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Reprogramming biosynthetic assembly-lines is a topic of intense interest. This is unsurprising as the scaffolds of most antibiotics in current clinical use are produced by such pathways. The modular nature of assembly-lines provides a direct relationship between the sequence of enzymatic domains and the chemical structure of the product, but rational reprogramming efforts have been met with limited success. To gain greater insight into the design process, we wanted to examine how Nature creates assembly-lines and searched for biosynthetic pathways that might represent evolutionary transitions. By examining the biosynthesis of the anti-tubercular wollamides, we uncover how whole gene duplication and neofunctionalization can result in pathway bifurcation. We show that, in the case of the wollamide biosynthesis, neofunctionalization is initiated by intragenomic recombination. This pathway bifurcation leads to redundancy, providing the genetic robustness required to enable large structural changes during the evolution of antibiotic structures. Should the new product be non-functional, gene loss can restore the original genotype. However, if the new product confers an advantage, depreciation and eventual loss of the original gene creates a new linear pathway. This provides the blind watchmaker equivalent to the design, build, test cycle of synthetic biology.
Lipopolysaccharide (LPS) is a major glycolipid component in the outer leaflet of the outer membrane of Gram-negative bacteria and known as endotoxin exhibited by the lipid A moiety, which serves as a membrane anchor. The effective permeability barrier properties of the outer membrane contributed by the presence of LPS in the extracellular layer of the outer membrane confer Gram-negative bacteria a high resistance against hydrophobic compounds such as antibiotics, bile salts and detergents to survive in harsh environments. The biogenesis of LPS is well studied in Escherichia coli (herewith E. coli) and the LPS transport (Lpt) is carried out by a transenvelope complex composed of seven essential proteins (LptABCDEFG), which are located in the three compartments of the cell such as the outer membrane, the inner membrane and the periplasm. The Lpt system also exists in Anabaena sp. PCC 7120 (herewith Anabaena sp.), however, homologues of LptC and LptE are still missing. BLAST search failed to identify a homologue of LptC, in contrast, the secondary structure analysis using the Pfam database based on the existing ecLptC secondary structure identified one open reading frame All0231 as the putative Anabaena sp. homologue of LptC, which is designated anaLptC. Despite the low sequence similarity, the secondary structure alignment between anaLptC and ecLptC using the HHpred server showed that both proteins share high secondary structural similarities. The genotypic analysis of the insertion mutant anaLptC did not identify a fully segregated genome and its phenotypic analysis revealed that it was sensitive against chemicals, suggesting that the analptC gene is essential for the growth of Anabaena sp. and involved in the outer membrane biogenesis. This is further supported by the observation of the small cell phenotype in the anaLptC mutant via transmission electron microscopy. Moreover, physical interactions between the anaLptC periplasmic domain with anaLptA as well as with anaLptF were established, indicating that the anaLptC periplasmic domain is correctly folded and alone functional and that the transmembrane helix is not required for the interaction with anaLptA and anaLptF. Furthermore, the reduction of the O-antigen containing LPS was observed in the insertion mutant anaLptC and the dissociation constant Kd of the anaLptC periplasmic domain for ecLPS was determined.The three-dimensional structure of the periplasmic domain of anaLptC was solved by X-ray crystallography with a resolution of 2.8 Å. The structural superposition between the ecLptC crystal structure (PDB number 3my2) and the crystal structure of anaLptC periplasmic domain obtained by this study showed the similarity in the folding of the two proteins with a Cα r.m.s.d value of about 1 Å and confirmed that the length of anaLptC is more than two times longer than that of ecLptC. The structural comparison also revealed that both structures share the typical β-jellyroll fold and conserved amino acids, which were shown in ecLptC to bind to LPS in vivo and found in anaLptC. Overall, these data strongly suggest that anaLptC is involved in the transport of LPS and support the model whereby the bridge spanning the inner membrane and the outer membrane would be assembled via interactions of the structurally conserved β-jellyroll domains shared by five (LptACDFG) out of seven Lpt proteins.
Chemical pollution is one of the main contributors to the degradation of lotic ecosystems and their biodiversity. Among chemicals driving lotic biodiversity decline are anthropogenic organic micropollutants (AOM), which affect the survival and functioning of freshwater organisms. Continuous exposure of freshwater organisms to AOM leads to adverse effects that sometimes cannot be traced with standard toxicity methods such as standard toxicity testing or biodiversity indices. Among these effects of AOM are selective or mutagenic effects that cause impaired species genetic diversity. Thus, the correlation between different levels of AOM and genetic diversity of species is still poorly understood. However, it can be explored by applying population genetics screening.
In Chapter 1 of this thesis, background information on environmental pollution, genetic screening, and the detection of evolutionary-relevant AOM effects in freshwater organisms are described and the thesis goals are identified. The main goal of the thesis is to study whether AOM exposure occurring in European rivers causes a significant evolutionary footprint in freshwater species and leads to a selection of more tolerant geno-and phenotypes. Therefore, population genetics indices together with high-resolution chemical exposure screening of a widespread indicator invertebrate species, Gammarus pulex (Linnaeus, 1758), living in polluted and pristine European rivers were investigated.
In Chapter 2, the development of a genetic screening method for G. pulex (microsatellites) is described. Due to genetic differentiation and the presence of morphologically cryptic lineages, the available sets of target loci do not enable a reliable population genetic characterization of G. pulex from central Germany. Thus, a novel set of microsatellite loci for a high-precision assessment of population genetic diversity was here applied. Eleven loci were first identified and thereafter amplified in G. pulex from three rivers. The new loci reliably amplified and indicated polymorphisms in the studied amphipods. The amplification resulted in the successful identification of genetically distinct populations of G. pulex from the analyzed rivers. Moreover, the microsatellite loci were amplified in other genetic lineages of G. pulex and another Gammarus species, G. fossarum, promising a broader applicability of the loci in related amphipod species.
