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Measuring connection to nature - a illustrated extension of the inclusion of nature in self scale
(2021)
The human-nature connection is an important factor that is frequently the subject of environmental education research and environmental psychology. Therefore, over the years, numerous measuring instruments have been established to quantitatively record a person’s connection to nature. However, there is no instrument specifically for children with cognitive limitations. For this reason, in this study, an established scale for connection to nature, the inclusion of nature in self scale (INS), was modified especially for the needs of this group. Study 1 investigated what students understand by the term “nature” in order to create an illustrated version of the INS. In study 2, the new instrument was tested on university students and compared with the original INS and the connectedness to nature scale (CNS). No significant differences between the original INS and the new developed scale were found (p = 0.247), from which it can be concluded that the illustrated INS (IINS) measures the connection to nature with similar accuracy as the original INS. In study 3, the instrument was tested together with other established nature connection instruments on the actual target group, students with disabilities. The correlation between the IINS, the CNS, and nature connectedness scale (NR) were in accordance with the expected literature values (rIINS-CNS = 0.570 & rIINS-NR = 0.605). The results of this study also prove effectiveness of the developed illustrated scale. This research thus provides a suitable measuring instrument for people with learning difficulties and can make a contribution to the investigation of human-nature connections and conservation education.
The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis
(2021)
The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T-cell-directed therapies fail to elicit effective anti-tumor immune responses. Here, we seek to evaluate the applicability of radio- immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapy- induced immune modulation resulted in an increase in cytotoxic T-cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experi- mental breast-to-brain metastasis. However, long-term efficacy was not observed. Recurrent brain metastases showed accumula- tion of blood-borne PD-L1+ myeloid cells after radio-immunother- apy indicating the establishment of an immune suppressive environment to counteract re-activated T-cell responses. This finding was further supported by transcriptional analyses indicat- ing a crucial role for monocyte-derived macrophages in mediating immune suppression and regulating T-cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases.
The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881–1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
Studium der Lebewesen : im Masterprogramm »Ökologie und Evolution« wird Diversität großgeschrieben
(2021)
Viele Gruppen der Lebewesen, insbesondere Insekten breiten sich durch steigende Temperaturen zunehmend in Gebieten aus, in denen sie ursprünglich nicht vorkommen(Novikov und Vaulin 2014; Bebber 2015). Hierbei ist die steigende Temperatur in
verschiedenen Gebieten der Hauptfaktor für Expansionen dieser Arten in Richtung des nördlichen Polarkreises. Einige dieser Arten sind sehr tolerant für verschiedene Variablen und können damit ihr Verbreitungsgebiet deutlich nach Norden hin ausdehnen. Aufgrund steigender Temperaturen werden jedoch andere Arten in ihrem Verbreitungsgebiet eingeschränkt oder ihre Verbreitung verschiebt sich in nördliche Richtung (Ogden und Lindsay 2016; Lawler et al. 2009). Auch für die Verbreitung von Krankheiten spielen Temperaturen, Ausbreitungen oder Verbreitungsverschiebungen eine wichtige Rolle (Mordecai et al. 2019).
So können, durch die Etablierung der passenden Vektoren, bisher nur in wärmeren Gebieten auftretende Krankheiten zukünftig auch in unseren Breitengraden eingeschleppt und
verbreitet werden. Bremsen, invasive Stechmücken aber auch einheimische Mücken tragen alle ein Potential,verschiedenste Krankheitserreger zu verbreiten, auch wenn die Eignung als
Vektor für jede Art unterschiedlich groß ausfällt und manche Arten daher kaum beobachtet und untersucht werden. Mit dem Augenmerk auf sich ändernde Verbreitungsgebiete hinsichtlich zukünftigen klimatischen Veränderungen und sich wandelnden anthropogenen Einflüssen sollten jedoch auch Arten mit bisher geringem Vektorpotential mit in Beobachtungsprogramme aufgenommen werden.
Wir untersuchten in Projekt I auf kontinentaler Skala die Verbreitung von sechs verschiedenen Bremsenarten und konnten sowohl Rückschlüsse auf eine mangelhafte Beobachtung der
Arten ziehen als auch Artpräferenzen hinsichtlich der Landschaftsnutzung, Auswirkungen des Klimas auf die Verbreitung der Art und bisher unbekannte Toleranzen hinsichtlich tiefen Temperaturen und äußerst verkürzten Wärmeperioden aufdecken. Eine Größenordnung niedriger wurde in Projekt II, basierend auf aktuellen und Vergangenen Klimadaten, die zukünftige und aktuelle Verbreitung einer invasiven, sich zukünftig ausbreitenden Stechmückenart innerhalb Deutschlands modelliert. Durch bisherig im Untersuchungsgebiet nur begrenztes Auftreten konnten noch keine Rückschlüsse auf die unterschiedlichen Präferenzen für das Habitat gezogen werden, es können jedoch für zukünftige Berechnungen Habitatpräferenzen aus anderen Gebieten hinzugezogen werden um die Art und ihre fortschreitende Ausbreitung genauer zu beobachten. Auf der kleinsten untersuchten Ebene konnten in Projekt III innerhalb eines Mikrohabitates verschiedenste Rückschlüsse auf limitierende oder förderliche abiotische Faktoren, die teilweise bisherig nicht oder nur geringfügig beobachtet wurden, gezogen werden. Ebenfalls konnten Auswirkungen der umgebenden Landschaft auf die Abundanzen der Tiere beobachtet werden. Mithilfe von verschiedenen Modellen und in Abhängigkeit von Klimakarten, Landbedeckungsdaten und Landnutzung sowie Eigenschaften und Toleranzen der untersuchten Arten lassen sich in verschiedenen Größenordnungen geeignete Habitate von einheimischen sowie invasiven Arten identifizieren und zukünftige Verbreitungen effizient vorhersagen.
Insgesamt können, basierend auf all diesen Daten, dadurch für alle untersuchten Faktoren Modelle auf andere Gebiete übertragen werden um somit potentielle Verbreitungen dort
vorherzusagen. Auf unseren Daten basierend können so zum Beispiel Modellierungen für die potentielle Ausbreitung der untersuchten Tabaniden innerhalb anderer Kontinente berechnet werden und Monitoringprogramme können die Ergebnisse unserer Studie als Startpunkt aufgreifen, um durch Beprobung an modellierten Standorten die Korrektheit unserer Modelle zu überprüfen und sowohl Landschaftstypen als auch Artzusammensetzung aufzunehmen um das Modell zu bestätigen oder zu verbessern. Die Modellierung der invasiven Art Aedes albopictus bietet die Möglichkeit, diese Art in Zukunft innerhalb der möglichen Ausbreitungskorridore genauer zu beobachten um ihre fortschreitende Verbreitung zu
verifizieren oder eventuelle Änderungen des klimatischen Verlaufes mit einzubinden und das Modell anzupassen. Die Untersuchung des Mikrohabitats von Culex pipiens pipiens und Culex torrentium bietet, auch hinsichtlich anderer Arten in diesem Habitat, eine potente Methode, Vorhersagen für Artvorkommen innerhalb anderer Unterirdischen Objekte zu berechnen. Hier können, bei ausreichend großer Datenlage, eine Vielzahl von Faktoren in die Auswertung mit einfließen.
Die durchgeführten Studien bestätigen die Notwendigkeit für verbesserte Monitoringkonzepte für alle vektorkompetenten Tiergruppen hinsichtlich der sich ändernden klimatischen Bedingungen, des globalen Handels und die sich wandelnde Nutzung der Landschaften durch den Menschen und darin begründete Veränderungen der Artenzusammensetzung eines Habitates, zeigen Möglichkeiten, diese Konzepte mit bisher
ungenutzten Daten aufzubauen und zu verbessern und können gleichzeitig zu deren Verbesserung herangezogen werden.
Gene conversion is defined as the non-reciprocal transfer of genetic information from one site to a homologous, but not identical site of the genome. In prokaryotes, gene conversion can increase the variance of sequences, like in antigenic variation, but can also lead to a homogenization of sequences, like in the concerted evolution of multigene families. In contrast to these intramolecular mechanisms, the intermolecular gene conversion in polyploid prokaryotes, which leads to the equalization of the multiple genome copies, has hardly been studied. We have previously shown the intermolecular gene conversion in halophilic and methanogenic archaea is so efficient that it can be studied without selecting for conversion events. Here, we have established an approach to characterize unselected intermolecular gene conversion in Haloferax volcanii making use of two genes that encode enzymes involved in carotenoid biosynthesis. Heterozygous strains were generated by protoplast fusion, and gene conversion was quantified by phenotype analysis or/and PCR. It was verified that unselected gene conversion is extremely efficient and it was shown that gene conversion tracts are much longer than in antigenic variation or concerted evolution in bacteria. Two sites were nearly always co-converted when they were 600 bp apart, and more than 30% co-conversion even occurred when two sites were 5 kbp apart. The gene conversion frequency was independent from the extent of genome differences, and even a one nucleotide difference triggered conversion.
Nature and its constituents are known to affect human well-being in positive and negative ways. Nature can be beneficial for humans by providing, for instance, food, recreation or inspiration. Natural disasters or transmitted diseases are, on the other hand, examples of nature’s detrimental or harmful contributions to human well-being. Such positive as well as negative effects have been termed Nature’s Contributions to People (NCP) by the Intergovernmental Science-Policy Platform for Biodiversity and Ecosystem Services (IPBES) and can be categorized into three different types of contributions: regulating, material and non-material NCP. While regulating and material NCP have been studied extensively, research on the non-material NCP is less common in comparison, especially regarding non-material NCP of biodiversity and wildlife. This dissertation therefore aims at shedding light on the non-material links between biodiversity, wildlife and human well-being. The thesis presents the results of three individual research studies in three separate chapters (CH1, 2 & 3).
In the first chapter (CH1) I conduct a systematic literature review on the non-material contributions of wildlife. Several previous reviews have published overviews on the non-material contributions of wildlife. However, only a few of these reviews examine both the positive and negative effects of wildlife in combination. These reviews usually cover few aspects of human well-being (e.g. recreation, health, psychological well-being) or just focus on a specific group of wildlife species (e.g. carnivores, scavengers). In addition, the pathways determining how wildlife affects human well-being are yet little understood. The aim of this review is therefore to create a holistic overview of the current knowledge on non-material contributions of wildlife (WCP), by summarising research on positive and negative effects and disentangling potential channels of human-wildlife experiences.
My results show that most studies in scientific literature report negative WCP. However, over the last decade the number of publications on positive WCP has increased, mainly in the Global North. This change in research focus, at the turn of the century, may be related to the newly emerging ideas and perspectives on nature during that time (e.g. Ecosystem Services and NCP). The results may also indicate different research interests across global regions and a focus on positive WCP (especially in the Global North). Surprisingly, the review identifies a lack of joint systematic assessments of positive and negative WCP across taxa, human well-being dimensions and ways (channels) of wildlife experiences. Studies show taxon-specific differences, with predominantly positive WCP reported for birds and predominantly negative WCP published for mammals and reptiles. Physical health was the most examined human well-being dimension, while many others, such as subjective well-being, social well-being, learning, identity or sense of place were rarely studied in comparison. The two channels of wildlife experiences that have been mainly studied or reported are Interaction and Knowing. While Interaction describes multisensory experiences in which people physically interact with wildlife. Knowing describes the metaphysical connection between humans and wildlife that arises through thinking or remembering experiences from wildlife encounters (including knowledge about wildlife).
To date, only few published studies examine the relationship between biodiversity and human well-being across larger spatial scales. For instance, little is known about how biodiversity is related to human well-being on the national or continental level. The second and third chapter (CH2 & 3) are thus comprised of two empirical case studies which examine the relationship between biodiversity and human well-being across Germany and Europe, respectively. As indicator for biodiversity, I use different species diversity measures including species richness and abundance. In the second chapter (CH2) I analyse the association between species richness and human health across Germany. The results demonstrate a significant positive relationship between plant and bird species richness and mental health while controlling for a multitude of socio-economic and demographic factors as well as other nature characteristics. In the third chapter (CH3) I conduct the first study on the relationship between species diversity and subjective well-being on the continental level. The results show that bird species richness (unlike mammal, megafauna and tree richness) is positively associated with life-satisfaction, a measure for subjective well-being across Europe. These results are robust while accounting for socio-economic and macro-economic factors. The results of both empirical studies are in correspondence with previous research, conducted on the local and national level.
Overall, my dissertation shows that wildlife and biodiversity greatly affect human well-being and provide substantial non-material NCP.
...
The geomagnetic field provides directional information for birds. The avian magnetic compass is an inclination compass that uses not the polarity of the magnetic field but the axial course of the field lines and their inclination in space. It works in a flexible functional window, and it requires short-wavelength light. These characteristics result from the underlying sensory mechanism based on radical pair processes in the eyes, with cryptochrome suggested as the receptor molecule. The chromophore of cryptochrome, flavin adenine dinucleotide (FAD), undergoes a photocycle, where radical pairs are formed during photo-reduction as well as during re-oxidation; behavioral data indicate that the latter is crucial for detecting magnetic directions. Five types of cryptochromes are found in the retina of birds: cryptochrome 1a (Cry1a), cryptochrome 1b, cryptochrome 2, cryptochrome 4a, and cryptochrome 4b. Because of its location in the outer segments of the ultraviolet cones with their clear oil droplets, Cry1a appears to be the most likely receptor molecule for magnetic compass information.
Microglia, the primary immune cells of the central nervous system, hold a multitude of tasks in order to ensure brain homeostasis and are one of the best predictors of biological age on a cellular level. We and others have shown that these long-lived cells undergo an aging process that impedes their ability to perform some of the most vital homeostatic functions such as immune surveillance, acute injury response, and clearance of debris. Microglia have been described as gradually transitioning from a homeostatic state to an activated state in response to various insults, as well as aging. However, microglia show diverse responses to presented stimuli in the form of acute injury or chronic disease. This complexity is potentially further compounded by the distinct alterations that globally occur in the aging process. In this review, we discuss factors that may contribute to microglial aging, as well as transcriptional microglia alterations that occur in old age. We then compare these distinct phenotypic changes with microglial phenotype in neurodegenerative disease.
Taxa under scrutiny in this thesis are Halophytophthora-like oomycetes. The genus Halophytophthora, proposed in 1990, is an assemblage of unrelated species grouped together on the basis habitat preference, i.e. the mangrove or saltmarsh biome, and morphological similarity to Phytophthora. The premise “Phytophthora-like species from the mangrove environment” became the genus concept for Halophytophthora and lasted for almost 2 decades which resulted to the addition of several species (i.e. H. elongata, H. exoprolifera, H. porrigovesica, H. kandeliae, H. masteri, and H. tartarea). At the onset of molecular phylogenetics, Halophytophthora was inferred as a highly polyphyletic taxon and the genus concept was found to be unsuitable. This thesis adds to this, since six Phytophthora spp. were isolated from the mangrove environment, two of which were found in the Philippines (Phytophthora elongata and Phytophthora insolita). After a thorough assessment of the morphologic and phylogenetic data of taxa included in this thesis, several taxonomic novelties were introduced – a new family (Salispinaceae), a new genus (Calycofera), new species (Calycofera cryptica, Phytopythium dogmae, Phytopythium leanoi, Salisapilia coffeyi, and Salispina hoi), and new combinations (Calycofera operculata, Salisapilia bahamensis, S. elongata, S. epistomia, S. masteri, S. mycoparasitica). In addition, Salisapiliaceae and Salisapilia were emended.
Research on Podospora anserina unraveled a network of molecular pathways affecting biological aging. In particular, a number of pathways active in the control of mitochondria were identified on different levels. A long-known key process active during aging of P. anserina is the age- related reorganization of the mitochondrial DNA (mtDNA). Mechanisms involved in the stabilization of the mtDNA lead to lifespan extension. Another critical issue is to balance mitochondrial levels of reactive oxygen species (ROS). This is important because ROS are essential signaling molecules, but at increased levels cause molecular damage. At a higher level of the network, mechanisms are active in the repair of damaged compounds. However, if damage passes critical limits, the corresponding pathways are overwhelmed and impaired molecules as well as those present in excess are degraded by specific enzymes or via different forms of autophagy. Subsequently, degraded units need to be replaced by novel functional ones. The corresponding processes are dependent on the availability of intact genetic information. Although a number of different pathways involved in the control of cellular homeostasis were uncovered in the past, certainly many more exist. In addition, the signaling pathways involved in the control and coordination of the underlying pathways are only initially understood. In some cases, like the induction of autophagy, ROS are active. Additionally, sensing and signaling the energetic status of the organism plays a key role. The precise mechanisms involved are elusive and remain to be elucidated.
Mitochondrial F1Fo-ATP-synthase dimers play a critical role in shaping and maintenance of mitochondrial ultrastructure. Previous studies have revealed that ablation of the F1Fo-ATP-synthase assembly factor PaATPE of the ascomycete Podospora anserina strongly affects cristae formation, increases hydrogen peroxide levels, impairs mitochondrial function and leads to premature cell death. In the present study, we investigated the underlying mechanistic basis. Compared to the wild type, we observed a slight increase in non-selective and a pronounced increase in mitophagy, the selective vacuolar degradation of mitochondria. This effect depends on the availability of functional cyclophilin D (PaCYPD), the regulator of the mitochondrial permeability transition pore (mPTP). Simultaneous deletion of PaAtpe and PaAtg1, encoding a key component of the autophagy machinery or of PaCypD, led to a reduction of mitophagy and a partial restoration of the wild-type specific lifespan. The same effect was observed in the PaAtpe deletion strain after inhibition of PaCYPD by its specific inhibitor, cyclosporin A. Overall, our data identify autophagy-dependent cell death (ADCD) as part of the cellular response to impaired F1Fo-ATP-synthase dimerization, and emphasize the crucial role of functional mitochondria in aging.
