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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.
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 division Ascomycota(Fungi) contains a large number of taxa known to reproduce only asexually by the formation of conidia or other non-motile propagules produced by mitotic cellular devisions. They are called anamorphic, mitosporic, asexual or conidial fungi and ecologically, they are often found associated with plant debris in different stages of decay. In general, saprobic anamorphs of ascomycetous affinities are poorly studied and their outstanding diversity is currently underexplored. Phylogenetic relationships are unknown for many of them and they are still largely underrepresented in the current phylogenetic classification system of Fungi, with many morphologically defined anamorphic taxa still awaiting taxonomic reassessment in the light of molecular approaches. The increasing usage of molecular markers combined with robust statistical methods has allowed their phylogenetic affinities to be revealed and to gradually incorporate many of them into the different taxonomic groups of the division Ascomycota. However, the phylogenetic placement and taxonomic status of a large number of saprobic taxa remain unresolved due to the lack of DNA sequence data.
The present dissertation aims to explore the rich but understudied diversity of those anamorphic fungi traditionally known as hyphomycetes that inhabit dead plant debris. It consists of five publications in which a polyphasic approach integrating morphological, developmental, cultural and molecular data was used to incorporate novel or incertae sedis taxa within Ascomycota and to make more sound decisions regarding their taxonomic status. Specific objectives include: 1. the collection, isolation and morphological characterization of selected anamorphic fungi representing putative new or interesting taxa of uncertain phylogenetic placement; 2. the generation of novel DNA sequence data to infer their phylogenetic relationships and to resolve their taxonomic affinities within Ascomycota; 3. the testing of any previously available morphologically based hypotheses on their putative position, generic placement or relationships with teleomorphic, pleomorphic or other anamorphic taxa; and 4. the determination of their generic validity, monophyly and taxonomic boundaries using molecular data and phylogenetic analyses methods.
Materials studied in these five projects consisted of specimens collected during field work carried out by the author or collaborators in different countries including USA, the Czech Republic and Panama between the years 2014 and 2017. The target substrates were dead leaves of different palm trees, dead wood and bark of pines and twigs or stems of unknown shrubs and woody vines that are all known to harbor a rich saprobic mycobiota. Putative novelties or anamorphic taxa with unknown or poorly studied phylogenetic affinities were selected for further morphological and molecular investigation. Micromorphological studies were based on fungal structures observed on natural substrate, herbarium specimens and in culture. DNA was extracted from cultures and PCR amplification followed by Sanger sequencing was carried out using relevant molecular markers employed in fungal phylogenetic studies. Newly obtained DNA sequence data were analyzed following a standard phylogenetic analysis pipeline and phylogenetic relationships were reconstructed using character-based methods such as Maximum Likelihood and Bayesian inference.
Conclusion is that anamorphic Ascomycota inhabiting dead plant debris represents a largely untapped source of biodiversity and information still in need of further exploration. A new capnodiaceous genus Castanedospora, seven new species named Taeniolella sabalicola, Hermatomyces bifurcatus, H. constrictus, H. megasporus, H. sphaericoides, H. verrucosus and Septonema lohmanii, and two new combinations, Castanedospora pachyanthicola and H. reticulatus, are proposed based on morphological and DNA sequence data. Molecular phylogenetics was confirmed as the tool of choice for the inference of relationships in novel or incertae sedis anamorphic fungi that are otherwise difficult to assess in the absence of a teleomorphic state. They were first resolved or revisited for several saprobic species such as Ernakulamia cochinensis, H. sphaericus, H. tucumanensis or Septonema fasciculare in a suitable framework for phylogenetic hypothesis testing. Molecular data allowed to fully incorporate all these taxa in Ascomycota, particularly within the classes Dothideomycetes and Sordariomycetes, and to provide a foundation for better taxonomic decisions on their classification. Large and polyphyletic genera such as Taeniolella, Sporidesmium and Septonema, partially treated in this work and containing mostly saprobic species of obscure affinities, remained in need of further investigation.
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.
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.
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.
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.