In Chapter 3, the effects of AOM on species genetic differentiation and sensitivity to toxic chemicals in a typical central European river with pristine and AOM-polluted sections was investigated. The river’s site-specific concentrations of AOM were assessed by chemical analysis of G. pulex tissue and water samples. To test, whether different levels of AOM in the river select for pollution-dependent genotypes, the genetic structure of G. pulex from the river was analyzed. Finally, the toxicokinetics of and sensitivity to the commonly used insecticide imidacloprid were determined for amphipods sampled at pristine and polluted sections to assess whether various levels of AOM in the river influence sensitivity of G. pulex to imidacloprid. The results indicated that different levels of AOM did not drive genetic divergence of G. pulex within the river but led to an increased sensitivity of exposed amphipods to imidacloprid. The amphipods living in polluted river sections were more sensitive to the insecticide due to chronic exposure to toxic levels of AOM.
In Chapter 4, the relationship between site-specific pollution levels of AOM and genetic diversity parameters of G. pulex was analyzed at the regional scale within six rivers in central Germany. The genetic structure of G. pulex in the studied area was tested for relatedness to the waterway distance between sites. Gammarus pulex genetic diversity parameters, including allelic richness and inbreeding rate, were tested against environmental pollution parameters using linear mixed-effect- and structural-equation models. According to the results, G. pulex genetic diversity parameters were significantly associated with the detected AOM levels. At sites with high concentrations of AOM and toxicity potential G. pulex showed reduced genetic diversity and increased rates of inbreeding. These results suggest that AOM play a major role in shaping the genetic diversity of G. pulex in rivers.
According to the findings presented here, the applied microsatellites can be used to successfully detect changes in genetic patterns in freshwater amphipods facing increased levels of AOM. The findings indicate that levels of AOM representative for European rivers do not lead to the separation of genotypes among G. pulex as the connectivity between sites majorly contributes to species’ genetic structure. However, the chronic exposure to increased levels of toxic AOM leads to a reduction of species genetic diversity and increases the sensitivity of G. pulex to the toxic chemical effects.
Prof. Karin Böhning-Gaese, seit 2010 Direktorin des Senckenberg Biodiversität und Klima Forschungszentrums in Frankfurt am Main und Professorin an der Goethe-Universität, wurde in den Rat für Nachhaltige Entwicklung berufen. Das 15-köpfige Gremium berät die Bundesregierung, erarbeitet Beiträge zur Fortentwicklung der Nachhaltigkeitsstrategie, veröffentlicht Stellungnahmen zu Einzelthemen und soll zur öffentlichen Bewusstseinsbildung und zur gesellschaftlichen Debatte über Nachhaltigkeit beitragen.
Mitglieder der ubiquitär verbreiteten Cryptochrom-Photolyase-Familie sind Blaulicht-absorbierende Flavoproteine mit hoher Sequenzhomologie aber diversen Funktionen. Photolyasen katalysieren die Reparatur UV-Licht-induzierter DNA-Schäden. Cryptochrome (CRYs) wirken als lichtunabhängige Transkriptionsrepressoren innerhalb des Kern-Oszillators der circadianen Uhr oder als primäre Photorezeptoren zur Synchronisation dieser mit dem äußeren Tag-Nacht-Rhythmus und steuern durch Regulation der Genexpression Wachstum und Entwicklung. Gemeinsames Strukturmerkmal aller CPF-Vertreter ist die Photolyase- homologe Region (PHR), die das Chromophor Flavinadenindinukleotid (FAD) bindet, das lichtabhängig zwischen den Redoxformen oxidiert (FADox), semireduziert (FAD●- bzw. FADH●) und vollreduziert (FADH-) wechseln kann und damit die CRY-Konformation und -Aktivität beeinflusst. Unterscheidungsmerkmale sind die spezifische C-terminale Erweiterung (CTE) sowie die Komposition der FAD-Bindetasche, die unterschiedliche FAD-Redoxformen stabilisiert. Die Mechanismen der CRY-Photosignaltransduktion sind nicht völlig erforscht.
CryP ist eines von vier CRYs in der Diatomee Phaeodactylum tricornutum und gehört zur bislang nicht charakterisierten Gruppe pflanzenähnlicher CRYs. In vorhergehenden Untersuchungen wurde für CryP eine nukleare Lokalisation und damit verbunden eine blaulicht- sowie dunkelabhängige Regulation der Transkription unterschiedlichster Gene gezeigt. Zudem reguliert CryP das Proteinlevel photosynthetischer Lichtsammelkomplexe. CryP interagiert mit bisher nicht charakterisierten Proteinen aus dem Bereich DNA und Regulation sowie Ribosomen und Translation. Heterolog exprimiertes und isoliertes CryP stabilisiert das Neutralradikal FADH● und das Antennenchromophor Methenyltetrahydrofolat (MTHF).
In vorliegender Dissertation wurde die Bedeutung des FAD-Redoxzustands und der C-terminalen Proteindomäne für Strukturänderungen hinsichtlich der Oligomerisierung und Konformation sowie für das CryP-Interaktionsverhalten untersucht. Hierzu wurden rekombinante CryP-Varianten heterolog isoliert, die Mutationen in für die FAD-Reduzierbarkeit entscheidenden Aminosäuren oder eine Deletion der CTE tragen.