Nomadic movements are often a consequence of unpredictable resource dynamics. However, how nomadic ungulates select dynamic resources is still understudied. Here we examined resource selection of nomadic Mongolian gazelles (Procapra gutturosa) in the Eastern Steppe of Mongolia. We used daily GPS locations of 33 gazelles tracked up to 3.5 years. We examined selection for forage during the growing season using the Normalized Difference Vegetation Index (NDVI). In winter we examined selection for snow cover which mediates access to forage and drinking water. We studied selection at the population level using resource selection functions (RSFs) as well as on the individual level using step-selection functions (SSFs) at varying spatio-temporal scales from 1 to 10 days. Results from the population and the individual level analyses differed. At the population level we found selection for higher than average NDVI during the growing season. This may indicate selection for areas with more forage cover within the arid steppe landscape. In winter, gazelles selected for intermediate snow cover, which may indicate preference for areas which offer some snow for hydration but not so much as to hinder movement. At the individual level, in both seasons and across scales, we were not able to detect selection in the majority of individuals, but selection was similar to that seen in the RSFs for those individuals showing selection. Difficulty in finding selection with SSFs may indicate that Mongolian gazelles are using a random search strategy to find forage in a landscape with large, homogeneous areas of vegetation. The combination of random searches and landscape characteristics could therefore obscure results at the fine scale of SSFs. The significant results on the broader scale used for the population level RSF highlight that, although individuals show uncoordinated movement trajectories, they ultimately select for similar vegetation and snow cover.
Identification and regulation of tomato Serine/Arginine-rich proteins under high temperatures
(2021)
Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.
The combined behaviours of individuals within insect societies determine the survival and development of the colony. For the western honey bee (Apis mellifera), individual behaviours include nest building, foraging, storing and ripening food, nursing the brood, temperature regulation, hygiene and defence. However, the various behaviours inside the colony, especially within the cells, are hidden from sight, and until recently, were primarily described through texts and line drawings, which lack the dynamics of moving images. In this study, we provide a comprehensive source of online video material that offers a view of honey bee behaviour within comb cells, thereby providing a new mode of observation for the scientific community and the general public. We analysed long-term video recordings from longitudinally truncated cells, which allowed us to see sideways into the cells in the middle of a colony. Our qualitative study provides insight into worker behaviours, including the use of wax scales and existing nest material to remodel combs, storing pollen and nectar in cells, brood care and thermoregulation, and hygienic practices, such as cannibalism, grooming and surface cleaning. We reveal unique processes that have not been previously published, such as the rare mouth-to-mouth feeding by nurses to larvae as well as thermoregulation within cells containing the developing brood. With our unique video method, we are able to bring the processes of a fully functioning social insect colony into classrooms and homes, facilitating ecological awareness in modern times. We provide new details and images that will help scientists test their hypotheses on social behaviours. In addition, we encourage the non-commercial use of our material to educate beekeepers, the media and the public and, in turn, call attention to the general decline of insect biomass and diversity.
Nematophilic bacteria as a source of novel macrocyclised antimicrobial non-ribosomal peptides
(2020)
A solution to ineffective clinical antimicrobials is the discovery of new ones from under-explored sources such as macrocyclic non-ribosomal peptides (NRP) from nematophilic bacteria. In this dissertation an antimicrobial discovery process –from soil sample to inhibitory peptide– is demonstrated through investigations on six nematophilic bacteria: Xenorhabdus griffiniae XN45, X. griffiniae VH1, Xenorhabdus sp. nov. BG5, Xenorhabdus sp. nov. BMMCB, X. ishibashii and Photorhabdus temperata. To demonstrate the first step of bacterium isolation and species delineation, endosymbionts were isolated from Steinernema sp. strains BG5 and VH1 that were isolated directly from soil samples in Western Kenya. After genome sequencing and assembly of novel Xenorhabdus isolates VH1 and BG5, species delineation was done via three overall genome relatedness indices. VH1 was identified as X. griffiniae VH1, BG5 as Xenorhabdus sp. nov. BG5 and X. griffiniae BMMCB was emended to Xenorhabdus sp. nov. BMMCB. The nematode host of X. griffiniae XN45, Steinernema sp. scarpo was highlighted as a putative novel species. To demonstrate the second step of genome mining and macrocyclic non-ribosomal peptide structure elucidation, chemosynthesis and biosynthesis, the non-ribosomal peptide whose production is encoded by the ishA-B genes in X. ishibashii was investigated. Through a combination of refactoring the ishA-B operon by a promoter exchange mechanism, isotope labelling experiments, high resolution tandem mass spectrometry analysis, bioinformatic protein domain analysis and chemoinformatic comparisons of actual to hypothetical mass spectrometry spectra, the structures of Ishipeptides were elucidated and confirmed by chemical synthesis. Ishipeptide A was a branch cyclic depsidodecapeptide macrocyclised via an ester bond between serine and the terminal glutamate. It chemosynthesis route was via a late stage macrolactamation and linearised Ishipeptide B was synthesised via solid phase iterative synthesis. Ishipeptides were not N-terminally acylated despite being biosynthesised from the IshA protein that had a C-starter domain. It was highlighted that more than restoration of the histidine active site of this domain is required to restore N-terminal acylation activity.
To demonstrate the final step of determination of antimicrobial activity, minimum inhibitory concentrations of Ishipeptides and Photoditritide from Photorhabdus temperata against fungi and bacteria were determined. None were antifungal while only the macrocyclic compounds were inhibitory, with Ishipeptide A inhibitory to Gram-positive bacteria at 37 µM. The cationic Photoditritide, a cyclic hexapeptide macrocyclised via a lactam bond between homoarginine and tryptophan, was 12 times more inhibitory (3.0 µM), even more effective than a current clinical compound, Ampicillin (4.2 µM). For both, macrocyclisation was hypothesised to contribute to antimicrobial activity. Ultimately, this dissertation demonstrated not only nematophilic bacteria as a source of novel macrocyclic antimicrobial non-ribosomal peptides but also a process of antimicrobial discovery–from soil sample to inhibitory peptide– from these useful bacteria genera. This is significant for the fight against antimicrobial resistance.
Organismic aging is known to be controlled by genetic and environmental traits. Pathways involved in the control of cellular metabolism play a crucial role. Previously, we identified a role of PaCLPP, a mitochondrial matrix protease, in the control of the mitochondrial energy metabolism, aging, and lifespan of the fungal aging model Podospora anserina. Most surprisingly, we made the counterintuitive observation that the ablation of this component of the mitochondrial quality control network leads to lifespan extension. In the current study, we investigated the role of energy metabolism of P. anserina. An age-dependent metabolome analysis of the wild type and a PaClpP deletion strain verified differences and changes of various metabolites in cultures of the PaClpP mutant and the wild type. Based on these data, we generated and analyzed a PaSnf1 deletion mutant and a ΔPaSnf1/ΔPaClpP double mutant. In both mutants PaSNF1, the catalytic α-subunit of AMP-activated protein kinase (AMPK) is ablated. PaSNF1 was found to be required for the development of fruiting bodies and ascospores and the progeny of sexual reproduction of this ascomycete and impact mitochondrial dynamics and autophagy. Most interestingly, while the single PaSnf1 deletion mutant is characterized by a slight lifespan increase, simultaneous deletion of PaSnf1 and PaClpP leads to a pronounced lifespan extension. This synergistic effect is strongly reinforced in the presence of the mating-type “minus”-linked allele of the rmp1 gene. Compared to the wild type, culture temperature of 35°C instead of the standard laboratory temperature of 27°C leads to a short-lived phenotype of the ΔPaSnf1/ΔPaClpP double mutant. Overall, our study provides novel evidence for complex interactions of different molecular pathways involved in mitochondrial quality control, gene expression, and energy metabolism in the control of organismic aging.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative movement disorder caused by expansion of CAG repeats in the ATXN2 gene beyond 33 units, while healthy individuals carry 22-23 repeats. First symptoms of SCA2 include uncoordinated movement, ataxic gait and slowing of the saccadic eye movements in line with the early pronounced atrophy of cerebellum, spinal cord and brainstem. Cerebellar Purkinje cells and spinal cord motor neurons are the most affected cells from ATXN2 expansions. Later on, patients manifest distal amyotrophy, problems in breathing and swallowing, depression and cognitive decline caused by widespread degeneration throughout the brain. The striking loss of mass in the brain, due to severe myelin fat atrophy, is accompanied by a similar reduction in the peripheral fat stores. After the devastating progression of disease, the severity and duration of which depends on the CAG repeat size, genetic background and environmental factors, patients succumb to SCA2 mostly because of respiratory failure at the terminal stage. Larger repeat sizes lead to an earlier manifestation of the disease and a more rapid progression. Aside from SCA2, intermediate-length and short pathogenic CAG expansions in ATXN2 between 26-39 repeats significantly increase the risk of developing other neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), fronto-temporal lobar dementia (FTLD) or Parkinson plus tauopathies like progressive supranuclear palsy (PSP) in various cohorts across the world.
Ataxin-2 (ATXN2) is a ubiquitously expressed cytosolic protein most famous for its involvement in neurodegenerative disease caused by the expanded poly-glutamine (polyQ) domain corresponding to a genomic (CAG)n tract. This N-terminal polyQ domain has no known function, other than increasing the aggregation propensity of mutant ATXN2 and facilitating interaction with other polyQ containing proteins, leading to their sequestration. The progressive accumulation of ATXN2 into cytosolic foci, and also that of its interaction partners over time, underlies the molecular pathomechanism. Next to polyQ domain, ATXN2 also contains a Like-Sm domain (Lsm), an Lsm-associated domain (LsmAD), multiple proline-rich domains (PRD) and a Poly(A)-Binding-Protein (PABP)-interacting motif (PAM2).
Through its Lsm/LsmAD domains, ATXN2 directly binds to a large number of transcripts, regulating their quality and translation rate. In a similar fashion, through its direct interaction with PABP via PAM2 motif, ATXN2 indirectly modifies the fate of even larger number of transcripts and global translation. Several PRDs scattered across the protein help ATXN2 associate with growth factor receptors and other endocytosis factors, modulating nutrient uptake and downstream signaling.
ATXN2 is a stress response factor. Therefore, its involvement in nutrient uptake plays a crucial part in cell’s capability to overcome non-permissive conditions. Upon nutrient deprivation, oxidative stress, proteotoxicity, heat stress or Ca2+ imbalance, ATXN2 relocalizes into cytosolic ribonucleoprotein particles known as stress granules (SGs), together with PABP, several eukaryotic translation initiation factors, many other RNA-binding proteins (RBP) with their target transcripts and the small ribosomal subunit. Collectively, they modulate the stability of the trapped transcripts, favoring the maturation and translation of IRES-dependent stress response proteins instead, according to the specific need. Many RBPs interact either directly or in an RNA-dependent manner in the SGs, and due to the large number of ALS-causing mutations identified in them (such as TDP-43, FUS, TIA-1, hnRNPA2/B1), SGs became a hot topic in neuropathology. Acute SGs serve to halt translation and growth, and to spend energy only for survival until stress disappears. However, chronic SG assembly eventually activates apoptotis leading to cell death. While the polyQ expansions in ATXN2 enhance SG stability, reduce their dissociation rate after stress, and lead to aberrant post-translational modifications of other SG components like TDP-43, complete loss of ATXN2 delays SG formation and results in easily dissolvable foci.
Most of the stressors that induce SG formation eventually converge on energetic deficit. Therefore, it is logical that the ultimate task of SGs is to stop further growth when it cannot be afforded. In yeast, the molecular mechanism underlying this growth arrest was explained as sequestration of the master growth regulator complex, Target-of-Rapamycin Complex 1 (TORC1), into SGs in an ATXN2-dependent manner. The repressor effect of ATXN2 on mammalian TORC1 (mTORC1) and global protein translation had already been documented in earlier studies; complete loss of ATXN2 function in knock-out mouse (Atxn2-KO) resulted in mTORC1 hyperactivity and transcriptional upregulation of multiple ribosomal subunits indicating an increased need for these machines. ...
The blood-brain barrier (BBB) protects the brain microenvironment from external damage. It is formed by endothelial cells (ECs) lining the brain vessels, expressing tight junctions and having reduced transcytosis, resulting in a very low paracellular and transcellular passage of substances, respectively (low permeability). The specific BBB phenotype is maintained by Wnt molecules secreted by astrocytes (ACs) that bind to receptors in ECs, and start a molecular cascade that leads to β-catenin translocating to the nucleus and activating the transcription of BBB genes.
An increasing number of studies report BBB dysfunction in Alzheimer’s disease (AD), although the topic is currently under debate. AD is a neurodegenerative condition characterized by brain depositions of Aβ aggregates and Tau neurofibrillary tangles. The aetiology of AD is unknown, although round 5% of all AD cases have a genetic origin. Mutations in APP or PSEN1/2 can lead to Aβ over-production and accumulation, causing familiar AD. There is no cure for AD, as all clinical trials failed during the past years. Consequently, I studied the role of the BBB in AD, aiming to investigate if a BBB dysfunction occurs in AD, and to identify by transcriptomic analysis novel gene regulations happening at the BBB in AD. The final objective was to evaluate the potential of identified BBB genes as therapeutical target.
I used transgenic mice expressing the human APP mutations Swiss, Dutch and Iowa under the control of the neuronal promoter Thy1 (Thy1-APPSwDI) as AD model. In this AD mouse model, I could detect Aβ deposits and memory loss by immunofluorescence (IF) and behavioural tests. Importantly, I identified an increase of BBB permeability to 3-4 kDa dextrans in 6 months, 9-12 months, and 18 months or older AD mice compared to age-matched control wild types (WT), indicating BBB dysfunction in AD mice.
In order to study the BBB transcriptional changes in AD, I sequenced the RNA from 6 and 18 months old AD and WT mouse brain microvessels (MBMVs), as well as of FACS-sorted ECs, mural cells (MuCs), ACs, and microglia (MG) in collaboration with GenXPro, a company specialized in 3’ RNA sequencing. Currently, no transcriptomic datasets of ECs and MuCs are publicly available, suggesting that this is the first study sequencing those cell types in the context of AD.
The analysis of sequencing data from MBMVs and ECs revealed a Wnt/β-catenin repression, and an increase of inflammatory genes like Ccl3 in ECs, that could explain the BBB dysfunction observed in AD mice. Furthermore, the sequencing data from MuCs identified a set of 11 genes strongly regulated in both 6 and 18 month AD groups. Three of those 11 genes are known to be involved in inflammatory processes, demonstrating that inflammation affects and plays an important role in MuCs and ECs during AD.
Thanks to published sequencing data, some up-regulated MG genes in AD are well known and recognized, such as Trem2 and Apoe. Those genes were found in the FACS-sorted MG data as well, validating the AD model and with it, the other novel sequenced datasets. Importantly, one of the most strongly AD-regulated genes in MBMV and MG samples was Dkk2, a member of the Dickkopf family of secreted proteins known to be involved in Wnt signalling modulation. Importantly, a dual luciferase reporter assay proved that Dkk2 is a Wnt inhibitor. A preliminary immunohistochemistry examination of DKK2 in human brain autopsy tissue from an AD patient and age-matched control revealed a stronger DKK2 immunoreactivity in the AD brain.
In order to answer the question whether a rescue of BBB function would ameliorate AD symptoms, I made use of a tamoxifen-inducible transgenic mouse line to activate the Wnt/β-catenin pathway specifically in ECs, leading to a gain of function (GOF) condition (Cdh5-CreERT2+/–/Ctnnb1(Ex3)fl/fl). This mouse line was then crossed with the AD line, creating AD/GOF and AD/control groups.
AD/GOF mice performed better in a Y-Maze memory test than AD/controls when the Wnt/β-catenin pathway was induced before AD onset, indicating a protective effect. Moreover, the finding implies that shielding BBB functioning in AD further protects the brain from AD toxic effects, suggesting an important role of brain vasculature in AD and its potential as therapeutic target.
Synaptic plasticity is the activity dependent alteration of the composition, form and strength of synapses and believed to be the underlying mechanism of learning and memory formation. While initial changes in synaptic transmission are caused by second messenger signaling pathways and rapid modifications in the cytoskeleton, to achieve stable and persistent changes at individual synapses, the expression of new mRNAs and proteins is required. The central dogma postulated that the cell body is the only source of newly synthesized proteins. For neurons, with their unique morphology, this meant that proteins would need be transported long distances, often hundreds of microns, to reach their destined locations in dendrites and at spines. To overcome this limitation, neurons have developed a strategy to regulate protein synthesis locally by distributing thousands of mRNAs into neuronal processes and use them for local protein synthesis. Ample research has demonstrated the importance of local protein synthesis to many forms of long-term synaptic plasticity. One potential regulator of mRNA localization and local translation in neurons are non-coding RNAs. Intensive work over the past decades has highlighted the importance of non-coding RNAs in many aspects of brain function. The aim of this thesis is to obtain a better understanding of the role of non-coding RNAs in synaptic function and plasticity in the murine hippocampus. For this, we focused our studies on two classes of non-coding RNAs.
In the first part of my thesis, I describe our efforts on characterizing circular RNAs, a novel and peculiar family of non-coding RNAs, in the murine hippocampus by combining high throughput RNA-Sequencing with fluorescence in situ hybridization. Furthermore, we investigated the mechanisms of circular RNA biogenesis in hippocampal neurons by temporarily inhibiting spliceosome activity and analyzing the differentially regulated circular RNAs.
