studied selection at the population level using resource selection functions (RSFs) as well as on the individual level using step-selection functions (SSFs) at varying spatio-temporal scales from 1 to 10 days. Results from the population and the individual level analyses differed. At the population level we found selection for higher than average NDVI during the growing season. This may indicate selection for areas with more forage cover within the arid steppe landscape. In winter, gazelles selected for intermediate snow cover, which may indicate preference for areas which offer some snow for hydration but not so much as to hinder movement. At the individual level, in both seasons and across scales, we were not able to detect selection in the majority of individuals, but selection was similar to that seen in the RSFs for those individuals showing selection. Difficulty in finding selection with SSFs may indicate that Mongolian gazelles are using a random search strategy to find forage in a landscape with large, homogeneous areas of vegetation. The combination of random searches and landscape characteristics could therefore obscure results at the fine scale of SSFs. The significant results on the broader scale used for the population level RSF highlight that, although individuals show uncoordinated movement trajectories, they ultimately select for similar vegetation and snow cover.
Identification and regulation of tomato Serine/Arginine-rich proteins under high temperatures
(2021)
Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.