Die Analyse der CryP-Oligomerisierungsstufe und Konformation erfolgte mittels Ko-Präzipitation, nativen und zweidimensionalen PAGEs sowie partieller Proteolyse. Dabei wurde heterolog isoliertes CryP in seinen drei Redoxformen oxidiert (mit FADox), semireduziert (mit FADH●) und vollreduziert (mit FADH-) sowie das um die CTE-verkürzte CryP-PHR verglichen. Für CryP wurde eine redoxunabhängige, PHR-vermittelte Di- und Tetramerisierung über elektrostatische Wechselwirkung der Monomere beobachtet. Die CTE bindet spezifisch und redoxunabhängig an die PHR in einem Bereich um die FAD-Bindetasche. Dies schließt eine großräumige Konformationsänderung zwischen PHR und CTE infolge einer FAD-Photoreduktion wie für pflanzliche und viele tierische CRYs als Aktivierungsmechanismus für CryP aus.
Interaktionsstudien mittels zweidimensionaler PAGE gaben Aufschluss über unterschiedliche Bindeverhalten der beiden betrachteten Interaktionspartner an CryP. Sowohl BolA, ein potentieller redoxregulierter Transkriptionsfaktor, als auch ID42612 mit unbekannter Funktion interagieren mit CryP unabhängig von der FAD-Redoxform. Dabei bindet BolA an die CTE des CryP-Dimers und -Monomers, während ID42612 einen Komplex mit dem CryP-Dimer bildet.
Mittels in vitro Absorptions- und Fluoreszenzspektroskopie wurde die FAD-Redoxchemie von CryP und CryP-PHR verglichen. Die beiden Varianten unterscheiden sich in der FAD-Photoreduzierbarkeit und -Oxidationskinetik. Das Volllängenprotein CryP kann ohne externes Reduktionsmittel zum semireduzierten FADH● phototreduziert werden, das im Gegensatz zu bekannten CRYs über Tage im Dunkeln stabil gegen aerobe Oxidation ist. Eine Belichtung mit Reduktionsmittel führt zur Bildung des vollreduzierten FADH-, das innerhalb von Minuten zu FADH● rückoxidiert. Das um die CTE verkürzte CryP-PHR kann nur mit externem Reduktionsmittel zu FADH● photoreduziert werden, der vollreduzierte Zustand wird nie erreicht. Die Stabilisierung von FADH● gegen aerobe Oxidation im CryP-Holoprotein ist vergleichbar zur FAD-Redoxchemie von Photolyasen. Verglichen mit sonstigen charakterisierten CRYs ist die Wichtigkeit der CTE für eine effiziente FAD-Photoreduktion und FADH●-Stabilisierung eine CryP-spezifische Charakteristik.
Neben der CTE trägt die zu FAD-N5 proximal gelegene Position zur FADH●-Stabilisierung bei, wie Absorptionsmessungen an CryP_N417C zeigten. CryP weist mit Asparagin die gleiche Konservierung an dieser Position wie Photolyasen auf und unterscheidet sich damit ebenfalls von klassischen CRYs.
Analysen zur cryp-Transkription mittels qRT-PCR zeigten eine rhythmische Expression mit maximalen Transkriptmengen in der Nacht und eine rasche photoinduzierte Herunterregulation der Transkription...
D-Galacturonic acid (GalA) is the major constituent of pectin-rich biomass, an abundant and underutilized agricultural byproduct. By one reductive step catalyzed by GalA reductases, GalA is converted to the polyhydroxy acid l-galactonate (GalOA), the first intermediate of the fungal GalA catabolic pathway, which also has interesting properties for potential applications as an additive to nutrients and cosmetics. Previous attempts to establish the production of GalOA or the full GalA catabolic pathway in Saccharomyces cerevisiae proved challenging, presumably due to the inefficient supply of NADPH, the preferred cofactor of GalA reductases. Here, we tested this hypothesis by coupling the reduction of GalA to the oxidation of the sugar alcohol sorbitol that has a higher reduction state compared to glucose and thereby yields the necessary redox cofactors. By choosing a suitable sorbitol dehydrogenase, we designed yeast strains in which the sorbitol metabolism yields a “surplus” of either NADPH or NADH. By biotransformation experiments in controlled bioreactors, we demonstrate a nearly complete conversion of consumed GalA into GalOA and a highly efficient utilization of the co-substrate sorbitol in providing NADPH. Furthermore, we performed structure-guided mutagenesis of GalA reductases to change their cofactor preference from NADPH towards NADH and demonstrated their functionality by the production of GalOA in combination with the NADH-yielding sorbitol metabolism. Moreover, the engineered enzymes enabled a doubling of GalOA yields when glucose was used as a co-substrate. This significantly expands the possibilities for metabolic engineering of GalOA production and valorization of pectin-rich biomass in general.
White stork (Ciconia ciconia) nestlings can provide quantitative information on the quality of the surrounding environment by indicating the presence of pollutants, as they depend on locally foraged food. This study represents the first comparison of biomarkers in two fractions of white stork nestling blood: plasma and S9 (the post-mitochondrial fraction). The aim of this study was to evaluate acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as to establish a novel fluorescence-based method for glutathione (GSH) and reactive oxygen species (ROS) detection in plasma and S9. Considering the enzymatic biomarkers, lower variability in plasma was detected only for AChE, as CES, GST, and GR had lower variability in S9. Enzyme activity was higher in plasma for AChE, CES, and GST, while GR had higher activity in S9. Regarding the fluorescence-based method, lower variability was detected in plasma for GSH and ROS, although higher GSH detection was reported in S9, and higher ROS was detected in plasma. The present study indicated valuable differences by successfully establishing protocols for biomarker measurement in plasma and S9 based on variability, enzyme activity, and fluorescence. For a better understanding of the environmental effects on nestlings’ physiological condition, biomarkers can be measured in plasma and S9.