Nematophilic bacteria as a source of novel macrocyclised antimicrobial non-ribosomal peptides
(2020)
A solution to ineffective clinical antimicrobials is the discovery of new ones from under-explored sources such as macrocyclic non-ribosomal peptides (NRP) from nematophilic bacteria. In this dissertation an antimicrobial discovery process –from soil sample to inhibitory peptide– is demonstrated through investigations on six nematophilic bacteria: Xenorhabdus griffiniae XN45, X. griffiniae VH1, Xenorhabdus sp. nov. BG5, Xenorhabdus sp. nov. BMMCB, X. ishibashii and Photorhabdus temperata. To demonstrate the first step of bacterium isolation and species delineation, endosymbionts were isolated from Steinernema sp. strains BG5 and VH1 that were isolated directly from soil samples in Western Kenya. After genome sequencing and assembly of novel Xenorhabdus isolates VH1 and BG5, species delineation was done via three overall genome relatedness indices. VH1 was identified as X. griffiniae VH1, BG5 as Xenorhabdus sp. nov. BG5 and X. griffiniae BMMCB was emended to Xenorhabdus sp. nov. BMMCB. The nematode host of X. griffiniae XN45, Steinernema sp. scarpo was highlighted as a putative novel species. To demonstrate the second step of genome mining and macrocyclic non-ribosomal peptide structure elucidation, chemosynthesis and biosynthesis, the non-ribosomal peptide whose production is encoded by the ishA-B genes in X. ishibashii was investigated. Through a combination of refactoring the ishA-B operon by a promoter exchange mechanism, isotope labelling experiments, high resolution tandem mass spectrometry analysis, bioinformatic protein domain analysis and chemoinformatic comparisons of actual to hypothetical mass spectrometry spectra, the structures of Ishipeptides were elucidated and confirmed by chemical synthesis. Ishipeptide A was a branch cyclic depsidodecapeptide macrocyclised via an ester bond between serine and the terminal glutamate. It chemosynthesis route was via a late stage macrolactamation and linearised Ishipeptide B was synthesised via solid phase iterative synthesis. Ishipeptides were not N-terminally acylated despite being biosynthesised from the IshA protein that had a C-starter domain. It was highlighted that more than restoration of the histidine active site of this domain is required to restore N-terminal acylation activity.
To demonstrate the final step of determination of antimicrobial activity, minimum inhibitory concentrations of Ishipeptides and Photoditritide from Photorhabdus temperata against fungi and bacteria were determined. None were antifungal while only the macrocyclic compounds were inhibitory, with Ishipeptide A inhibitory to Gram-positive bacteria at 37 µM. The cationic Photoditritide, a cyclic hexapeptide macrocyclised via a lactam bond between homoarginine and tryptophan, was 12 times more inhibitory (3.0 µM), even more effective than a current clinical compound, Ampicillin (4.2 µM). For both, macrocyclisation was hypothesised to contribute to antimicrobial activity. Ultimately, this dissertation demonstrated not only nematophilic bacteria as a source of novel macrocyclic antimicrobial non-ribosomal peptides but also a process of antimicrobial discovery–from soil sample to inhibitory peptide– from these useful bacteria genera. This is significant for the fight against antimicrobial resistance.
The compound class of the fabclavines was described as secondary or specialized metabolites (SM) for Xenorhabdus budapestensis and X. szentirmaii. Their corresponding structure was elucidated by NMR and further derivatives could be identified in both strains. Biochemically, fabclavines are hybrid SMs derived from two non-ribosomal-peptide-synthetases (NRPS), one type I polyketide-synthase (PKS) and polyunsaturated fatty acid (PUFA) synthases. In detail, a hexapeptide is connected via partially reduced polyketide units to an unsual polyamine. Structurally, they are related to the (pre-)zeamines, described for Serratia plymuthica and Dickeya zeae. Fabclavines exhibit a broad-spectrum bioactivity against a variety of different organisms like Grampositive and Gram-negative bacteria, fungi, protozoa but also against eukaryotic celllines.
In this work, the fabclavine biosynthesis was elucidated and assigned to two independently working assembly lines. The NRPS-PKS-pathway is initiated by the first NRPS FclI via generation of a tetrapeptide, which is elongated by the second NRPS FclJ, leading to a hexapeptide. Alternatively, FclJ can also act as direct start of the biosynthesis, resulting in the final formation of shortened fabclavine derivatives with a diinstead of a hexapeptide. In both cases, the peptide moiety is transferred to the iterative type I PKS FclK, leading to an elongation with partially reduced polyketide units. The resulting NRPS-PKS-intermediate is still enzyme-bound. The PUFA-homologues FclC, FclD and FclE in combination with FclF, FclG and FclH belong to the polyamine-forming pathway. Briefly, repeating decarboxylative Claisen thioester condensation reactions of acyl-coenzym A building blocks lead to the generation of an acyl chain in a PKS- or fatty acid biosynthesis-like manner. The corresponding β-keto-groups are either completely reduced or transaminated in a specific and repetitive way, resulting in the concatenation of so-called amine-units. The final β-keto-group is reduced to a hydroxy-group and the intermediate is reductively released by the thioester reductase FclG. A subsequent transamination step leads to the final polyamine. The NRPS-PKS- as well as the polyamine-pathway are connected by FclL. This condensation domain-like protein catalyzes the condensation of the polyamine with the NRPS-PKS-part, which results in the release of the final fabclavine. The results are described in detail in the first publication (first author).
Fabclavine biosynthesis gene cluster (BGC) are widely spread among the genus Xenorhabdus and Photorhabdus. In Xenorhabdus strains a high degree of conservation regarding the BGC synteny as well as the identity of single proteins can be observed. However, Photorhabdus strains harbor only the PUFA-homologues. While in Photorhabdus no product could be detected, our analysis revealed that the Xenorhabdus strains produce a large chemical diversity of different derivatives. Briefly, the general backbone of the fabclavines is conserved and only four chemical moieties are variable: The second and last amino acids of the NRPS-part, the number of incorporated polyketide units as well as the number of amine units in the polyamine. In combination with the elucidated biosynthesis, these variables could be assigned to single biosynthesis components as diversity mechanisms. Together with the 10 already described derivatives, a total of 32 derivatives could be detected. Interestingly, except for taxonomic closely related strains, all analyzed strains produce their own set of derivatives. Finally, we could confirm that the fabclavines are the major bioactive compound class in the analyzed strains under laboratory conditions. The results are described in detail in the second publication (first author).
Together with our collaboration partner Prof. Selcuk Hazir a potent bioactivity against Enterococcus faecalis, which is associated with endodontic infections, could be contributed to X. cabanillasii. Here, we could confirm that this bioactivity can be assigned to the fabclavines. The results are described in detail in the third publication(co-author).
Among the genus Xenorhabdus, X. bovienii represents an exception as its NRPS and PKS genes of the fabclavine BGC are missing or truncated, resulting in the exclusive production of polyamines. Furthermore, its PUFA-homologue FclC harbors an additional dehydratase (DH) domain. Upon extensive analysis a yet unknown deoxy-polyamine was identified and assigned to this additional domain. Finally, the DH domain was transferred into other polyamine pathways. Regardless of an in cis or in trans integration, the chimeric pathways produced deoxy-derivatives of its naturally occurring polyamines, suggesting that this represents another diversification mechanism. The results are described in detail in the attached manuscript (first author).
Monoterpenes and their monoterpenoid derivatives form a subclass of terpene(oid)s. They are widely used in medicines/pharmaceuticals, as flavor and fragrance compounds, or in agriculture and are also considered as future biofuels. However, for many of these substances, the extraction from natural sources poses challenges such as occurring at low concentrations in their raw material or because the natural sources are diminishing. Furthermore, many of the structurally more complex terpenoids cannot be chemically synthesized in an economic way. Therefore, microbial production provides an attractive alternative, taking advantage of the often distinct regio- and stereoselectivity of enzymatic reactions. However, monoterpenes and monoterpenoids are challenging products for industrial biotechnology processes due to their pronounced cytotoxicity, which complicates the production in microorganisms compared to longer-chain terpenes (sesquiterpenes, diterpenes, etc.).
The aim of this thesis was to generate a biotechnological complement to fossil-resources-based chemical processes for industrial monoterpenoid production. Therefore, a starting point for the further development of a microbial cell factory based on the microbe Pseudomonas putida KT2440 was aimed to be created. This production organism should be able to conduct a whole- cell biocatalysis to selectively oxyfunctionalize monoterpene hydrocarbons using renewable industrial by-products and waste streams as raw material for monoterpenoid production (Figure 1). As a model substance, the production of (-)-menthol should be addressed due to its industrial significance. (-)-Menthol is one of the world’s most widely-used flavor and fragrance compounds by volume as well as a medical component, having an annual production volume of over 30,000 tons. An approach for (-)-menthol production from renewable resources could be a biotechnological(-chemical) two-step conversion (Figure 1), starting from (+)-limonene, a by-product of the citrus fruit processing industry.
The thesis project was divided into three parts. In the first part, enzymes (limonene-3- hydroxylases) were to be identified that can convert (+)-limonene into the precursor of (-)-menthol, (+)-trans-isopiperitenol. To counteract product toxicity, in the second part, the tolerance of the intended production organism P. putida KT2440 towards monoterpenes and their monoterpenoid derivatives should be increased. Finally, in the third part, the identified hydroxylase enzymes would be expressed in the improved P. putida KT2440 strain to create a whole-cell biocatalyst for the first reaction step of a two-step (-)-menthol production, starting from (+)-limonene.
To achieve these objectives, different genetic/molecular biology and analytical methods were applied. In this way, two cytochrome P450 monooxygenase enzymes from the fungi Aureobasidium pullulans and Hormonema carpetanum could be identified and functionally expressed in Pichia pastoris, which can catalyze the intended hydroxylation reaction on (+) limonene with high stereo- and regioselectivity. A further characterization of the enzyme from A. pullulans showed that apart from (+) limonene the protein can also hydroxylate ( ) limonene, - and -pinene, as well as 3-carene.
Furthermore, within this thesis, mechanisms of microbial monoterpenoid resistance of P. putida could be identified. It was shown that the different monoterpenes and monoterpenoids tested have very different toxicity levels and that mainly the Ttg efflux pumps of P. putida GS1 are responsible for the tolerance to many of these compounds. Based on these results, a P. putida KT2440 strain with increased resistance to various monoterpenoids, including isopiperitenol, could then be generated, which can be used as a host organism for the further development of monoterpenoid-producing cell factories.
While within the scope of this work the heterologous expression of the fungal gene in prokaryotic cells in a functional form could not be realized despite different approaches, the identified enzymes, the monoterpenoid-tolerant P. putida strain and a plasmid developed for heterologous gene expression in P. putida provide a starting point for the further design of a microbial cell factory for biotechnological monoterpenoid production.
Evidence is increasingly pointing towards a significant global decline in biodiversity. The drivers of this decline are numerous, including habitat change and overexploitation, rapid deforestation, pollution, exotic species and disease, and finally climate change as an emerging driver of biodiversity change (Nakamura, et al., 2013; Hancocks, 2001; Pereira, Navarro & Martins, 2012). Raising public awareness of the need to conserve biological diversity is essential to safeguard the richness of life forms all over the world (Lindemann-Matthies, 2002). In this regard, institutions such as science museums, zoos and aquariums have the potential to play an important role (Rennie & Stocklmayer, 2003). Especially, zoos can provide a productive learning environment (Miles & Tout, 1992), facilitating the promotion of public conservation awareness and the adoption of pro-environmental behaviours that would reduce negative human impacts on biodiversity (Barongi, et al., 2015).
Based on these concepts, my study contributes to the developing field of visitor studies. Taking as reference non-zoo visitors and zoo visitors, I have focused on reviewing some aspects of conservation education, such as people's awareness of conservation, people's interest in animals and people's feelings towards animals and attitudes towards zoos. The study identified differences between non-regular and regular zoo visitors in interests in animals, as well as visitor attitudes towards conservation issues and zoos. Therefore, the present study indicated that positive emotional reactions and, in particular, a perceived sense of connection to the animal were linked and depended on the frequency of zoo visits. It was as well remarkable, that conservation awareness was influenced by the interest in animals, the interest in visiting zoos, the attitudes towards these institutions, and the age and the country of origin. All these variables had a greater effect in the conservation consciousness of the participants. Additionally interestingly, the main reason for visiting zoos in every country was to learn something about animals. This highlights the educational role of zoos and broadly supports the idea that people want to visit zoos to learn something about animals, in turn facilitating pro-conservation learning and changes in attitude. They are uniquely positioned to interact with visitors, communities, and society and to contribute by providing an informative and entertaining environment. Visiting zoos could led to contribute to promoting animal connectedness and interest in species.
Carotinoide sind Pigmente, die in Pflanzen, Algen, einigen Pilzen und Bakterien vorkommen. Sie spielen eine wichtige Rolle bei der Photosynthese durch Absorption von Licht und beim Lichtschutz. Sie sind verantwortlich für die braunen, roten, orangen und gelben Farben von Obst, Gemüse, Herbstblättern und die Farbe einiger Blumen und Algen. Tiere können keine Carotinoide synthetisieren, daher ist ihre Anwesenheit auf die Nahrungsaufnahme zurückzuführen. Carotinoide sind Tetraterpenoide (40C), die aus Isoprenoidmolekülen (5C) synthetisiert werden. Der Methylerythritol-phosphatweg ist der Carotinoid-Vorläuferweg, der die Isoprenoideinheiten bildet. Carotinoide haben aufgrund ihrer gesundheitlichen Vorteile das Interesse der Nutrazeutika-Industrie geweckt.
Fucoxanthin ist ein Carotinoid, das nur in Kieselalgen, Braunalgen, Haptophyten und einigen Dinoflagellaten vorkommt. Aufgrund seiner Vorteile zur Vorbeugung von Krebs, kognitiven Erkrankungen und Fettleibigkeit sowie seiner antioxidativen Eigenschaften ist Fucoxanthin ein sehr interessantes Molekül fur die Nutrazeutikabranche.
Fucoxanthin hat eine komplexe chemische Struktur mit einer Allenbindung und einer Epoxyketogruppe. Daher wäre seine chemische Synthese kompliziert, da es auch eine stereokontrollierte Synthese erfordert86. Aus diesem Grund ist die Extraktion aus Makroalgen oder Mikroalgen die Methode der Wahl für die kommerzielle Herstellung von Fucoxanthin.
In dieser Arbeit bestand das Ziel darin, die Fucoxanthin-Produktivität in Kieselalgen mit gentechnischen Methoden zu steigern, damit die Zellen mehr Fucoxanthin produzieren. Zu diesem Zweck wurde der Effekt der Insertion zusätzlicher Kopien von Genen in das Genom untersucht, die für geschwindigkeitsbestimmende oder Schlüsselenzyme im Carotinoid- und MEP-Weg kodieren.
Zu Beginn wurden diese Effekte bei einzelnen Mutanten beobachtet. Letztendlich ist es jedoch das Ziel, eine Mutante zu erzeugen, die mehrere geschwindigkeitsbestimmende Enzyme überexprimiert, um auf diese Weise Engpässe zu vermeiden. In früheren Studien erreichten Eilers et al.54 durch die einmalige Überexpression der psy- und dxs-Gene in der Kieselalge P. tricornutum einen 2.4- und 1.8-fachen Anstieg der Fucoxanthin-Spiegel.
In dieser Arbeit führte die Insertion zusätzlicher Kopien der Gene idi und pds2 nicht dazu, dass die Zellen mehr Fucoxanthin produzieren. Im Gegensatz dazu erreichten die Mutanten mit zusätzlichen Kopien der Gen ggpps und mit zusätzlichen Kopien sowohl von psy als auch von dxs seine um 28% bzw. 10% höhere Fucoxanthin-Produktivität pro Million Zellen. Bei diesen Mutanten ist die Gesamtproduktivität jedoch geringer als beim Wildtyp, da ihr Wachstum langsamer als beim Wildtyp ist.
Unter Berücksichtigung der besten Zielgene wurden Mutanten erzeugt, die gleichzeitig zusätzliche Kopien von psy, dxs und ggpps enthielten. Die Mutanten hatten unter sehr niedriegen Lichtbedingungen eine um bis zu 61% höhere Produktivität pro Million Zellen als der Wildtyp. Ausnahmsweise wurden diese Mutanten bei sehr schwachem Licht (10 µE m-2 s-1) gezüchtet, da sie sehr gestresst waren und als Zellklumpen wuchsen. Obwohl die Gesamt-Fucoxanthin-Spiegel in diesen Mutanten unter diesen Bedingungen höher sind als im Wildtyp, sind sie daher niedriger als die Fucoxanthin-Spiegel bei den in anderen Experimenten verwendeten Lichtbedingungen (50 µE m-2 s-1). Als Ergebnis dieser Experimente kann gesagt werden, dass die Belastung der Zellen nach den genetischen Veränderungen untersucht werden muss, da dies zu einer Abnahme der Biomasse und folglich zu einer Abnahme der Fucoxanthinproduktion führt. Alternativ könnte auch eine 2-Stufen-Kultur etabliert werden, in der in einem ersten Schritt eine hohe Biomasse erreicht wird und im zweiten Schritt die Expression der interessierenden Gene induziert wird.
Aufgrund der antioxidativen Eigenschaften von Carotinoiden besteht eine übliche Strategie zur Akkumulation von Carotinoiden darin, die Zellen unter oxidative Stressbedingungen zu setzen. Diese Strategie ist jedoch nicht wirksam für die Anreicherung von Fucoxanthin unter hohen Salzkonzentrationen oder hohen Lichtbedingungen. Bessere Versuchspläne könnten jedoch eine 2-Stufen-Kultur oder adaptive Laborbedingungen gewesen sein.