In welchen Situationen steht ein Tier unter Stress und wie beeinflusst Stress dessen Wohlbefinden? Dies sind die Kernfragen, mit denen Zoos konfrontiert sind, wenn es darum geht, den Bedürfnissen ihrer Tiere gerecht zu werden. Die Beantwortung dieser Fragen ist jedoch angesichts der großen individuellen Variabilität des Inputs, der Stress hervorrufen kann,und des Outputs, der das Wohlbefinden bestimmt, eine Herausforderung. Um diese Herausforderung zu meistern, brauchen Zoos Kenntnisse darüber, welche Haltungsbedingungen und Managementsituationen Verhaltens-, physiologische oder emotionale Veränderungen hervorrufen, sowohl positive als auch negative. Dies trifft insbesondere auf Arten zu, die aufgrund ihrer Biologie und des großen öffentlichen Interesses große Anforderungen an das Management in Menschenobhut stellen, wie den Afrikanischen Elefanten. Die vorliegende Arbeit hatte daher das Ziel, unter Berücksichtigung der individuellen Variation die Auswirkungen bestimmter Managementsituationen auf physiologischen Stress und das Wohlbefinden der Tiere zu evaluieren.
Für diese Arbeit wurden zehn Afrikanische Elefanten aus drei Zoos im Rahmen eines Experiments in 2016 und 2017 mehrmals untersucht. Dieses Experiment umfasste zum einen die Messung von physiologischem Stress auf der Basis der Konzentration des „Stresshormons“ Cortisol im Speichel der Elefanten. Zu diesem Zweck wurden an bestimmten Tagen und zu folgenden Zeitpunkten Speichelproben entnommen: morgens, nachmittags vor und mehrmals nach einer von zwei Managementsituationen (positives Verstärkungstraining [PRT] und neuartiges Enrichmentobjekt [NOV]). Zum anderen diente die Exposition gegenüber dem neuartigen Enrichmentobjekt als sogenannter Novel Object Test. Dieser Standardtest der Persönlichkeitsforschung bei Tieren deckte bei anderen Arten konsistente Verhaltensunterschiede zwischen Individuen auf. Um zu untersuchen, ob dies auch auf Afrikanische Elefanten zutrifft, wurden die individuellen Verhaltensreaktionen auf das neuartige Objekt aufgezeichnet. Darüber hinaus wurden unabhängig von dem Experiment vor und nach einem Transport jeweils morgens und nachmittags Speichelproben von dem transferierten Tier und von zwei Tieren im Bestimmungszoo gesammelt, um den Effekt dieses potenziellen Stressors auf die individuellen Cortisolspiegel zu untersuchen.
Publikation A zeigt, dass die Elefanten unter den Bedingungen des Routinemanagements (das heißt dem routinemäßigen Tagesablauf der Tierpflege) am Morgen signifikant höhere Cortisolwerte im Speichel aufwiesen als am Nachmittag. Diese diurnale Variation der Cortisolsekretion ist typisch für tagaktive Arten und wurde daher auch für die untersuchten Elefanten erwartet. Unter Stressbedingungen wurde weder ein signifikanter Unterschied zwischen den Cortisolspiegeln vor und nach dem Transport noch zwischen den Cortisolwerten am Morgen und am Nachmittag festgestellt. Der prozentuale Unterschied zwischen dem morgendlichen und nachmittäglichen Cortisolspiegel war jedoch beim transferierten Tier nach dem Transport wesentlich geringer als vor dem Transport, was möglicherweise auf eine Stressreaktion auf den Transport und die Eingewöhnung im neuen Zoo hindeutet. Darüber hinaus zeigten sich deutliche Cortisolanstiege unmittelbar nach der ersten Zusammenführung des transferierten Tiers mit dem Bullen im neuen Zoo. Dieses Ergebnis demonstriert zum einen, dass Cortisol physiologischen Stress widerspiegelt. Zum anderen zeigt es die Notwendigkeit, zeitnah nach einem Stressor Speichelproben zu entnehmen, was nach dem Transport nicht möglich war.
Die Studie in Manuskript B zeigt unterschiedliche durchschnittliche Zeitverläufe der Cortisolantworten im Speichel auf die Managementsituationen PRT und NOV. PRT könnte aufgrund des beobachteten cortisolsenkenden und damit potenziell stresspuffernden Effekts förderlich für das Wohlbefinden sein. NOV induzierte im Mittel eine moderate, kurzfristige Cortisolantwort. Dies deutet darauf hin, dass die Tiere geringem physiologischem Stress ausgesetzt waren, mit dem sie jedoch erfolgreich umgehen konnten. Außerdem bestand eine bemerkenswerte individuelle Variation in den Cortisolverläufen in derselben Situation. Die Unterschiede im Cortisolspiegel zwischen den Tieren hingen mit dem Alter (bei NOV) und dem Zoo (bei PRT) zusammen. Der Effekt des Geschlechts und des Haltungssystems auf den Cortisolspiegel war hingegen variabel. Die Ergebnisse der Studie zeigen, dass die individuelle Variation der Cortisolsekretion unbedingt berücksichtigt werden muss, um physiologischen Stress zuverlässig zu erkennen.
Die Studie in Manuskript C ergab, dass sich die untersuchten Tiere im Novel Object Test konsistent in ihrem Verhalten gegenüber einem neuartigen Objekt unterschieden. Dieses Ergebnis zeigt, dass der Novel Object Test auch bei Elefanten genutzt werden kann, um die Persönlichkeit der Tiere zu untersuchen...
In the framework of the PNRA (Italian National Antarctic Research Program) project CARBONANT focusing on biogenic carbonates and held in January–February 2002, several Ross Sea banks were sampled to obtain samples of biogenic carbonates. In the Mawson Bank, species belonging to the isopod genus Chaetarcturus Brandt, 1990 were recorded, including a specimen that did not match any described species. In this paper we describe Chaetarcturus cervicornis sp. n., which is characterized by supraocular spines and two pairs of tubercle-like protrusions on the cephalothorax. The new species is very similar to C. bovinus (Brandt & Wägele, 1988) and C. adareanus (Hodgson, 1902), but has a clearly different spine pattern. The study of the species of the genus Chaetarcturus in the Ross Sea contributes to increase our knowledge on the diversity of the Antarcturidae in the Southern Ocean. Ross Sea banks seem to hold an interesting and not-well-known fauna, deserving attention in future research.
Nonmycorrhizal root-colonizing fungi are key determinants of plant growth, driving processes ranging from pathogenesis to stress alleviation. Evidence suggests that they might also facilitate host access to soil nutrients in a mycorrhiza-like manner, but the extent of their direct contribution to plant nutrition is unknown. To study how widespread such capacity is across root-colonizing fungi, we surveyed soils in nutrient-limiting habitats using plant baits to look for fungal community changes in response to nutrient conditions. We established a fungal culture collection and used Arabidopsis thaliana inoculation bioassays to assess the ability of fungi to facilitate host’s growth in the presence of organic nutrients unavailable to plants. Plant baits captured a representation of fungal communities extant in natural habitats and showed that nutrient limitation has little influence on community assembly. Arabidopsis thaliana inoculated with 31 phylogenetically diverse fungi exhibited a consistent fungus-driven growth promotion when supplied with organic nutrients compared to untreated plants. However, direct phosphorus measurement and RNA-seq data did not support enhanced nutrient uptake but rather that growth effects may result from changes in the plant’s immune response to colonization. The widespread and consistent host responses to fungal colonization suggest that distinct, locally adapted nonmycorrhizal fungi affect plant performance across habitats.
IMPORTANCE: Recent studies have shown that root-associated fungi that do not engage in classical mycorrhizal associations can facilitate the hosts’ access to nutrients in a mycorrhiza-like manner. However, the generality of this capacity remains to be tested. Root-associated fungi are frequently deemed major determinants of plant diversity and performance, but in the vast majority of cases their ecological roles in nature remain unknown. Assessing how these plant symbionts affect plant productivity, diversity, and fitness is important to understanding how plant communities function. Recent years have seen important advances in the understanding of the main drivers of the diversity and structure of plant microbiomes, but a major challenge is still linking community properties with function. This study contributes to the understanding of the cryptic function of root-associated fungi by testing their ability to participate in a specific process: nutrient acquisition by plants.
Cardiolipin, the mitochondria marker lipid, is crucially involved in stabilizing the inner mitochondrial membrane and is vital for the activity of mitochondrial proteins and protein complexes. Directly targeting cardiolipin by a chemical-biology approach and thereby altering the cellular concentration of “available” cardiolipin eventually allows to systematically study the dependence of cellular processes on cardiolipin availability. In the present study, physics-based coarse-grained free energy calculations allowed us to identify the physical and chemical properties indicative of cardiolipin selectivity and to apply these to screen a compound database for putative cardiolipin-binders. The membrane binding properties of the 22 most promising molecules identified in the in silico approach were screened in vitro, using model membrane systems finally resulting in the identification of a single molecule, CLiB (CardioLipin-Binder). CLiB clearly affects respiration of cardiolipin-containing intact bacterial cells as well as of isolated mitochondria. Thus, the structure and function of mitochondrial membranes and membrane proteins might be (indirectly) targeted and controlled by CLiB for basic research and, potentially, also for therapeutic purposes.
Alternative splicing (AS) is a major mechanism for gene expression in eukaryotes, increasing proteome diversity but also regulating transcriptome abundance. High temperatures have a strong impact on the splicing profile of many genes and therefore AS is considered as an integral part of heat stress response. While many studies have established a detailed description of the diversity of the RNAome under heat stress in different plant species and stress regimes, little is known on the underlying mechanisms that control this temperature-sensitive process. AS is mainly regulated by the activity of splicing regulators. Changes in the abundance of these proteins through transcription and AS, post-translational modifications and interactions with exonic and intronic cis-elements and core elements of the spliceosomes modulate the outcome of pre-mRNA splicing. As a major part of pre-mRNAs are spliced co-transcriptionally, the chromatin environment along with the RNA polymerase II elongation play a major role in the regulation of pre-mRNA splicing under heat stress conditions. Despite its importance, our understanding on the regulation of heat stress sensitive AS in plants is scarce. In this review, we summarize the current status of knowledge on the regulation of AS in plants under heat stress conditions. We discuss possible implications of different pathways based on results from non-plant systems to provide a perspective for researchers who aim to elucidate the molecular basis of AS under high temperatures.