Eine andere mögliche Strategie zur Erhöhung des Fucoxanthinspiegels wäre die Durchführung einer zufälligen Mutagenese der Zellen. Auf diese Weise sind keine Vorkenntnisse über den Carotinoidsyntheseweg und seine Regulation erforderlich und es kann zu Veränderungen in Genen führen, die keine offensichtlichen Ziele sind.
Experimente mit zufälliger Mutagenese erfordern ein Hochdurchsatz-Screeningsystem, da Hunderte oder sogar Tausende von Mutanten erhalten werden. Eine mögliche Strategie, um die Kultivierung der hohen Anzahl von Mutanten zu vereinfachen, ist die Einkapselung dieser Mutanten in Alginatkügelchen. Auf diese Weise können alle Mutanten in demselben Gefäß kultiviert werden. Die eingekapselten Zellen können dann beispielsweise mit einem Durchflusszytometer auf große Partikel durch Fluoreszenz- oder Absorptionsmessungen gescreent werden.
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A recent global meta‐analysis reported a decrease in terrestrial but increase in freshwater insect abundance and biomass (van Klink et al., Science 368, p. 417). The authors suggested that water quality has been improving, thereby challenging recent reports documenting drastic global declines in freshwater biodiversity. We raise two major concerns with the meta‐analysis and suggest that these account for the discrepancy with the declines reported elsewhere. First, total abundance and biomass alone are poor indicators of the status of freshwater insect assemblages, and the observed differences may well have been driven by the replacement of sensitive species with tolerant ones. Second, many of the datasets poorly represent global trends and reflect responses to local conditions or nonrandom site selection. We conclude that the results of the meta‐analysis should not be considered indicative of an overall improvement in the condition of freshwater ecosystems.
Primary determinants of communities in deadwood vary among taxa but are regionally consistent
(2020)
The evolutionary split between gymnosperms and angiosperms has far‐reaching implications for the current communities colonizing trees. The inherent characteristics of dead wood include its role as a spatially scattered habitat of plant tissue, transient in time. Thus, local assemblages in deadwood forming a food web in a necrobiome should be affected not only by dispersal ability but also by host tree identity, the decay stage and local abiotic conditions. However, experiments simultaneously manipulating these potential community drivers in deadwood are lacking. To disentangle the importance of spatial distance and microclimate, as well as host identity and decay stage as drivers of local assemblages, we conducted two consecutive experiments, a 2‐tree species and 6‐tree species experiment with 80 and 72 tree logs, respectively, located in canopy openings and under closed canopies of a montane and a lowland forest. We sampled saproxylic beetles, spiders, fungi and bacterial assemblages from logs. Variation partitioning for community metrics based on a unified framework of Hill numbers showed consistent results for both studies: host identity was most important for sporocarp‐detected fungal assemblages, decay stage and host tree for DNA‐detected fungal assemblages, microclimate and decay stage for beetles and spiders and decay stage for bacteria. Spatial distance was of minor importance for most taxa but showed the strongest effects for arthropods. The contrasting patterns among the taxa highlight the need for multi‐taxon analyses in identifying the importance of abiotic and biotic drivers of community composition. Moreover, the consistent finding of microclimate as the primary driver for saproxylic beetles compared to host identity shows, for the first time that existing evolutionary host adaptions can be outcompeted by local climate conditions in deadwood.
Acetobacterium woodii utilizes the Wood‐Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non‐acetogenic growth on 1,2‐propanediol (1,2‐PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2‐PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC‐encapsulated propanol‐generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH‐dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2‐PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.
Plastid DNA sequence data have been traditionally widely used in plant phylogenetics because of the high copy number of plastids, their uniparental inheritance, and the blend of coding and non-coding regions with divergent substitution rates that allow the reconstruction of phylogenetic relationships at different taxonomic ranks. In the present study, we evaluate the utility of the plastome for the reconstruction of phylogenetic relationships in the pantropical plant family Ochnaceae (Malpighiales). We used the off-target sequence read fraction of a targeted sequencing study (targeting nuclear loci only) to recover more than 100 kb of the plastid genome from the majority of the more than 200 species of Ochnaceae and all but two genera using de novo and reference-based assembly strategies. Most of the recalcitrant nodes in the family’s backbone were resolved by our plastome-based phylogenetic inference, corroborating the most recent classification system of Ochnaceae and findings from a phylogenomic study based on nuclear loci. Nonetheless, the phylogenetic relationships within the major clades of tribe Ochnineae, which comprise about two thirds of the family’s species diversity, received mostly low support. Generally, the phylogenetic resolution was lowest at the infrageneric level. Overall there was little phylogenetic conflict compared to a recent analysis of nuclear loci. Effects of taxon sampling were invoked as the most likely reason for some of the few well-supported discords. Our study demonstrates the utility of the off-target fraction of a target enrichment study for assembling near-complete plastid genomes for a large proportion of samples.
This work deals with the characterization of three different type II polyketide synthase systems (PKS II) from the Gram-negative bacteria Xenorhabdus and Photorhabdus.
Particular attention was paid to a biochemically underexplored class of aryl polyene (APE) pigments. Bioinformatic analysis of enzymes involved in the biosynthesis and the in vitro reconstruction proved that the synthesis of APEs involves an unusual fatty acid-like elongation mechanism. Furthermore, the discovery of unexpected protein-protein interactions provided new insights into the multienzyme complex formation of this unusual PKS II system. Through collaboration with the groups from Prof. Michael Groll and junior Prof. Nina Morgner, two protein complexes were structurally solved and several native protein multimerization events were identified and allowed us to suggest a possible protein-interaction network. The results are summarized in publication ‘An Uncommon Type II PKS Catalyzes Biosynthesis of Aryl Polyene Pigments’ (first author; J. Am. Chem. Soc.).
In addition to in vitro-analysis, in vivo-studies were used to investigate the APE compound produced by X. doucetiae in more detail. The activation of the silent biosynthetic gene cluster (BGC) led to the detection of the APE compound in the homologous host. Further combination of homologous expression and targeted deletions of the APE BGC revealed an APE-lipid-like structure. MS-based analyses and purification of intermediates allowed us to deduce structural building blocks of the APE-lipid, which is composed of an APE structural core, a glucosamine residue and an unusual long-chain fatty acid with unusual conjugated double bonds and a phosphoethanolamine head group. In combination with the above stated in vitro-data, we assumed a plausible biosynthetic mechanism of the APE-lipid. The results are summarized in the section ‘Additional Results: Tracing the Full-length APE’.
The biosynthesis of isopropylstilbene (IPS) has already been well-studied by the Bode laboratory and the group of Prof. Ikuro Abe. Studies with Photorhabdus laumondii TT01 by the Bode group revealed the distributed locations and functions of the genes involved in biosynthesis, which originate from two pathways. Particularly, the Bode group first demonstrated that an unusual ketosynthase/cyclase (StlD) catalyzes the condensation of 5-phenyl-2,4-pentadienoyl-ACP and isovaleryl-beta-ketoacyl-ACP via a Michael addition. Such a pathway for stilbene formation is distinct from those widespread in plants. The Abe group solved the structure and biochemical mechanism of StlD and further investigated the aromatization reaction of the aromatase StlC. However, the generation of the required cinnamoyl-precursor 5-phenyl-2,4-pentadienoyl-ACP as a Michael acceptor for this cyclization reaction remained elusive. In this work, we were able to reconstitute the synthesis of the Michael acceptor in vitro, by the action of enzymes from the fatty acid biosynthesis. With the knowledge about the crucial cross-talk from primary and specialized metabolism, we further determined the minimal endowment for stilbene production in a heterologous host. Here, the discovered AasS enzyme StlB is responsible for the generation of cinnamoyl-ACP and among others, plFabH plays a key role as gatekeeper enzyme for further processing. With this information in hand, we were able to obtain IPS production in E. coli. These results are presented in the manuscript ‘Biosynthesis of the Multifunctional Isopropylstilbene in Photorhabdus laumondii Involves Cross-talk Between Specialized and Primary Metabolism’ (co-first author, manuscript).
The biosynthesis of the orange-to-red-pigmented anthraquinones (AQs) is the best-studied type II PKS system according to preliminary results. While several investigations by Brachmann et al. discovered the BGC and the overall product spectrum of the main AQ-256 and its methylated derivatives, data of Quiqin Zhou (Bode group) performed biochemical in vitro analysis paired with in vivo heterologous expression of the ant-genes antA-I. This led to the identification of shunt products that indicated an AQ-scaffold derived from an octaketide intermediate that gets shortened to a heptaketide by the hydrolase AntI, resulting in the main anthraquinone AQ-256. This PKS-shortening mechanism was further confirmed by the protein crystal structure of AntI by the Groll group (publication, minor contributions, co-author, Chem Sci. ‘Molecular Mechanism of Polyketide Shortening in Anthraquinone Biosynthesis of Photorhabdus luminescens’). Further substrate analysis of the P. luminescens AQ-producer and mutants revealed an inhibitory effect of cinnamic acid against the hydrolase AntI. Cinnamic acid might therefore be involved in regulation of AQ biosynthesis (‘Anthraquinone Production is Influenced by Cinnamic Acid’, first author, manuscript).
Biochemical analysis from Quiqin Zhou with the minimal PKS of the AQ-synthase further revealed the exclusive activation of the AQ-ACP by the PPTase AntB. The PPTase is insoluble alone but gets stabilized by the CoA-ligase, most likely inactive, working as a chaperone. Thus, the minimal PKS endowment to produce the octaketide scaffold compromises, besides the ACP, the KS:CLF heterodimer and the MCAT, the co-occurrence of the PPTase AntB and the CoA-ligase AntG. For the first time, X-ray crystallography depicted a minimal PKS in action, by obtaining the structural data of native complexes from an ACP:KS:CLF, the KS:CLF alone and an ACP:MCAT in their non-active and active forms. It was possible to confirm a KS-bound hexaketide, which was built upon heterologous expression of the KS:CLF. Mutagenesis with amino-acids proposed to be involved in protein-protein interactions in the ACP:KS:CLF complex revealed some interesting protein-interaction sites. Additionally, an induced-fit mechanism of the MCAT with the ACP during the malonylation reaction confirmed a monodirectional transfer reaction (‘Structural Snapshots of the Minimal PKS System Responsible for Octaketide Biosynthesis’ co-author, manuscript under review).
Human GLUT2 and GLUT3, members of the GLUT / SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.
In recent years, reports of elephants causing damage in rural villages by destroying houses and foraging on stored food have been increasing, but little is known about the determinants and magnitude of this damage. In this study, we have examined the extent of property damage by elephants (Loxodonta africana and Elephas maximus), in one African and two Asian study areas over a six‐year period. A total of 1,172 damaged constructions were observed on site, involving detailed damage assessment by trained enumerators and standardized interviews with witnesses. Depending on the study area, between 67.1 and 86.4% of damage events were attributed to single, individual elephants or pairs of males. The majority of properties were damaged in search for food (62.5–76.7% respectively). Property damage caused higher mean losses than crop damage on farmland in all study areas. Results suggest that property damage by elephants has been largely underestimated and needs to form a focus in future human–elephant conflict research. We suggest a need to reduce the attractiveness of villages by storing food in locked and safe places, away from sleeping areas and to foster the development of elephant safe stores, appropriate to the particular cultural background of the target area.
As fossil resources are diminishing, environmental concerns arise and chemical synthesis often involves expensive catalysts or extensive extraction procedures, the demand for production of industrially relevant compounds from renewable resources increases. In this context, engineering microorganisms for production of specialty chemicals, such as 3-alkylphenols, presents an attractive, environmental-friendly approach. 3-alkylphenols have various applications: due to their antiseptic and stabilizing properties many 3-alkylphenols, including 3-methylphenol (3-MP), are utilized as additives in disinfectant reagents and biological products, while they can be also implemented as platform chemicals for production of lubricating oil additives or flavors. Some 3-akylphenols have potential for transmission control of the disease sleeping sickness that is transmitted by tsetse flies in sub-saharan Africa, since 3-ethylphenol (3-EP) and 3-propylphenol (3-PP) and to a lesser degree 3-MP were found to attract tsetse flies and improved catch rates in impregnated tsetse fly traps. Microbial fermentation of 3-alkylphenols would provide a simple and inexpensive way for local communities in Africa to produce these compounds and prepare their own tsetse fly traps.
Some molds synthesize 3-MP as an intermediate during biosynthesis of the mycotoxin patulin. However, the heterologous host Saccharomyces cerevisiae has advantageous traits for industrial application, since it is well characterized, robust, simple to handle and easily genetically accessible. In this thesis, genetical engineering approaches were utilized to establish the yeast S. cerevisiae for biotechnological production of 3-alkylphenols. As a proof of concept, the iterative polyketide synthase from Penicillium patulum, 6-methylsalicylic acid synthase (MSAS), and 6-methylsalicylic acid (6-MSA) decarboxylase PatG from Aspergillus clavatus were heterologously expressed in S. cerevisiae resulting in the first reported de novo biosynthesis of 3-MP via 6-MSA in yeast from sugars (Hitschler & Boles, 2019). It was shown that codon-optimization and genomic integration of heterologous genes, high initial cell densities and a balanced expression of PatG were beneficial for heterologous production of up to 589 mg/L 3-MP in S. cerevisiae. However, toxicity of 3-MP limited higher product accumulation.
Different in vivo detoxification strategies were implemented to face this bottleneck. Growth tests revealed that 3-methylanisole (3-MA) is less toxic to the yeast cells than 3-MP. Expression of an orcinol-O-methyltransferase from chinese rose hybrids (OOMT2) was combined with in situ extraction converting the toxic 3-MP product into the volatile 3-MA and accumulating up to 211 mg/L 3-MA in the dodecane phase. Alternatively, up to 533 mg/L 3-MP glucoside were synthesized by expression of a UDP-glycosyltransferase (UGT72B27) from Vitis vinifera in the 3-MP producing strain, revealing saccharose as beneficial carbon source and ethanol growth phase as essential for high 3-MP production, although 3-MP conversions were not yet complete. Both detoxification strategies allowed circumvention of the toxicity imposed limited product accumulation. This was demonstrated when both detoxification strategies were combined with redirection of the carbon flux through deletion of phosphoglucose isomerase gene PGI1 and feeding a mixture of fructose and glucose leading to majorly improved product formation, with up to 899 mg/L 3-MA/3-MP and 873 mg/L 3-MP/3-MP glucoside, compared to less than 313 mg/L product titers in the wild type controls (Hitschler & Boles, 2020).
For provision of the tsetse fly attractants 3-EP from propionyl-CoA and 3-PP from butyryl-CoA, the substrate promiscuities of MSAS and PatG were exploited. However, slower formation rates with the alternative substrates propionyl-CoA and butyryl-CoA suggested that competing formation of 6-MSA from the preferred priming unit acetyl-CoA was dominating in vivo. Indeed, 3-EP or 3-PP formation was not observed in 3-MP producing yeast strains. Assuming that intracellular levels of propionyl-CoA and butyryl-CoA were limiting 3-EP and 3-PP formation, different strategies were implemented to raise the supply of these alternative priming units and successfully compete with acetyl-CoA for MSAS priming.
Supplementation of propionate increased propionyl-CoA levels by endogenous pathways sufficiently to enable 3-EP formation in yeast mediated by MSAS and PatG. Deletion of the 2-methylcitrate synthases CIT2 and CIT3 revealed that degradation of propionyl-CoA was not limiting 3-EP formation at this stage. In order to raise propionyl-CoA levels further, a heterologous propionyl-CoA synthase (PrpE) was expressed in the 3-MP producing yeast strain leading to up to 12.5 mg/L 3-EP with propionate feeding and blockage of degradation. Moreover, PrpE enabled also 3-EP formation without propionate supplementation suggesting that an endogenous supply of propionate existed that was reactivated by PrpE. As threonine or 2-ketobutyrate feeding increased 3-EP titers in combination with PrpE, this indicated that threonine degradation via 2-ketobutyrate was responsible for the endogenous propionate supply. Moreover, expression of branched-chain ketoacid dehydrogenase complex from Pseudomonas putida combined with PrpE provided propionyl-CoA from endogenous 2-ketobutyrate and raised 3-EP titers up to 5.9 mg/L compared to 2.8 mg/L with only PrpE indicating a potential route for optimization of 3-EP titers independent of propionate or threonine feeding.
For 3-PP production from butyryl-CoA, a heterologous ‘reverse ß-oxidation’ pathway was introduced in the 3-MP producing yeast strain providing sufficient butyryl-CoA for biosynthesis of up to 2 mg/L 3-PP. Degradation of the precursor via ß-oxidation was slightly limiting, since deletion of fatty acyl-CoA oxidase POX1 increased 3-PP titers slightly to 2.6 mg/L.
As the concentrations of 3-alkylphenols are close to the concentrations implemented in tsetse fly traps, the engineered yeast strains have the potential for simple and inexpensive on-site production of 3-alkylphenols as tsetse fly attractants by local rural communities in Africa. In spite of this success, 3-MP remained the main product in the developed yeast strains. Since 3-EP and 3-PP are more efficient tsetse fly attractants, a shift in substrate specificities of MSAS and PatG is desirable for a more favorable 3-EP/3-MP and 3-PP/3-MP product ratio regarding tsetse fly attraction. During rational engineering of MSAS, the MSASQ625A/I752V mutant showed a beneficial shift of product ratios with up to 11 mg/L 3-EP/63 mg/L 3-MP and 4.5 mg/L 3-PP/116 mg/L 3-MP, compared to a higher proportion of 3-MP with up to 343 mg/L, 11 mg/L 3-EP and 1.5 mg/L 3-PP in the wild type controls. Further engineering of MSAS and PatG might majorly improve production of 3-EP and 3-PP.