First-principle metabolic modelling holds potential for designing microbial chassis that are resilient against phenotype reversal due to adaptive mutations. Yet, the theory of model-based chassis design has rarely been put to rigorous experimental test. Here, we report the development of Saccharomyces cerevisiae chassis strains for dicarboxylic acid production using genome-scale metabolic modelling. The chassis strains, albeit geared for higher flux towards succinate, fumarate and malate, do not appreciably secrete these metabolites. As predicted by the model, introducing product-specific TCA cycle disruptions resulted in the secretion of the corresponding acid. Adaptive laboratory evolution further improved production of succinate and fumarate, demonstrating the evolutionary robustness of the engineered cells. In the case of malate, multi-omics analysis revealed a flux bypass at peroxisomal malate dehydrogenase that was missing in the yeast metabolic model. In all three cases, flux balance analysis integrating transcriptomics, proteomics and metabolomics data confirmed the flux re-routing predicted by the model. Taken together, our modelling and experimental results have implications for the computer-aided design of microbial cell factories.
Patients harboring mutations in the gene DEPDC5 often display variations of neurological diseases including epilepsy, autism spectrum disorders (ASD) and other neuro-architectural alterations. DEPDC5 protein has been identified as an amino acid sensor responsible for negatively regulating the mechanistic target of rapamycin (mTOR), a central regulator in cell growth and cell homeostasis. Often, mutations of the DEPDC5 protein result in mTOR hyperactivity leading to abnormal neuronal phenotypes and the generation of excitatory/inhibitory imbalances in animal models. Complete knockout (KO) of DEPDC5 results in death shortly after birth, while inhibition of mTOR activity recovers postnatal death (Marsan et al. 2016). However, heterozygous DEPDC5-KOs in animals have been variable in their disease phenotypes during adulthood indicating developmental differences between subspecies and early development mechanisms which could be impactful on the outcome of the diseases.
To understand the mechanisms underlying DEPDC5 mutations during early development, a novel primary human neural progenitor cell line extracted from fetal tissue was characterized during proliferation and differentiation. CRISPR-Cas9 induced mutations of the DEPDC5 gene resulted in hyperphosphorylation of mTOR signaling processes and rapid expansion of the neuronal population during differentiation. Analysis of transcriptome data identified deregulation amongst p53 signaling, ribosome biogenesis, nucleotide and lipid synthesis as well as protein degradation pathways due to loss of DEPDC5. Disease gene datasets identified a correlation between Tuberous Sclerosis mutations as being more closely associated with DEPDC5 mutations while also finding overlap with some ASD and epilepsy genes. By using the mTOR inhibitor rapamycin, a substantial amount of the deregulated gene network was recovered while also reversing rapid neuronal differentiation caused by loss of DEPDC5. Though we saw increased dendritic arborization and subsequent decreases in dendrite lengths and soma sizes, rapamycin failed to recover these effects suggesting mTOR independent processes produced by DEPDC5-KO. This study provides new insights on the relationship between mutations in DEPDC5 and the functional, genomic and deregulatory networks it intertwines in humans and highlights that the DEPDC5 associated pathomechanisms are not fully related to mTOR hyperactivation, but include independent processes. This also sheds light on the question why rapamycin treatment only partially restores DEPDC5 related phenotypes and gives insight on treatments for DEPDC5 patients.
Oaks may contribute to the stabilization of European forests under climate change. We utilized two common gardens established in contrasting growth regimes, in Greece (Olympiada) and Germany (Schwanheim), to compare the diurnal photosynthetic performance of a Greek and an Italian provenance of two Mediterranean oaks (Quercus pubescens and Q. frainetto) during the 2019 growing season. Although the higher radiation in the southern common garden led to a strong midday depression of chlorophyll a fluorescence parameters (maximum quantum efficiency of PSII, performance index on absorption basis), comparable light-saturated net photosynthetic rates were achieved in both study areas. Moreover, both species and provenances exhibited analogous responses. Q. pubescens had enhanced chlorophyll a fluorescence traits but similar photosynthetic rates compared to Q. frainetto, whereas the provenances did not differ. These findings indicate the high photosynthetic efficiency of both oaks under the current climate in Central Europe and their suitability for assisted migration schemes.
The Brachybasidiaceae are a family of 22 known species of plant-parasitic microfungi belonging to Exobasidiales, Basidiomycota. Within this family, species of the largest genus Kordyana develop balls of basidia on top of stomatal openings. Basidial cells originate from fungal stroma filling substomatal chambers. Species of Kordyana typically infect species of Commelinaceae. During fieldwork in the neotropics, fungi morphologically similar to Kordyana spp. were found on Goeppertia spp. (syn. Calathea spp., Marantaceae), namely on G. panamensis in Panama and on G. propinqua in Bolivia. These specimens are proposed as representatives of a genus new to science, Marantokordyana, based on the distinct host family and molecular sequence data of ITS and LSU rDNA regions. The specimens on the two host species represent two species new to science, M. oberwinkleriana on G. panamensis and M. boliviana on G. propinqua. They differ by the size and shape of their basidia, molecular sequence data of ITS and LSU rDNA regions, and host plant species. In the past, the understanding of Brachybasidiaceae at order and family level was significantly improved by investigation realized by Franz Oberwinkler and his collaborators at the University of Tübingen, Germany. On species level, however, our knowledge is still very poor due to incomplete species descriptions of several existing names in literature, scarceness of specimens, as well as sequence data lacking for many taxa and for further barcode regions. Especially species of Kordyana and species of Dicellomyces are in need of revision.