In summary, this thesis successfully established the yeast S. cerevisiae as cell factory for production of different 3-alkylphenols optimizing expression of the heterologous production pathway, elucidating means to detoxify products and establishing different approaches to increase intracellular levels of acyl-CoA precursors. The engineered yeast strains can be potentially implemented for simple and inexpensive fermentation of tsetse fly attractants in Africa.
Diese Arbeit behandelt die Rolle der Proteinkinasen IKKe und TBK1 in der Progression von humanen malignene Melanomen und die Rolle von alpha-Synuclein in der Schmerzwahrnehmung von Mäusen.
Beobachten, untersuchen, experimentieren: Wie soll das gehen unter Pandemiebedingungen? Wenn auch die Chancen der digitalen Medien nun gezwungenermaßen zusehends sichtbar wurden, so sind gerade für angehende Biologielehrkräfte Primärerfahrungen mit originalen Organismen und das Einüben naturwissenschaftlicher Arbeitsmethoden sehr wichtig...
Schistosomiasis is a severe neglected tropical disease caused by trematodes and transmitted by freshwater snails. Snails are known to be highly tolerant to agricultural pesticides. However, little attention has been paid to the ecological consequences of pesticide pollution in areas endemic for schistosomiasis, where people live in close contact with non-sanitized freshwaters. In complementary laboratory and field studies on Kenyan inland areas along Lake Victoria, we show that pesticide pollution is a major driver in increasing the occurrence of host snails and thus the risk of schistosomiasis transmission. In the laboratory, snails showed higher insecticide tolerance to commonly found pesticides than associated invertebrates, in particular to the neonicotinoid Imidacloprid and the organophosphate Diazinon. In the field, we demonstrated at 48 sites that snails were present exclusively in habitats characterized by pesticide pollution and eutrophication. Our analysis revealed that insensitive snails dominated over their less tolerant competitors. The study shows for the first time that in the field, pesticide concentrations considered “safe” in environmental risk assessment have indirect effects on human health. Thus we conclude there is a need for rethinking the environmental risk of low pesticide concentrations and of integrating agricultural mitigation measures in the control of schistosomiasis.
Background: Within the last decades, there has been increasing research on the occurrence of chemicals of emerging concern (CECs) in aquatic ecosystems due to their potential adverse effects on freshwater organisms and risk to human health. However, information on CECs in freshwater environments in sub-Saharan countries is very limited. Here, we investigated the occurrence of CECs in snails and sediments collected from 48 sites within the Lake Victoria South Basin, Kenya, which have been previously investigated for water contamination. Samples were analyzed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) with a target list of 429 compounds.
Results: In total, 30 compounds have been detected in snails and 78 in sediment samples, compared to 79 previously identified compounds in water. By extending the monitoring of CECs to snails and sediments, we found 68 compounds that were not previously detected in water. These compounds include the anti-cancer drug anastrozole, detected for the first time in the Kenyan environment. Individual compound concentrations were detected up to 480 ng/g wet weight (N-ethyl-o-toluenesulfonamide) in snails and 110 ng/g organic carbon (pirimiphos-methyl) in sediments. Higher contaminant concentrations were found in agricultural sites than in areas not impacted by anthropogenic activities. Crustaceans were the organisms at greatest toxic risk from sediment contamination [toxic unit (TU) up to 0.99] with diazinon and pirimiphos-methyl driving this risk. Acute and chronic risks to algae were driven by diuron (TU up to 0.24), whereas fish were found to be at low-to-no acute risk (TU up to 0.007).
Conclusions: The compound classes present at the highest frequencies in all matrices were pesticides and biocides. This study shows substantial contamination of surface water in rural western Kenya. By filling data gaps on contamination of sediments and aquatic biota, our study reveals that CECs pose a substantial risk to environmental health in Kenya demanding for monitoring and mitigation.
In the past decades, the use and production of chemicals has been on the rise globally due to increasing industrialization and intensive agriculture; resulting in the occurrence and ecotoxicological risks of chemicals of emerging concern (CECs) in the aquatic compartments. Risks include changes in community structure resulting in the dominance of one species and ecosystem imbalance. When dominant disease-causing organisms are in the environment, the disease transmission is increased. For example, host snails for the schistosomiasis, a human trematode disease, are known to be tolerant to pesticide
exposure compared to the predators. This would therefore result in an increased abundance of snails which consequently increase the disease transmission in the human population.
Kenya, being a low income country faces a lot of challenges with provision of clean water, diseases and sanitation facilities, and increasing population which results in intensive agriculture coupled with pesticide use. Although a lot of research has been carried out on the environmental occurrence and risk of CECs (Chapter 1), most of these studies have been done in developed countries with limited information from Africa. Additionally, research in Africa focused on urban areas with limited number of compounds analyzed and mostly in the water phase, and inadequate information on the effects of CECs on the aquatic organisms. In order to reduce this knowledge gap, this dissertation focused on identification and quantification of CECs present in water, sediment and snails from western Kenya, and the contribution of pesticides to the transmission of schistosomiasis.
Chapter 2 gives a summary of the results and discussion of the dissertation. In Chapter 3, a comprehensive chemical analysis was carried out on 48 water samples to identify compounds, spatial patterns and associated risks for fish, crustacean and algae using toxic unit (TU) approach. A total of 78 compounds were detected with pesticides and biocides being the compounds most frequently detected. Spatial pattern analysis revealed limited compound grouping based on land use. Acute risk for crustaceans and algae were driven by one to three individual compounds. These compounds responsible for toxicity were prioritized as candidate compounds for monitoring and regulation in Kenya.
In Chapter 4, an extension of Chapter 3 was done to cover the CECs present in snails and sediment from the 48 sites. A total of 30 compounds were found in snails and 78 in sediments with 68 additional compounds being found which were not previously detected in water. Higher contaminant concentrations were found in agricultural sites than in areas without anthropogenic activities. The highest acute toxicity (TU 0.99) was determined for crustaceans based on compounds in sediment samples. The risk was driven by diazinon and pirimiphos-methyl. Acute and chronic risks to algae were driven by diuron whereas fish were found to be at low to no acute risk.
In Chapter 5, the effect of pesticide contamination on schistosomiasis transmission was evaluated by applying complimentary laboratory and field studies. In the field studies, the ecological mechanisms through which pesticides and physical chemical parameters affect host snails, predators and competitors were investigated. Pesticide data was obtained from the results in chapter 3. The overall distribution of grazers and predators was not affected by pesticide pollution. However, within the grazers, pesticide pollution increased dominance of host snails. On the contrary, the host-snail competitors were highly sensitive to pesticide exposure. For the laboratory studies, macroinvertebrates including Schistosoma-host snails, competitors and predators were exposed to 6 concentrations levels of imidacloprid and diazinon. Snails showed higher insecticide tolerance compared to competitors and predators. Finally, Chapter 6 summarizes the conclusions of this dissertation, placing it in a broader
context. In this dissertation, a comprehensive chemical characterization and risk assessment of CECs has been carried out in freshwater systems; together with the effects of pesticides on schistosomiasis transmission in rural western Kenya. Results of this dissertation showed that rural areas are contaminated posing a risk to aquatic organisms which contribute to schistosomiasis transmission. This shows the need for regular monitoring and policy formulation to reduce pollutant emissions which contributes negatively to both ecological and human health effects.
Abstract
Divergence is mostly viewed as a progressive process often initiated by selection targeting individual loci, ultimately resulting in ever increasing genomic isolation due to linkage. However, recent studies show that this process may stall at intermediate stable equilibrium states without achieving complete genomic isolation. We tested the extent of genomic isolation between two recurrently hybridizing nonbiting midge sister taxa, Chironomus riparius and Chironomus piger, by analyzing the divergence landscape. Using a principal component‐based method, we estimated that only about 28.44% of the genomes were mutually isolated, whereas the rest was still exchanged. The divergence landscape was fragmented into isolated regions of on average 30 kb, distributed throughout the genome. Selection and divergence time strongly influenced lengths of isolated regions, whereas local recombination rate only had minor impact. Comparison of divergence time distributions obtained from several coalescence‐simulated divergence scenarios with the observed divergence time estimates in an approximate Bayesian computation framework favored a short and concluded divergence event in the past. Most divergence happened during a short time span about 4.5 million generations ago, followed by a stable equilibrium between mutual gene flow through ongoing hybridization for the larger part of the genome and isolation in some regions due to rapid purifying selection of introgression, supported by high effective population sizes and recombination rates.
Impact Summary
The process of speciation has fascinated biologists from early on. Prevailing theory suggested that gene flow among populations is the main obstacle for their divergence. Recently, it became clear that speciation with gene flow is possible under certain circumstances. However, it remains unclear how the divergence process proceeds in time, how widespread the phenomenon is, and whether it always and inevitably leads to complete isolation. Comparing the genomes of individuals of two regularly hybridizing sister taxa of nonbiting midges, we could show that they diverged during a short period millions of generations ago. Their divergence process apparently ceased before the entire genome was mutually isolated. The taxa remain distinct since, even though they share most of their genome. Our findings thus extend our view of the nature of species and the temporal dynamics of their divergence and describe novel approaches to analyze both current and past divergence processes.
Currently, the genus Polypedates comprises 26 species distributed in South, Southeast, and East Asia. Because of their relatively low dispersal capability and intolerance to seawater, this genus is ideal for the study of terrestrial range evolution that extends into the island archipelagos of southeastern Asia. In this study, based on data compiled for Polypedates from previous studies and partial mitochondrial and nuclear genes collected in this study, we performed systematic biogeographical analysis. We confirmed a Sundaland origin for the extant genus and showed northward dispersal into mainland Southeast Asia and Asia, which coincided with the timing of paleoclimatic change from the Oligocene to Middle Miocene. Climate fluctuations had a profound impact on species diversification within the genus Polypedates. Furthermore, the Red River did not mediate species exchange between Southeast Asia and mainland Asia until the end of the Miocene, with the sudden onset of northward dispersal in several clades independently at that time. Alternatively, the lineage of widespread insular P. leucomystax strongly supports the hypothesis of terrestrial connection between island archipelagos of Southeast Asia during the Mid-Pleistocene paleoclimate fluctuations. Our biogeographical analysis also supports the recent introduction of P. leucomystax to the Philippines and Ryukyus, as previously suggested.
This manuscript-based thesis is divided into four chapters. Chapter one is an introduction to lichens and the Antarctic. It introduces the goal of the thesis and the problems related with lichen systematics and the lack of knowledge about Antarctic lichens. The Antarctic is one of the last wildernesses, isolated from the other continents by the Antarctic Circumpolar Current, the Subantarctic Front, the Antarctic Polar Front, and the Drake Passage. Terrestrial life in Antarctica is restricted to widely separated and small ice-free areas that cover only 0.3% of the continent. Colonization of the Antarctic is a challenge for many taxa and is related to their ability for long-range dispersal and their adaptation to the harsh climate. Antarctic terrestrial ecosystems are significantly threatened by climate change, invasive species, and their interactions. Glacial retreat caused by higher than average temperatures exposes new habitats that can be easily colonized from local biota, but non-native species can also be favored by the new climatic conditions. In addition, propagule movement mediated by humans can introduce new species or change the population structure of many taxa. The terrestrial biota is comprised almost exclusively by “lower organisms” (invertebrates, bryophytes, algae, lichenized fungi, and microorganisms). Lichens are the dominant component, and the most important primary producers. Lichens are symbiotic associations consisting of a fungus (mycobiont) and one or more photosynthetic (photobiont) partners. They can disperse sexually or vegetatively. There are several problems related to the symbiotic nature of lichens that do not facilitate easy identification; although molecular data offers additional evidence, species delimitation in lichens is still not straightforward. The true number of species is underestimated due to the presence of cryptic species and species pairs. Recommended universal fungal barcode sequences (e. g. ITS) sometimes fail to delimit species pairs. Thus, it is necessary to identify fast-evolving markers that allow for the delimitation of closely related species before proceeding with the analysis of lichen populations. The goal of this thesis is to elucidate the so far unknown genetic structure among Antarctic lichen populations because of the immediate consequences for conservation strategies. The thesis focuses not only on patterns of differentiation and gene flow, but also investigates the question of human-mediated propagule transfer into Antarctica and among Antarctic sites. This project provides data on the genetic structure of Antarctic lichens that is urgently needed to develop conservation strategies in the face of global warming and increased human activities in the region. Due to the fact that it is not possible to apply all of the unspecific fingerprinting methods to lichens, microsatellites or simple sequence repeats (SSRs) are one of the best tools to investigate the genetic structure of lichen populations. SSRs offer the possibility to discriminate the lichen partners, but species-specific microsatellites have been developed for only a few species. Regarding the Antarctic, only one species has been studied with SSRs.
The second chapter describes new methods and tools to delimit closely related species of lichens and provides fast evolving markers to characterize their genetic structure. The chapter introduces the lichen species analysed in this thesis and the problems related to their correct identification by morphological methods and molecular data. Chapter two explains the sampling methods for lichen populations and the localities from small areas in which the species pairs occur together. Then the methods used to generate and validate fungal specific microsatellites that cross-amplify species pairs are described. This chapter focuses on the species pair Usnea antarctica and U. aurantiacoatra because they are the most common lichens in the Maritime Antarctic. An internal transcribed spacer (ITS) marker do not discriminate between these species, and some authors have suggested to synonymize them. Unpublished results from another Antarctic species pair, Placopsis antarctica and P. contortuplicata, are included to confirm the capability of SSRs to discriminate closely related lichen species. This thesis is the first study to generate SSRs that cross amplify species pairs, using BLAST to compare one genome against the other to obtain markers with the same length in flanking regions. The de novo developed SSRs are able to discriminate the two closely related species, and can detect variability at the population level. In the end of the chapter, ITS sequences, microsatellites, and SNPs are used to delimit the species of Usnea antarctica and U. aurantiacoatra. The chapter exposes the importance of a correct species delimitation and the ability of SSRs and SNPs to delimit the Antarctic Usnea species pair compared with the recommended universal fungal barcode sequence ITS. ...
Good quality data on precipitation are a prerequisite for applications like short-term weather forecasts, medium-term humanitarian assistance, and long-term climate modelling. In Sub-Saharan Africa, however, the meteorological station networks are frequently insufficient, as in the Cuvelai-Basin in Namibia and Angola. This paper analyses six rainfall products (ARC2.0, CHIRPS2.0, CRU-TS3.23, GPCCv7, PERSIANN-CDR, and TAMSAT) with respect to their performance in a crop model (APSIM) to obtain nutritional scores of a household’s requirements for dietary energy and further macronutrients. All products were calibrated to an observed time series using Quantile Mapping. The crop model output was compared against official yield data. The results show that the products (i) reproduce well the Basin’s spatial patterns, and (ii) temporally agree to station records (r = 0.84). However, differences exist in absolute annual rainfall (range: 154 mm), rainfall intensities, dry spell duration, rainy day counts, and the rainy season onset. Though calibration aligns key characteristics, the remaining differences lead to varying crop model results. While the model well reproduces official yield data using the observed rainfall time series (r = 0.52), the products’ results are heterogeneous (e.g., CHIRPS: r = 0.18). Overall, 97% of a household’s dietary energy demand is met. The study emphasizes the importance of considering the differences among multiple rainfall products when ground measurements are scarce.
The Global South is facing severe challenges in ensuring livelihood security due to climate change impacts, environmental degradation and population growth as well as changing lifestyles. These complex problems cannot be solely solved by single scientific disciplines – they require transdisciplinary research (TDR). Stakeholders from civil society, the corporate sector, government and science need to pool their knowledge to find solutions for sustainable transformations. In Namibia, we have been involved in TDR projects on water supply, and sanitation services as well as livestock management in rangeland systems. In this paper, we review two TDR projects that differ in multiple ways and hence allow us to carve out structural differences and critically discuss research outcomes, lessons learned and the challenge of North–South collaborations. Our review builds upon published and unpublished project documents as well as expert interviews with Namibian and German researchers who were involved in the projects. Our results show that TDR can be put into practice in different ways, depending on the research focus and the period available. The TDR phases of problem framing, inter- and transdisciplinary integration were implemented with different tools and foci points. We discuss the role of project length and funding conditions for project success and outcome generation. In addition, we critically consider the role of Namibian and German researchers in these international collaborations. The conclusions we draw touch upon the points of preparatory research funding, the equal acknowledgement of Global South contributions to joint research projects and the explicit handling of TDR components in project work. Significance: • The current social-ecological challenges are complex and require TDR as a mode of knowledge coproduction, particularly in a development context. • Inter- and transdisciplinary integration are critical processes for a project to be successful and require the allocation of adequate time and monetary resources. • Longer-term projects with a funded preparatory research phase constitute a structural model for TDR as project outcomes can evolve over time. • Global South researchers carry a hidden burden in international collaborations that has to be adequately acknowledged upfront in project planning and final products.
In times of global warming caused by the extensive use of fossil fuels, the need to capture gaseous carbon compounds is growing bigger. Several groups of microorganisms can fix the greenhouse gas CO2. Out of these, acetogenic bacteria are role models in their ability to reduce CO2 with hydrogen to acetate, which makes acetogens prime candidates for genetic modification towards biotechnological production of value-added compounds from CO2, such as biofuels. However, growth of acetogens on gaseous substrates is strongly energy-limited, and successful metabolic engineering requires a detailed knowledge of the bioenergetics. In 1939, Clostridium aceticum was the first acetogen to be described. A recent genomic study revealed that this organism contains cytochromes and therefore may use a proton gradient in its respiratory chain. We have followed up these studies and will present data that C. aceticum does not use a H+ but a Na+ gradient for ATP synthesis, established by a Na+-Rnf. Experimental data and in silico analyses enabled us to propose the biochemistry and bioenergetics of acetogenesis from H2 + CO2 in C. aceticum.
1. Recent research highlights the ecological importance of individual variation in behavioural predictability. Individuals may not only differ in their average expression of a behavioural trait (their behavioural type) and in their ability to adjust behaviour to changing environmental conditions (individual plasticity), but also in their variability around their average behaviour (predictability). However, quantifying behavioural predictability in the wild has been challenging due to limitations of acquiring sufficient repeated behavioural measures.
2. We here demonstrate how common biologging data can be used to detect individual variation in behavioural predictability in the wild and reveal the coexistence of highly predictable individuals along with unpredictable individuals within the same population.
3. We repeatedly quantified two behaviours—daily movement distance and diurnal activity—in 62 female brown bears Ursus arctos tracked across 187 monitoring years. We calculated behavioural predictability over the short term (50 consecutive monitoring days within 1 year) and long term (across monitoring years) as the residual intra-individual variability (rIIV) of behaviour around the behavioural reaction norm. We tested whether predictability varies systematically across average behavioural types and whether it is correlated across functionally distinct behaviours, that is, daily movement distance and amount of diurnal activity.
4. Brown bears showed individual variation in behavioural predictability from predictable to unpredictable individuals. For example, the standard deviation around the average daily movement distance within one monitoring year varied up to fivefold from 1.1 to 5.5 km across individuals. Individual predictability for both daily movement distance and diurnality was conserved across monitoring years. Individual predictability was correlated with behavioural type where individuals which were on average more diurnal and mobile were also more unpredictable in their behaviour. In contrast, more nocturnal individuals moved less and were more predictable in their behaviour. Finally, individual predictability in daily movement distance and diurnality was positively correlated, suggesting that individual predictability may be a quantitative trait in its own regard that could evolve and is underpinned by genetic variation.
5. Unpredictable individuals may cope better with stochastic events and unpredictability may hence be an adaptive behavioural response to increased predation risk.
Freshwater is one of the most fundamental resources for life and is the habitat for a wide diversity of species. One of the most diverse aquatic insect taxa is Trichoptera Kirby, 1813, caddisflies. These semi-aquatic insects have aquatic larvae and terrestrial adults and are found all around the globe in freshwater habitats. Water is also one of the most important natural resources for the human population, but alarmingly, freshwaters are among the most threatened natural habitats. Thus, the monitoring and preservation of the quality of freshwater habitats should have a high priority. In order to track changes in the biota a baseline reference is necessary, but freshwater biodiversity is under-studied in many parts of the Earth such as the biodiversity hotspots of the Himalaya and the Hengduan Mountains. This thesis treats the trichopteran genus Himalopsyche Banks, 1940 (Rhyacophilidae) which has its diversity center in the Himalayas and the Hengduan Mountains. Himalopsyche larvae are large and conspicuous and only occur in clean, unpolluted streams. This makes Himalopsyche potentially suited as indicator organisms for freshwater quality monitoring, but taxonomic knowledge is yet insufficient. Based on samples from a field survey in the Hengduan Mountains targeting both larvae and adults I uncovered three new Himalopsyche species which are described in this thesis (Chapter II), and with the aid of molecular data I associated larvae of Himalopsyche to adult species (Chapter I). The molecular association enabled the first comparative morphological study of Himalopsyche species in the larval stage, and the morphological study in Chapter II revealed that there are four distinct larval types of Himalopsyche. However, no diagnostic characters to identify Himalopsyche larvae to species level were found. To understand Himalopsyche larval morphology from an evolutionary perspective, I reconstructed the first molecular phylogeny of the genus (Chapter III). This demonstrated that each larval type corresponds to a deep phylogenetic split, indicating that larval types evolved early in Himalopsyche evolution and remained constant since. Based on the phylogenetic results as well as larval and adult morphology, I re-defined five species groups of Himalopsyche: H. kuldschensis Group, H. lepcha Group, H. navasi Group, H. phryganea Group, and H. tibetana Group. The species groups differ with respect to their diversity centers. The monotypic H. lepcha Group resides in the Himalayas, and the monotypic H. phryganea Group inhabits Western Nearctic. The H. kuldschensis and H. tibetana Groups are geographically overlapping with distributions in the Himalayas, but the distribution of H. kuldschensis Group stretches more to the west to include the Tian Shan, and the H. tibetana Group is more concentrated around the eastern Himalayas and the Hengduan Mountains. The H. navasi Group has a more eastern distribution than most Himalopsyche including isolated areas such as Japan and Indonesia. The earliest split in Himalopsyche divides the H. navasi Group from remaining Himalopsyche, suggesting a more eastern area of origin of Himalopsyche than its current diversity center, with subsequent radiations in the Himalayas and Hengduan Mountains. In addition to the three chapters, in this thesis I discuss further aspects of Himalopsyche biology including genital evolution, species complexes, and Himalopsyche ecology.
Xenorhabdus and Photorhabdus are bacterial genera that live in symbiosis with entomopathogenic nematodes of the genera Steinernema and Heterorhabditis, respectively. These nematodes infect insect larvae through the trachea and then enter the hemocoel. Once inside the hemocoel, the nematodes release the bacteria through their intestine. Thereafter, the bacteria become active and kill the larvae within 48 h. During this process, the immune system of the insect host is compromised by molecules produced and secreted by the bacteria. This illustrates that the bacteria possess not only a large arsenal of biological weaponry such as antibiotics and fungicides but also lipases, proteases, etc. Therefore, they are not only able to kill the insect but also protect the cadaver from other food competitors.
During the past decades, a large number of natural products have been identified from Xenorhabdus and Photorhabdus. However, the targets and functions for many of these biological molecules are still unknown. Therefore, the goal of the doctoral thesis is to elucidate the modes of action of these natural products from Xenorhabdus and Photorhabdus with the main focus on non-ribosomal peptides (NRPs). The work can be divided into two parts. Initially, it starts with the synthesis of natural compounds and various chemically modified derivatives. Besides that, a number of peptides were synthesized for other projects to either verify their structures or quantify the amount produced by the bacteria. Then, secondary analysis methods are applied and provide additional insight into the modes of action of these compounds.
During the thesis, I carried out peptide synthesis either manually or with an automatic synthesizer system from Biotage. Here, the Fmoc-protecting group strategy was preferred in most cases. Natural products, such as silathride, xenoautoxin, phenylethylamide, tryptamide, rhabdopeptide, 3-hydroxyoctanoic acid, and PAX, were produced during this process. Furthermore, new peptide derivatives derived from synthetic NRPS approaches using the XU concept or SYNZIP were generated as standards.
Most of these natural compounds were experimentally verified by MIC tests (broth microdilution, plate diffusion) to be biologically active. For example, silathride, phenylethylamide, and tryptamide showed quorum quenching effects when tested against Chromobacterium violaceum. Initial results from collaborators (PD Dr. Nadja Hellmann/Mainz) showed that tryptamide and phenylethylamide interact with membrane or membrane proteins.
(R)-3-hydroxyoctanoic acid was synthesized to verify the molecule structure of phototemtide A, a cyclic lipopeptide with antiprotozoal activity. The rhabdopeptides are another class, which showed remarkable antiprotozoal effects. However, their mode of action was unknown. These compounds are relatively short peptide sequences, which contain hydrophobic residues, such as valine, leucine, or phenylalanine. Moreover, they possess N methylation, resulting in a rod-shaped highly hydrophobic structure. In this work, I synthesized eight new derivatives of rhabdopeptides for photo-affinity labeling (PAL). These molecules should react covalently under UV-light irradiation with the biological target of the peptides. In addition, these derivatives can be enriched in a pull-down assay using click chemistry. Afterward, analytic methods such as mass detection (proteome analysis) can be applied to elucidate the protein targets.
The PAX peptides derivatives are well-known to have anti-microbial activities and believed to be secreted into the environment by the producing bacteria. However, I found that the majority of these peptides are located in the cell pellet fraction and not in the supernatant. This has been shown through quantification using HPLC MS. New PAX derivatives were synthesized, which carry a moiety suitable for covalent modification using click-chemistry, therefore being functionalizable with a fluorescence dye. In collaboration with Dr. Christoph Spahn (Prof. Dr. Mike Heilemann group), we used confocal, as well as super-resolution microscopy, in particular, single-molecule localization microscopy (SMLM) to investigate the spatial distribution of clickable PAX molecules and revealed that they localize at the bacterial membrane. Furthermore, bioactivity assays revealed that the promotor exchanged X. doucetiae PAX mutants, which do not produce PAX molecules without chemical induction (hereby termed as pax-), were more susceptible to several insect AMPs tested. Based on these findings, a new dual mechanism of action for PAX was proposed. Besides the previously shown antimicrobial activity, these molecules with a positive net charge of +5 (pH = 7) would bind to the negatively charged bacterial surface. Hereby, the surface charge (typically negative) would be inversed resulting in a protective effect for Xenorhabdus against other positively charged AMPs. Furthermore, PAX was investigated as AMP against E. coli to study its antimicrobial mechanism of action. Here, the results show that PAX can disrupt the E. coli membrane at higher concentrations (> 30 µg/ml), enter the cytosol, and lead to reorganization of subcellular structures, such as the nucleoid during this process.
Another aspect of secondary analysis is the application of proteomic analysis. Therefore, I induced X. nematophila, X. szentirmaii, and P. luminescens with insect lysate. These samples were analyzed using HPLC-MS/MS (Q Exactive) together with a database approach (Maxquant/Andromeda). The results showed that in all strains the lipid degradation and the glyoxylate pathway were induced. This is in line with the given insect lysate diet, which mostly contained lipids. Moreover, several interesting unknown peptides and proteins were also upregulated and might get into the focus of future research.
The genus Giraffa likely evolved around seven million years ago in Indo-Asia and spread over the Arabian-African land bridge into Eastern Africa. The oldest fossil of the African lineage was found in Kenya and dated to 7-5.4 Mya. Beside modern giraffe, four additional African species have likely existed (G. gracilis, G. pygmaea, G. stillei, and G. jumae). Based on their morphological similarities, G. gracilis is often considered to be the closest relative of the modern giraffe. Nevertheless, the phylogeny within the genus Giraffa is largely unresolved.
Modern giraffe (Giraffa sp.) have been neglected by the scientific community for a long time and still very little is known about their biology. Traditionally, present-day giraffe have been considered a single species (G. camelopardalis) which is divided into six to eleven subspecies, with nine subspecies being the most accepted classification. This classification was based on morphological differences and geographic ranges. However, recent genetic analyses found hidden diversity within Giraffa and proposed four genetically distinct giraffe species (G. camelopardalis, G. reticulata, G. tippelskirchi, G. giraffa) with presumably little gene flow among them.
Gene flow on a population level is the exchange of genetic information among populations facilitated by the migration of individuals between populations. Additionally, it is an important criterion to delineate species, because many species concepts, especially the Biological Species Concept, rely on the concept of reproductive isolation. Yet, new genetic methods are identifying an increasing number of species that show signs of introgressive hybridization or gene flow among them. Therefore, strict reproductive isolation cannot always be applied to delineate species, especially in young, probably still diverging, species such as giraffe.
Therefore, giraffe are ideal study organisms to investigate the level of gene flow in recently diverged species with adjacent or potentially overlapping ranges. Furthermore, their recent classification as “Vulnerable” by the IUCN and their unreliable distribution maps require the genetic evaluation of their population structure, distribution and conservation status.
In Publication 1 (Winter et al. (2018a), Ecological Genetics and Genomics, 7–8, 1–5), I studied the distribution and matrilineal population structure of Angolan giraffe (G. giraffa angolensis) using sequences from the cytochrome b gene (1,140 bp) and the mitochondrial control region for individuals from across their known range and beyond, and additionally including individuals from all known giraffe species and subspecies. The reconstruction of a phylogenetic tree and a mitochondrial haplotype network allowed to identify the most easterly known natural population of Angolan giraffe, a population that was previously assigned to their sister-subspecies South African giraffe (G. giraffa giraffa), indicating the limit of classification by morphology and geography. Furthermore, the analyses show that Namibia’s iconic desert-dwelling giraffe population is genetically distinct, even from the nearest population at Etosha National Park, suggesting very limited, if any, natural exchange of matrilines. Yet, no geographic barriers are known for this region that would prevent genetic exchange. Therefore, the two populations are likely on different evolutionary trajectories. Limited individuals with an Etosha haplotype further suggest that translocation of Etosha giraffe into the desert population had only a minor impact on the local population. Two separate haplogroups within Etosha National Park suggest an “out of Etosha” radiation of Angolan giraffe to the East followed by a later back-migration.
In Publication 2 (Winter et al. (2018b), Ecology and Evolution, 8(20), 10156–10165), I investigated the genetic population structure of giraffe across their range (n = 137) with focus on the amount of gene flow among the proposed giraffe species with a 3-fold increased set of nuclear introns (n = 21). Limited gene flow of less than one effective migrant per generation, even between the closely related northern (G. camelopardalis) and reticulated giraffe (G. reticulata) further supports the existence of four giraffe species by a different methodology, gene flow. This is significant because most species concepts build on reproductive isolation. Furthermore, this result is corroborated by four distinct major clades in a phylogenetic tree analysis, and distinct clusters in Principal Component Analysis and STRUCTURE analysis. All these analyses suggest a low level of genetic exchange among the four giraffe species and, therefore, a high degree of reproductive isolation in accordance with the Biological Species Concept (BSC). In Addition, only a single individual in 137 was identified as being potential of natural hybrid origin, which promotes the four-species concept further. ...
The Mediterranean realm, comprising the Mediterranean and Macaronesian regions, has long been recognized as one of the world’s biodiversity hotspots, owing to its remarkable species richness and endemism. Several hypotheses on biotic and abiotic drivers of species diversification in the region have been often proposed but rarely tested in an explicit phylogenetic framework. Here, we investigate the impact of both species-intrinsic and -extrinsic factors on diversification in the species-rich, cosmopolitan Limonium, an angiosperm genus with center of diversity in the Mediterranean. First, we infer and time-calibrate the largest Limonium phylogeny to date. We then estimate ancestral ranges and diversification dynamics at both global and regional scales. At the global scale, we test whether the identified shifts in diversification rates are linked to specific geological and/or climatic events in the Mediterranean area and/or asexual reproduction (apomixis). Our results support a late Paleogene origin in the proto-Mediterranean area for Limonium, followed by extensive in situ diversification in the Mediterranean region during the late Miocene, Pliocene, and Pleistocene. We found significant increases of diversification rates in the “Mediterranean lineage” associated with the Messinian Salinity Crisis, onset of Mediterranean climate, Plio-Pleistocene sea-level fluctuations, and apomixis. Additionally, the Euro-Mediterranean area acted as the major source of species dispersals to the surrounding areas. At the regional scale, we infer the biogeographic origins of insular endemics in the oceanic archipelagos of Macaronesia, and test whether woodiness in the Canarian Nobiles clade is a derived trait linked to insular life and a biotic driver of diversification. We find that Limonium species diversity on the Canary Islands and Cape Verde archipelagos is the product of multiple colonization events followed by in situ diversification, and that woodiness of the Canarian endemics is indeed a derived trait but is not associated with a significant shift to higher diversification rates. Our study expands knowledge on how the interaction between abiotic and biotic drivers shape the uneven distribution of species diversity across taxonomic and geographical scales.
To support future research based on natural sciences collection data, DiSSCo (Distributed System of Scientific Collections) – the European Research Infrastructure for Natural Science Collections – adopts Digital Object Architecture as the basis for its planned data infrastructure. Using the outputs of one Research Data Alliance (RDA) interest group (IG) and five working groups (WGs) we show how RDA recommendations and supporting documents have been applied to the various stages of the DiSSCo data lifecycle.
Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO2-based bioeconomy and a H2-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H2 cycle and thus prime candidates as driving forces in a H2- and CO2-bioeconomy. Naturally, they convert two molecules of CO2 via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO2, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.
Cell fate clusters in ICM organoids arise from cell fate heredity and division: a modelling approach
(2020)
During the mammalian preimplantation phase, cells undergo two subsequent cell fate decisions. During the first decision, the trophectoderm and the inner cell mass are formed. Subsequently, the inner cell mass segregates into the epiblast and the primitive endoderm. Inner cell mass organoids represent an experimental model system, mimicking the second cell fate decision. It has been shown that cells of the same fate tend to cluster stronger than expected for random cell fate decisions. Three major processes are hypothesised to contribute to the cell fate arrangements: (1) chemical signalling; (2) cell sorting; and (3) cell proliferation. In order to quantify the influence of cell proliferation on the observed cell lineage type clustering, we developed an agent-based model accounting for mechanical cell–cell interaction, i.e. adhesion and repulsion, cell division, stochastic cell fate decision and cell fate heredity. The model supports the hypothesis that initial cell fate acquisition is a stochastically driven process, taking place in the early development of inner cell mass organoids. Further, we show that the observed neighbourhood structures can emerge solely due to cell fate heredity during cell division.
Reprogramming of tomato leaf metabolome by the activity of heat stress transcription factor HsfB1
(2020)
Plants respond to high temperatures with global changes of the transcriptome, proteome, and metabolome. Heat stress transcription factors (Hsfs) are the core regulators of transcriptome responses as they control the reprogramming of expression of hundreds of genes. The thermotolerance-related function of Hsfs is mainly based on the regulation of many heat shock proteins (HSPs). Instead, the Hsf-dependent reprogramming of metabolic pathways and their contribution to thermotolerance are not well described. In tomato (Solanum lycopersicum), manipulation of HsfB1, either by suppression or overexpression (OE) leads to enhanced thermotolerance and coincides with distinct profile of metabolic routes based on a metabolome profiling of wild-type (WT) and HsfB1 transgenic plants. Leaves of HsfB1 knock-down plants show an accumulation of metabolites with a positive effect on thermotolerance such as the sugars sucrose and glucose and the polyamine putrescine. OE of HsfB1 leads to the accumulation of products of the phenylpropanoid and flavonoid pathways, including several caffeoyl quinic acid isomers. The latter is due to the enhanced transcription of genes coding key enzymes in both pathways, in some cases in both non-stressed and stressed plants. Our results show that beyond the control of the expression of Hsfs and HSPs, HsfB1 has a wider activity range by regulating important metabolic pathways providing an important link between stress response and physiological tomato development.