Non-ribosomal peptide synthetases (NRPSs) are modular biosynthetic megaenzymes producing many important natural products and refer to a specific set of peptides in bacteria’s and fungi’s secondary metabolism. With the actual purpose of providing advantages within their respective ecological niche, the bioactivity of the structurally highly diverse products ranges from, e.g., antibiotic (e.g., vancomycin) to immunosuppressive (e.g., cyclosporin A) to cytostatic (e.g., echinomycin or thiocoralin) activity.
An NRPS module consists of at least three core domains that are essential for the incorporation of specific substrates with the 'multiple carrier thiotemplate mechanism' into a growing peptide chain: an adenylation (A) domain selects and activates a cognate amino acid; a thiolation (T) domain shuffles the activated amino acid and the growing peptide chain, which are attached at its post-translationally 4ʹ-phosphopantetheine (4'-PPant) group, between the active sites; a condensation (C) domain links the upstream and downstream substrates. NRPS synthesis is finished with the transfer of the assembled peptide to the C-terminal chain-terminating domain. Accordingly, the intermediate is either released by hydrolysis as a linear peptide chain or by an intramolecular nucleophilic attack as a cyclic peptide.
The NRPS’s modular character seems to imply straightforward engineering to take advantage of their features but appears to be more challenging. Since the pioneering NRPS engineering approaches focused on the reprogramming and replacement of A domains, several working groups developed advanced methods to perform a complete replacement of subdomains or single or multiple catalytic domains.
The first part of this work focusses parts of the publication with the title 'De novo design and engineering of non-ribosomal peptide synthetases', which follows up assembly line engineering with the development of a new guideline. Thereby, the pseudodimeric V-shaped structure of the C domain is exploited to separate the N-terminal (CDSub) and C-terminal (CASub) subdomains alongside a four-AA-long linker. This results in the creation of self-contained, catalytically active CASub-A-T-CDSub (XUC) building blocks. As an advantage over the previous XU concept, the characteristics (substrate- and stereoselectivity) assigned to the C domain subunits are likewise exchanged, and thus, no longer represent a barrier. Furthermore, with the XUC concept, no important interdomain interfaces are disrupted during the catalytic cycle of NRPS, allow to expect much higher production titers. Moreover, the XUC concept shows a more flexible application within its genus origin of building blocks to create peptide libraries. Additionally, with this concept only 80 different XUC building blocks are needed to cover the entire proteinogenic amino acid spectrum.
The second part of this work addresses the influence of the C domain on activity and specificity of A domains. In a comprehensive analysis, a clear influence of different C domains on the in vitro activation rate and the in vivo substrate spectrum could be observed. Further in situ and in silico characterizations indicate that these influences are neither the result of the respective A domains promiscuity nor the C domain’s proofreading, but due to an 'extended gatekeeping' function of the C domain. This novel term of an 'extended gatekeeping' function describes the very nature of interfaces that C domains can form with an A domain of interest. Therefore, the C-A interface is assumed to have a more significant contribution to a selectivity filter function.
The third part of this work combines the NRPS engineering with phylogenetic/evolutionary perspectives. At first, the C-A interface could be precisely defined and further identified to encode equivalent information corresponding to the complete C-A didomain. Moreover, the comparison of NRPSs topology reveals hints for a co-evolutionary relatedness of the C-A didomain and could be shown to reassemble even after separation. In this regard, based on a designed CAopt.py algorithm, the reassembling-compatibility of hybrid interfaces could be determined by scoring of the co-expressed NRPS hybrids. This algorithm also enables the randomization of the interface sequences, thus, leading to the identification of more functional interface variant, which cause significantly higher peptide production and could even be applied to other native and hybrid interfaces.
Ischemic heart disease caused by occlusion of coronary vessels leads to the death of downstream tissues, resulting in a fibrotic scar that cannot be resolved. In contrast to the adult mammalian heart, the adult zebrafish heart can regenerate following injury, enabling the study of the underlying cellular and molecular mechanisms. One of the earliest responses that take place after cardiac injury in adult zebrafish is coronary revascularization. Previous transcriptomic data from our lab show that vegfc, a well-known regulator of lymphatic development, is upregulated early after injury and peaks at 96 hours post cryoinjury, coinciding with the peak of coronary endothelial cell proliferation. To test the hypothesis that vegfc is involved in coronary revascularization, I examined its expression pattern and found that it is expressed by coronary endothelial cells after cardiac damage. Using a loss-of-function approach to block Vegfc signaling, I found that it is required for coronary revascularization during cardiac regeneration. Notably, blocking Vegfc signaling resulted in a significant reduction in cardiomyocyte regeneration. Using transcriptomic analysis, I identified the extracellular matrix component gene emilin2a and the chemokine gene cxcl8a as effectors of Vegfc signaling. During cardiac regeneration, cxcl8a is expressed in epicardium-derived cells, while the gene encoding its receptor cxcr1 is expressed on coronary endothelial cells. I found that overexpressing emilin2a increases coronary revascularization, and induces cxcl8a expression. Using loss-of-function approaches, I observed that both cxcl8a and cxcr1 are required for coronary revascularization after cardiac injury.
Altogether, my findings indicate that Vegfc acts as an angiocrine factor that plays an important role in regulating cardiac regeneration in zebrafish. Mechanistically, Vegfc promotes the expression of emilin2a, which promotes coronary proliferation, at least in part by enhancing Cxcl8a-Cxcr1 signaling. This study helps in understanding the mechanisms underlying coronary revascularization during cardiac regeneration, with promising therapeutic applications for human heart regeneration.
Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade.