Mutualistic interactions between plants and animals can affect both plant and animal communities, and potentially leave imprints on plant demography. Yet, no study has simultaneously tested how trait variation in plant resources shapes the diversity of animal consumers, and how these interactions influence seedling recruitment. Here, we analyzed whether (i) phylogenetic diversity and functional diversity of fruiting plants were correlated with the corresponding diversity of frugivorous birds, and (ii) whether phylogenetic diversity and functional identity of plant and bird communities influenced the corresponding diversity and identity of seedling communities. We recorded mutualistic interactions between fleshy-fruited plants and frugivorous birds and seedling communities in 10 plots along an elevational gradient in the Colombian Andes. We built a phylogeny for plants/seedlings and birds and measured relevant morphological plant and bird traits that influence plant-bird interactions and seedling recruitment. We found that phylogenetic diversity and functional diversity of frugivorous birds were positively associated with the corresponding diversities of fruiting plants, consistent with a bottom-up effect of plants on birds. Moreover, the phylogenetic diversity of seedlings was related to the phylogenetic diversity of plants, but was unrelated to the phylogenetic diversity of frugivorous birds, suggesting that top-down effects of animals on seedlings were weak. Mean seed mass of seedling communities was positively associated with the mean fruit mass of plants, but was not associated with the mean avian body mass in the frugivore communities. Our study shows that variation in the traits of fleshy-fruited plants was associated with the diversity of frugivorous birds and affected the future trajectory of seedling recruitment, whereas the morphological traits of animal seed dispersers were unrelated to the phylogenetic and functional structure of seedling communities. These findings suggest that bottom-up effects are more important than top-down effects for seed-dispersal interactions and seedling recruitment in diverse tropical communities.
In biological systems (cell culture media, cells, body fluids), drugs/toxicants are usually not freely dissolved but partially bound to biomolecules; only a fraction of the chemical is free/unbound (fu). To predict pharmacological effects and toxicity, it is important that the fu of the drug is known. As the differences between free and nominal concentrations are determined by test system parameters (e.g., the protein and lipid content, and the type of surface material), comparison of nominal concentrations between two different new approach methods (NAM) may lead to faulty conclusions. The same problem exists when in vitro concentrations are compared to those in human subjects. Therefore, the respective fu of a chemical in a test system needs to be determined for in vitro-to-in vivo extrapolations (IVIVE). Besides direct measurements, prediction models can help to obtain fu. Here we describe a simplified approach to approximate fu and provide background information on the underlying assumptions. Comparative predictions and measurements of fu of various drugs are shown to exemplify the approach. Basic input data, like protein and lipid concentrations, are also provided. Beyond such test systems data, the only required chemical-specific inputs are the lipophilicity of the candidate drug and its ionization state, as determined by the dissociation constants of its acidic or basic groups. This overview is intended to be used by any lab scientist without specific toxicokinetics training to obtain an estimate of fu in a given cell culture medium.
The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.
Biominerals fossilisation: fish bone diagenesis in plio–pleistocene african hominid sites of Malawi
(2020)
Fish fossilisation is relatively poorly known, and skeletal element modifications resulting from predation, burial and diagenesis need to be better investigated. In this article, we aim to provide new results about surface, structural and chemical changes in modern and fossil fish bone. Fossil samples come from two distinct localities of roughly the same age in the Pliocene–Pleistocene Chiwondo Beds adjacent to Lake Malawi. Optical and scanning electron microscope (SEM) observations, energy dispersive spectroscopy (EDS) analyses and Fourier transform infrared (FTIR) spectrometry were carried out on three categories of fish bones: (i) fresh modern samples collected in the lake, (ii) extracted from modern fish eagle regurgitation pellets, and (iii) fossils from Malema and Mwenirondo localities. A comparison of these data allowed us to detect various modifications of bone surfaces and structure as well as composition changes. Some differences are observed between fresh bones and modern pellets, and between pellets and fossils. Moreover, fossil fish bone surface modifications, crystallinity, and chemical composition from Malema and Mwenirondo differ despite their chronological and spatial proximities (2.5–2.4 Ma, 500 m). In both sites, the post-predation modifications are strong and may hide alterations due to the predation by bird of prey such as the fish eagle. The combination of the used methods is relevant to analyses of diagenetic alterations in fish bones.
A new cyclic lipopeptide, phototemtide A (1), was isolated from Escherichia coli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR‐ESI‐MS and NMR experiments. The absolute configurations of amino acids and 3‐hydroxyoctanoic acid in 1 were determined by using the advanced Marfey's method and comparison after total synthesis of 1, respectively. Additionally, three new minor derivatives, phototemtides B–D (2–4), were identified by detailed HPLC–MS analysis. Phototemtide A (1) showed weak antiprotozoal activity against Plasmodium falciparum, with an IC50 value of 9.8 μm. The biosynthesis of phototemtides A–D (1–4) was also proposed.
A novel role for mutant mRNA degradation in triggering transcriptional adaptation to mutations
(2020)
Robustness to mutations promotes organisms’ well-being and fitness. The increasing number of mutants in various model organisms, and humans, showing no obvious phenotype (Bouche and Bouchez, 2001; Chen et al., 2016b; Giaever et al., 2002; Kok et al., 2015) has renewed interest into how organisms adapt to gene loss. In the presence of deleterious mutations, genetic compensation by transcriptional upregulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems (El-Brolosy and Stainier, 2017; Rossi et al., 2015; Tondeleir et al., 2012); however, the molecular mechanisms underlying this response remained unclear. To investigate this phenomenon, I develop and study multiple models of transcriptional adaptation in zebrafish and mouse cell lines. I first show that transcriptional adaptation is not caused by loss of protein function, indicating that the trigger lies upstream, and find that the response involves enhanced transcription of the related gene(s). Furthermore, I observe a correlation between levels of mutant mRNA degradation and upregulation of related genes. To investigate the role of mutant mRNA degradation in triggering the response, I generate mutant alleles that do not transcribe the mutated gene and find that they fail to induce a transcriptional response and display stronger phenotypes. Transcriptome analysis of alleles displaying mutant mRNA degradation revealed upregulation of a significant proportion of genes displaying sequence similarity with the mutated gene’s mRNA, suggesting a model whereby mRNA degradation intermediates induce transcriptional adaptation via sequence similarity. Further mechanistic analyses suggested RNA-decay factors-dependent chromatin remodeling, and repression of antisense RNAs to be implicated in the response. These results identify a novel role for mutant mRNA degradation in buffering against mutations. Besides, they hold huge implications on understanding disease-causing mutations and shall help in designing mutations that lead to minimal transcriptional adaptation-induced compensation, facilitating studying gene function in model organisms.
Climate change is influencing some environmental variables in the Southern Ocean (SO) and this will have an effect on the marine biodiversity. Peracarid crustaceans are one of the dominant and most species-rich groups of the SO benthos. To date, our knowledge on the influence of environmental variables in shaping abundance and species composition in the SO’s peracarid assemblages is limited, and with regard to ice coverage it is unknown. The aim of our study was to assess the influence of sea ice coverage, chlorophyll-a, and phytoplankton concentrations on abundance, distribution and assemblage structure of peracarids. In addition, the influence of other physical parameters on peracarid abundance was assessed, including depth, temperature, salinity, sediment type, current velocity, oxygen, iron, nitrate, silicate and phosphate. Peracarids were sampled with an epibenthic sledge (EBS) in different areas of the Atlantic sector of the SO and in the Weddell Sea. Sampling areas were characterized by different regimes of ice coverage (the ice free South Orkney Islands, the seasonally ice-covered Filchner Trough and the Eastern Antarctic Peninsula including the Prince Gustav Channel which was formerly covered by a perennial ice shelf). In total 64766 individuals of peracarids were collected and identified to order level including five orders: Amphipoda, Cumacea, Isopoda, Mysidacea, and Tanaidacea. Amphipoda was the most abundant taxon, representing 32% of the overall abundances, followed by Cumacea (31%), Isopoda (29%), Mysidacea (4%), and Tanaidacea (4%). The Filchner Trough had the highest abundance of peracarids, while the South Orkney Islands showed the lowest abundance compared to other areas. Ice coverage was the main environmental driver shaping the abundance pattern and assemblage structure of peracarids and the latter were positively correlated with ice coverage and chlorophyll-a concentration. We propose that the positive correlation between sea ice and peracarid abundances is likely due to phytoplankton blooms triggered by seasonal sea ice melting, which might increase the food availability for benthos. Variations in ice coverage extent and seasonality due to climate change would strongly influence the abundance and assemblage structure of benthic peracarids.
The species composition of local communities varies in space, and its similarity generally decreases with increasing geographic distance between communities, a phenomenon known as distance decay of similarity. It is, however, not known how changes in local species composition affect ecological processes, that is, whether they lead to differences in the local composition of species' functional roles. We studied eight seed‐dispersal networks along the South American Andes and compared them with regard to their species composition and their composition of functional roles. We tested (1) if changes in bird species composition lead to changes in the composition of bird functional roles, and (2) if the similarity in species composition and functional‐role composition decreased with increasing geographic distance between the networks. We also used cluster analysis to (3) identify bird species with similar roles across all networks based on the similarity in the plants they consume, (i) considering only the species identity of the plants and (ii) considering the functional traits of the plants. Despite strong changes in species composition, the networks along the Andes showed similar composition of functional roles. (1) Changes in species composition generally did not lead to changes in the composition of functional roles. (2) Similarity in species composition, but not functional‐role composition, decreased with increasing geographic distance between the networks. (3) The cluster analysis considering the functional traits of plants identified bird species with similar functional roles across all networks. The similarity in functional roles despite the high species turnover suggests that the ecological process of seed dispersal is organized similarly along the Andes, with similar functional roles fulfilled locally by different sets of species. The high species turnover, relative to functional turnover, also indicates that a large number of bird species are needed to maintain the seed‐dispersal process along the Andes.
Au Tchad, à cause de ses retombées financières une attention particulière est prêtée aux arbres à karité (Vitella- ria paradoxa C.F.Gaertn.). Cependant, cette culture est menacée par les plantes vasculaires parasites de la famille des Lo- ranthaceae. La présente étude a été effectuée dans 3 sites dans la région du Mandoul pour évaluer l’ampleur des attaques de Loranthaceae (gui africain) sur des arbres en fonction des classes de circonférence du tronc à 1,5 cm du sol. Elle a consisté à dénombrer sur une de surface, les arbres à karité infestés et les touffes de parasites rencontrées sur ces arbres, afin de dé- terminer leur taux et leur intensité d’infestation. Les résultats obtenus montrent que Tapinanthus dodoneifolus (DC) Danser a été trouvée comme la seule espèce de Loranthaceae qui parasite les arbres karité étudiés dans la zone d’étude. Le taux moyen d’infestation estimé à 73% augmente avec l’âge des arbres karité. La moyenne d’intensité de l’infestation/arbre (2,75 touffes à Békôh, 2,27 à Yomi and 2,04 à Bébopen) montre que Tapinanthus dodoneifolus constitue une réelle menace pour les peu- plements de karité dans la zone d’étude. Il reste à rechercher le seuil d’infestation qui provoque une réduction significative de la fructification. Pour l’instant, bien que pénible à cause de la hauteur des arbres adultes, la lutte mécanique contre les Ta- pinanthus par la coupe systématique des branches infestées est urgente dans les parcs à karité dans cette zone d’étude.
Déterminants de l’utilisation de Acacia auriculiformis comme bois d’œuvre en Afrique de l’Ouest
(2020)
Acacia auriculiformis, un bois énergie, suscite de plus en plus des intérêts de bois d’œuvre au niveau des industriels de bois au Bénin. L’appréciation des performances de l’espèce dans les usines et en plantation est déterminante pour la vul- garisation de l’espèce comme alternative pour mitiger la déforestation en lien avec la demande en bois d’œuvre. L’objectif principal de ce travail est donc d’évaluer les conditions entourant l’adoption de Acacia auriculiformis comme espèce de bois d’œuvre au Bénin, Afrique de l’Ouest. Au total, 154 usines de bois et 25 plantations ont été enquêtées dans les zones abritant les plantations à A. auriculiformis. A. auriculiformis est l’espèce la plus fréquente dans les usines de bois (81%) suivie de Afzelia africana (55%), Tectona grandis (47%) et Khaya senegalensis (47%). Les superficies des plantations à A. auriculi- formis ont augmenté entre 1999 et 2019. Les connaissances sur l’utilisation de ce bois sont variables dans la zone d’étude. Le bois de A. auriculiformis est apprécié comme bois d’œuvre parce qu’il présente une couleur esthétique, un séchage rapide, une facilité de mise en œuvre, une imprégnabilité élevée, une densité moyenne à élevée et un bel aspect après mise en œuvre. Cependant, son bois fournit beaucoup de sciure, a beaucoup de nœuds et présente une déformabilité moyenne. Sa disponibili- té et son accessibilité sont les principaux facteurs justifiant la préférence de l’espèce par les industriels de bois d’œuvre. Cette forme d’utilisation de l’espèce est également remarquée au Togo, en Côte d’Ivoire. L’espèce présente une bonne perspective d’utilisation comme bois d’œuvre.
Ipomoea beninensis Akoègn., Lisowski & Sinsin (Convolvulaceae) is the only endemic plant known for Benin. To date, no data exist on its usages, distribution, abundance, and threats. An improved understanding of indigenous know- ledge and of local practices can provide insight into how the species could be sustainably conserved. We interviewed 114 local residents for collecting ethnobotanical and ethnoecological data in six sites known to host the species. Data were pro- cessed by calculation of descriptive statistics and variance and multivariate analyses. A total of twelve uses were reported. Among them, treatment of varicella (19%), malaria (18%) and fodder (17%) were the most recurrent. These mainly involve use of the species rootstock. Almost all respondents mentioned decline of the species in natural habitats. None of them was aware about the endemic status of the species. Consequently, negative practices toward the protection of I. beninensis were prevalent among local residents. Several conservation measures are proposed to ensure the longterm survival of I. beninensis.
Neural oscillations are at the core of important computations in the mammalian brain. Interactions between oscillatory activities in different frequency bands, such as delta (1–4 Hz), theta (4–8 Hz) or gamma (>30 Hz), are a powerful mechanism for binding fundamentally distinct spatiotemporal scales of neural processing. Phase-amplitude coupling (PAC) is one such plausible and well-described interaction, but much is yet to be uncovered regarding how PAC dynamics contribute to sensory representations. In particular, although PAC appears to have a major role in audition, the characteristics of coupling profiles in sensory and integration (i.e. frontal) cortical areas remain obscure. Here, we address this question by studying PAC dynamics in the frontal-auditory field (FAF; an auditory area in the bat frontal cortex) and the auditory cortex (AC) of the bat Carollia perspicillata. By means of simultaneous electrophysiological recordings in frontal and auditory cortices examining local-field potentials (LFPs), we show that the amplitude of gamma-band activity couples with the phase of low-frequency LFPs in both structures. Our results demonstrate that the coupling in FAF occurs most prominently in delta/high-gamma frequencies (1-4/75-100 Hz), whereas in the AC the coupling is strongest in the delta-theta/low-gamma (2-8/25-55 Hz) range. We argue that distinct PAC profiles may represent different mechanisms for neuronal processing in frontal and auditory cortices, and might complement oscillatory interactions for sensory processing in the frontal-auditory cortex network.
Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding.
Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar‐based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2‐dependent CO2 reductase and furthermore belong to different classes of ‘Rnf’‐ and ‘Ech‐acetogens’. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood–Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g−1 h−1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO‐based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.
Large carnivores often impact human livelihoods and well‐being. Previous research has mostly focused on the negative impacts of large carnivores on human well‐being but has rarely considered the positive aspects of living with large carnivores. In particular, we know very little on people's direct experiences with large carnivores like personal encounters and on people's awareness and tolerance toward their exposure to large carnivores. Here, we focus on the wolf (Canis lupus), and report on a phone survey in Germany. We examined whether encounters with wolves were positive or negative experiences and quantified people's awareness and tolerance related to their exposure to wolves. We found that the majority of people reported positive experiences when encountering wolves, regardless of whether wolves were encountered in the wild within Germany, in the wild abroad, or in captivity. The frequency of encounters did not affect the probability to report positive, neutral, or negative experiences. Moreover, people in Germany expressed a high tolerance of living in close vicinity to wolves. These findings are novel and important because they highlight the positive aspects of living in proximity with large carnivores in human‐dominated landscapes.
Even one century after Santiago Ramón y Cajal’s groundbreaking contribu- tions to neuroscience, one of the most fundamental questions in the field is still largely open, namely understanding how the shape of a dendrite is adapted to its specific biological function. A systematic investigation of this problem is challenging both technically and conceptually because neurons have diverse genetic, molecular, morphological, connectional and functional properties.
In the light of the preceding, dendritic arborisation (da) neurons of the Drosophila melanogaster larva PNS have proven to be an excellent model system for the study of such growth and patterning processes. Structure and function in these cell classes are intimately intertwined, as class type-specific dendritic arbour differentiation processes are required to satisfy a given phys- iological need. Also, there is a remarkable genetic toolkit that enables one to selectively and reproducibly label, image and manipulate each one of these sensory neuron classes. In this thesis, I address the aforementioned open problem by linking single-cell patterning, information processing and wiring optimisation in sensory da neurons to behaviour in Drosophila larva.