Die Vorläuferform der eukaryotischen mRNA (prä-mRNA) durchläuft, eine Reihe von Prozessierungs-Schritte, die schließlich zu der Synthese einer „reifen“ und Exportkompetenten mRNA führt. prä-mRNA Spleißen ist ein essentieller Teilschritt dieser Reifung bei der intragene Sequenzen, sogenannte Introns, von der prä-mRNA entfernt werden, während Exons legiert werden. Das prä-mRNA Spleißen wird durch das Spleißosom katalysiert. Dieser Mega-Dalton Komplex, besteht aus fünf Sub-Komplexen, die sich wiederum aus katalytisch aktiven „kleinen nukleären Ribonukleinsäuren“ (snRNAs) und einer Vielzahl von proteinogenen Faktoren zusammensetzen. Diese Subkomplexe, bezeichnet als snRNPs (small nuclear Ribonucleoprotein Particles), binden die prä-mRNA an charakteristischen Sequenzen und richten die prä-mRNA durch eine Reihe von Konformations-Änderungen so aus, dass benachbarte Exons in Kontakt treten und über eine biochemische Ligations-Reaktion verbunden werden können.
Die Exon- bzw Intronerkennung der snRNPs wird durch zahlreiche Spleißfaktoren reguliert. Eine Proteinfamilie, die essentiell für die Regulierung des Spleißens ist, sind Serin/Arginin-reiche Proteine (SR-Proteine). Diese binden vorzugsweise an das 3‘ oder 5’ Ende von Exons, rekrutieren snRNPs und stimulieren dadurch die Exon-Inklusion. Durch diese Stimulierung können Spleiß-Events reguliert und gezielt spezifische Exons ausgeschlossen oder eingeschlossen werden. Dieser Prozess, der als alternatives Spleißen (AS) bezeichnet wird, tritt in 95% des menschlichen Transkriptoms auf und erweitert die Diversität eines Organismus, da verschiedene Transkripte von demselben Gen erzeugt werden können und folglich die Translation unterschiedlicher Proteine mit distinkten Funktionen ermöglicht wird.
Darüber hinaus verfügt die Zelle durch das AS über eine weitere posttranskriptionale Genregulationsebene, die insbesondere unter zellulären Stressbedingungen zur Expression von alternativen Protein-Isoformen von der Zelle genutzt wird. Eine in medizinischer Hinsicht besonders relevante Stressbedingung ist die sogenannte Hypoxie, die eine Sauerstoff-Unterversorgung von Zellen oder Gewebebereichen beschreibt. Hypoxie bzw. hypoxische Bereiche finden sich in Krebszellen und treten in 90% aller soliden Tumoren auf. Als Teil der Hypoxie Stress-Antwort, verfügt die Zelle über einen Adaptations-Mechanismus, der durch Hypoxieinduzierbare Faktoren (HIF) vermittelt wird. Diese Faktoren induzieren die Transkription zahlreicher Gene und stimulieren die Expression von Stressfaktoren, die an der zellulären Adaption der Hypoxie beteiligt sind. Einer dieser Faktoren ist der vaskuläre endotheliale Wachstumsfaktor A (VEGFA), welcher unter hypoxischen Bedingungen sekretiert wird und dadurch die Proliferation von Endothelzellen, die Neubildung von Blutgefäßen und damit die Vaskularisation des hypoxischen Bereichs stimuliert.
Die zelluläre Anpassung ist jedoch nicht nur auf die transkriptionelle Regulation des HIF-vermittelten Hypoxie Signalwegs beschränkt, sondern wird auf multiplen Genexpressions-Ebenen reguliert. Obwohl bekannt ist, dass tausende Transkripte unter hypoxischen Bedingungen alternativ gespleißt werden, sind die Faktoren, die die zelluläre Stress-Antwort durch AS regulieren, sowie deren molekularer Mechanismus jedoch weitestgehend unbekannt.
Diese Arbeit umfasst die Identifizierung und Charakterisierung von AS Events, sowie den Einfluss und die Regulation von Spleißfaktoren auf AS unter hypoxischen Bedingungen. Hierzu führten wir globale Genexpressions- und AS-Analysen in HeLaKarzinomzelllinien unter Normoxie (21% O2) und Hypoxie (0.2% O2) durch und zeigen, dass 7962 Gene nach 24h Hypoxie unterschiedlich exprimiert werden. Über AS-Analysen konnten 4434 Transkripte identifiziert werden, die bei Hypoxie über AS reguliert sind. Dabei trat „Exon-Skipping“ als das am häufigsten auftretende AS-Events auf. Über PCR basierte Validierungs-Experimente konnten 5 regulierte Transkripte nachgewiesen werden. Dabei weisen Exon 3 und 4 in BORA, Exon 6 in MDM4 und Exon 4-5 in CSSP1 Exon-Skipping Events auf, während Exon-Inklusionen in CEP192 Exon 28 und in der 3’UTR von EIF4A2 validiert werden konnten.
Darüber hinaus wurde im Rahmen der AS-Analyse die Regulation des sogenannten „backsplicings“ bei Hypoxie untersucht. Im Gegensatz zum linearen Spleißens, wird beim backsplicing das 5’Ende und das 3’Ende von Exons verbunden, was die Bildung von sogenannten zirkulären RNAs (circRNAs) zufolge hat. Obwohl nur wenige Funktionen dieser RNA-Klasse bekannt sind, wurde die Regulation von circRNAs während der Zell-Differenzierung sowie in diversen Krebszellen beschrieben. Dabei können circRNAs als microRNA- oder Protein-Schwämme fungieren oder dienen als Protein-Interaktion Plattform und regulieren dabei die Genexpression.