In particular, I study Class I ventral peripherical dendritic arborisation (c1vpda) neurons. These are a class of proprioceptive neurons that relay information on the position of the larva’s body back to the CNS during crawling behaviour to assure proper locomotion. Their stereotypical comb- like shaped dendritic branches spread along the body-wall, and they get noticeably deformed during crawling behaviour. The bending of the den- dritic branches is hypothesised to be a possible mechanism to transduce the mechanosensory inputs arising from cuticle folding. Interestingly, c1vpda neurons do not necessarily satisfy optimal wiring constraints since they are required to pattern into a specific shape to fulfil their function. Therefore, I considered the da system to study how the specific functional requirements may be combined with optimal wiring constraints during development.
Although the molecular machinery of dendrite patterning in c1vpda neurons is well studied, the precise elaboration of the comb-like shaped dendrites of these cells remains elusive. Moreover, even though a lot of work has been put into the description and quantification of growth processes of the nervous system, there are still few solid and standardised models of arbour staging and patterning. Importantly, the defining parameters that determine the dendrite elaboration program that in turn is responsible for creating the final arbour morphology are still unknown. As a result, unraveling possible universal stages of dendrite elaboration shared between different model systems and cell types is challenging.
Thus, in order to understand the development of the fine regulation of branch outgrowth that leads to the observed terminal arbour morphology in the mature cell, I collected in vivo, long-term, non-invasive high temporal res- olution time-lapse recordings of dendritic trees during the differentiation process in the embryo and its maturation phase in the larva. For further analysis, I developed new algorithms that quantified the structural changes in dendrite morphology in the time-lapse videos. My approach provides a framework to analyse such developmental data, or any dataset comprising continuous morphological dynamical processes in an unbiased way. Using these newly developed methods, I examined the development of a sample of c1vpda cells and identified five stages of differentiation in these data: initial stem polarization, extension, pruning, stabilization, and isometric stretching during larval stages.
The beginning of the growth process is marked by the polarisation of the main stem. Subsequently, during the extension phase, branches emerge interstitially from the existing main stem. Later, higher-order branches sprout from pre-existing lateral branches, increasing arbour complexity. This is followed by a pruning stage where developmental intermediate dendritic branches are removed. This step leads to a spatial rearrangement of the dendritic tree. The end of the pruning step is followed by a stabilisation period where arbour morphology remains virtually unaltered in the embryo. After hatching, c1vpda dendrites experience an isometric scaling, with their branching complexity and pattern being invariant across all larval stages.
After dissecting the c1vpda dendrites spatiotemporal differentiation process, I established a link between dendritic shape and behaviour. I measured intra- cellular Ca++ activity in the dendrite branches of l1 larvae during forward locomotion, while simultaneously recording branch deformation using a dual genetic line. I reported that post-embryonic c1vpda dendrites Ca++ responses increased in freely crawling larvae. Furthermore, I showed strong correlations between Ca++ signal and deformation of the comb-like dendritic ranches during body-wall contractions.
Then, using a geometrical model, I provided evidence that the pruning stage could reorganise the dendrite morphology to maximise mechanosensory re- sponses during body wall contraction. I showed that the angle orientation of each side branch correlates with the bending curvature and thus with the me- chanical displacement of the cell membrane during locomotion. During the pruning phase, I observed a preferential reduction of less efficient branches with low bending curvature, influencing the mechanisms of dendritic sig- nal integration of c1vpda sensory neurons. I proceeded to quantify branch dynamics at single tip resolution during pruning, providing evidence that a simple random pruning mechanism is sufficient to remodel the tree structure compatible with the observed way.
I used these time-lapse data to constrain a new computational noisy growth model with random pruning based on optimal wiring principles. This model is able to generate highly realistic synthetic c1vpda morphologies. The model furthermore requires few parameters to generate highly accurate temporal development trajectories and morphologies at single-cell level. Utilising this data and model enabled me to investigate upon the hypothesis that a noisy dendrite growth and random pruning mechanism synergise to achieve den- dritic trees efficient in terms of both wiring and function. My findings show how single neurons can create functionally specialised dendrites while min- imising wiring costs, elucidating how general principles of self-organisation may be involved in the generation of these structures.
Atelopus is a species-rich group of Neotropical bufonids. Present knowledge on bioacoustics in this genus is relatively poor, as vocalisations have been described in only about one fifth of the ca. 100 species known. All studied members of the genus produce vocalisations although, with a few exceptions, most species lack a middle ear. Nonetheless, hearing has been demonstrated even in earless Atelopus making bioacoustics in these toads an inspiring research field. So far, three structural call types have been identified in the genus. As sympatry is uncommon in Atelopus, calls of the same type often vary little between species. Based on recordings from the 1980s, we describe vocalisations of three Venezuelan species (A. carbonerensis, A. mucubajiensis, A. tamaense) from the Cordillera de Mérida, commonly known as the Andes of Venezuela and the Tamá Massif, a Venezuelan spur of the Colombian Cordillera Oriental. Vocalisations correspond, in part, to the previously identified call types in Atelopus. Evaluation of the vocalisations of the three species presented in this study leads us to recognise a fourth structural call type for the genus. With this new addition, the Atelopus acoustic repertoire now includes (1) pulsed calls, (2) pure tone calls, (3) pulsed short calls and (4) pure tone short calls. The call descriptions provided here are valuable contributions to the bioacoustics of these Venezuelan Atelopus species, since all of them have experienced dramatic population declines that limit possibilities of further studies.
The growing number of infections with multi-resistant bacteria or the current COVID-19 pandemic put compounds with therapeutic properties into the public focus. Non-ribosomal peptides (NRPs) are natural products that are already marketed as antibiotics, cytotoxic agents or immunosuppressants. Their biological activities rely on the structural diversity including non-proteinogenic amino acids (AAs), heterocycles or modifications like methylation or acylation.
The biosynthesis of NRPs is carried out by non-ribosomal peptide synthetases (NRPSs). These multifunctional megaenzymes show a modular architecture like in an assembly-line. Each module is thereby responsible for the incorporation and modification of one AA and therefore contains different catalytic domains. The adenylation (A) domain recognizes and activates its specific substrate in an ATP-dependent manner which is transferred to a 4’-phosphopantetheine cofactor post-translationally attached to the thiolation (T) domain. Peptide bond formation between two T domain bound substrates catalysed by the condensation (C) domain transfers the growing peptide chain to the following module. Such a C-A-T module can be extended with optional domains to integrate structural diversity and a terminal thioesterase (TE) domain usually releases the peptide via hydrolysis or intramolecular attack of nucleophiles. Inspired by the modular architecture, NRPS engineering deals with the modification of NRPs in order to increase biological activities, circumvent bacterial resistances or create de novo peptides. This can be achieved by mutasynthesis or modification of the substrate binding pocket as well as single and multiple domain substitution. However, the few successful approaches led to impaired enzymes and did not establish a general applicable guideline. In the first publication as part of this work, the development of such a guideline comprising three rules is addressed. First, the A-T-C tridomain named exchange unit (XU) is seen as a catalytic unit instead of a module. When using them as building blocks, the C domain’s specificity for the AA of the following XU has to be considered as second rule. Third, a conserved WNATE motif within the C-A linker depicts the fusion point of the XUs. Upon heterologous expression of the cloned plasmids in E. coli and high performance liquid chromatography coupled mass spectrometry-based analysis of the extracts, the ambactin-producing NRPS from Xenorhabdus was reprogrammed with one and two XUs. This only leads to a moderate loss of production titre or an even higher one when the AA configuration was changed by introducing a dual condensation/epimerization (C/E) domain. The pentamodular GameXPeptide-producing NRPS was reconstructed using up to five XUs of four different NRPSs and even completely de novo synthetases were created. The second publication describes the exchange unit condensation domain (XUC) concept and relies on a fusion point between the two subdomains (N-terminal CDsub and C-terminal CAsub) of the C domain’s V-shaped pseudodimeric structure which generates A-T didomains with flanking CAsub and CDsub. These hybrid C domain-forming building blocks depict an improvement to the XU concept by avoiding the drawback of C domain specificity. This allows a more flexible NRPS engineering that can e.g. enable peptide library design. Furthermore, beside a combination of both concepts within one NRPS and a transfer to Bacillus NRPSs, the use of XUC with relaxed A domain specificity allowed further peptide modifications by introducing non-natural AAs. The third publication deals with aldehyde and alcohol-generating reductase (R) domains which depict an alternative for peptide release in NRPSs. A promoter exchange in X. indica identified a pyrazine-producing NRPS with a minimal architecture of an A, T and R domain and was therefore termed ATRed. R domains were additionally used in engineered NRPSs to produce pyrazinones and derivatives thereof by XU substitution although most constructs failed to show production. Beyond that, an R domain has been shown to replace a TE domain in wild type synthetases leading to slightly modified NRPs and the postulated biosynthesis was incidentally revised. Furthermore, an NRPS with terminal R domain was engineered to produce a free peptide aldehyde, which are known to be potent proteasome inhibitors. For the above mentioned ATReds, the presence of up to three coding regions was further identified in 20 different Xenorhabdus strains but only six of them were verified to produce pyrazines. All ATReds share variable sequence similarities among each other and were subsequently divided into three subtypes. One subtype is supposed to perform the pyrazine biosynthesis via a non-canonical catalytic triad.
Background: The gut microbiome can influence life history traits associated with host fitness such as fecundity and longevity. In most organisms, these two life history traits are traded-off, while they are positively linked in social insects. In ants, highly fecund queens can live for decades, while their non-reproducing workers exhibit much shorter lifespans. Yet, when fertility is induced in workers by death or removal of the queen, worker lifespan can increase. It is unclear how this positive link between fecundity and longevity is achieved and what role the gut microbiome and the immune system play in this. To gain insights into the molecular regulation of lifespan in social insects, we investigated fat body gene expression and gut microbiome composition in workers of the ant Temnothorax rugatulus in response to an experimental induction of fertility and an immune challenge.
Results: Fertile workers upregulated several molecular repair mechanisms, which could explain their extended lifespan. The immune challenge altered the expression of several thousand genes in the fat body, including many immune genes, and, interestingly, this transcriptomic response depended on worker fertility. For example, only fertile, immune-challenged workers upregulated genes involved in the synthesis of alpha-ketoglutarate, an immune system regulator, which extends the lifespan in Caenorhabditis elegans by down-regulating the TOR pathway and reducing oxidant production. Additionally, we observed a dramatic loss in bacterial diversity in the guts of the ants within a day of the immune challenge. Yet, bacterial density did not change, so that the gut microbiomes of many immune challenged workers consisted of only a single or a few bacterial strains. Moreover, the expression of immune genes was linked to the gut microbiome composition, suggesting that the ant host can regulate the microbiome in its gut.
Conclusions: Immune system flare-ups can have negative consequence on gut microbiome diversity, pointing to a previously underrated cost of immunity. Moreover, our results provide important insights into shifts in the molecular regulation of fertility and longevity associated with insect sociality.
A tale of two seasons: The link between seasonal migration and climatic niches in passerine birds
(2020)
The question of whether migratory birds track a specific climatic niche by seasonal movements has important implications for understanding the evolution of migration, the factors affecting species' distributions, and the responses of migrants to climate change. Despite much research, previous studies of bird migration have produced mixed results. However, whether migrants track climate is only one half of the question, the other being why residents remain in the same geographic range year-round. We provide a literature overview and test the hypothesis of seasonal niche tracking by evaluating seasonal climatic niche overlap across 437 migratory and resident species from eight clades of passerine birds. Seasonal climatic niches were based on a new global dataset of breeding and nonbreeding ranges. Overlap between climatic niches was quantified using ordination methods. We compared niche overlap of migratory species to two null expectations, (a) a scenario in which they do not migrate and (b) in comparison with the overlap experienced by closely related resident species, while controlling for breeding location and range size. Partly in accordance with the hypothesis of niche tracking, we found that the overlap of breeding versus nonbreeding climatic conditions in migratory species was greater than the overlap they would experience if they did not migrate. However, this was only true for migrants breeding outside the tropics and only relative to the overlap species would experience if they stayed in the breeding range year-round. In contrast to the hypothesis of niche tracking, migratory species experienced lower seasonal climatic niche overlap than resident species, with significant differences between tropical and nontropical species. Our study suggests that in seasonal nontropical environments migration away from the breeding range may serve to avoid seasonally harsh climate; however, different factors may drive seasonal movements in the climatically more stable tropical regions.
Trypanosoma cruzi, the causative agent of Chagas disease, colonizes the gut of triatomine insects, including Rhodnius prolixus. It is believed that this colonization upsets the microbiota that are normally present, presumably switching the environment to one more favorable for parasite survival. It was previously thought that one particular bacterium, Rhodococcus rhodnii, was essential for insect survival due to its ability to produce vital B-complex vitamins. However, these bacteria are not always identified in great abundance in studies on R. prolixus microbiota. Here we sequenced the microbiota of the insect anterior midgut using shotgun metagenomic sequencing in order to obtain a high-resolution snapshot of the microbes inside at two different time points and under two conditions; in the presence or absence of parasite and immediately following infection, or three days post-infection. We identify a total of 217 metagenomic bins, and recovered one metagenome-assembled genome, which we placed in the genus Dickeya. We show that, despite Rhodococcus being present, it is not the only microbe capable of synthesizing B-complex vitamins, with the genes required for biosynthesis present in a number of different microbes. This work helps to gain a new insight into the microbial ecology of R. prolixus.
Bioaerosols are considered to play a relevant role in atmospheric processes, but their sources, properties and spatiotemporal distribution in the atmosphere are not yet well characterized. In the Amazon Basin, primary biological aerosol particles (PBAP) account for a large fraction of coarse particulate matter, and fungal spores are among the most abundant PBAP there as well as in other vegetated continental regions. furthermore, PBAP could also be important ice nuclei in Amazonia. Measurement data on the release of fungal spores under natural conditions, however, are sparse. Here we present an experimental approach to analyze and quantify the spore release from fungi and other spore producing organisms under natural and laboratory conditions. For measurements under natural conditions, the samples were kept in their natural environment and a setup was developed to estimate the spore release numbers and sizes together with the microclimatic factors temperature and air humidity, as well as the mesoclimatic parameters net radiation, rain, and fog occurrence. For experiments in the laboratory, we developed a cuvette to assess the particle size and number of newly released fungal spores under controlled conditions, simultaneously measuring temperature and relative humidity inside the cuvette. Both approaches were combined with bioaerosol sampling techniques to characterize the released particles by microscopic methods. For fruiting bodies of the basidiomycetous species, Rigidoporus microporus, the model species for which these techniques were tested, the highest frequency of spore release occurred in the range of 62 and 96 % relative humidity. The results obtained for this model species reveal characteristic spore release patterns linked to environmental or experimental conditions, indicating that the moisture status of the sample may be a regulating factor, while temperature and light seem to play a minor role for this species. The presented approach enables systematic studies aimed at the quantification and validation of spore emission rates and inventories, which can be applied to a regional mapping of cryptogamic organisms under given environmental conditions.
Bioaerosols are considered to play a relevant role in atmospheric processes, but their sources, properties, and spatiotemporal distribution in the atmosphere are not yet well characterized. In the Amazon Basin, primary biological aerosol particles (PBAPs) account for a large fraction of coarse particulate matter, and fungal spores are among the most abundant PBAPs in this area as well as in other vegetated continental regions. Furthermore, PBAPs could also be important ice nuclei in Amazonia. Measurement data on the release of fungal spores under natural conditions, however, are sparse. Here we present an experimental approach to analyze and quantify the spore release from fungi and other spore-producing organisms under natural and laboratory conditions. For measurements under natural conditions, the samples were kept in their natural environment and a setup was developed to estimate the spore release numbers and sizes as well as the microclimatic factors temperature and air humidity in parallel to the mesoclimatic parameters net radiation, rain, and fog occurrence. For experiments in the laboratory, we developed a cuvette to assess the particle size and number of newly released fungal spores under controlled conditions, simultaneously measuring temperature and relative humidity inside the cuvette. Both approaches were combined with bioaerosol sampling techniques to characterize the released particles using microscopic methods. For fruiting bodies of the basidiomycetous species, Rigidoporus microporus, the model species for which these techniques were tested, the highest frequency of spore release occurred in the range from 62 % to 96 % relative humidity. The results obtained for this model species reveal characteristic spore release patterns linked to environmental or experimental conditions, indicating that the moisture status of the sample may be a regulating factor, whereas temperature and light seem to play a minor role for this species. The presented approach enables systematic studies aimed at the quantification and validation of spore emission rates and inventories, which can be applied to a regional mapping of cryptogamic organisms under given environmental conditions.
Photorhabdus and Xenorhabdus bacteria live in a highly specific symbiosis with nematodes that belong to the genus of Heterorhabditis and Steinernema, respectively. These cruiser type nematodes actively search for soil-dwelling insects and infect them via natural openings. Inside of the insect, the bacteria are released into the hemocoel where they start producing an array of secondary metabolites to bypass the insect immune system and kill the prey within 48 hours. Many of those natural products possess bioactivities against other bacteria, fungi, protozoa or insects, which makes them interesting candidates for pharmaceutical applications. Even though advanced molecular biological methods in combination with bioinformatics tools can now be used to predict biosynthetic gene clusters (BGCs) and their products, there are still many BGCs with unknown products. Even for the plethora of natural products that were successfully identified in the last couple of years, the exact ecological function often remains elusive, as laboratory conditions can vary considerably from the natural environment of the bacteria. Knowledge about the natural conditions that stimulate, or repress production of certain natural products and their underlying regulatory mechanisms yield new approaches for natural product research and enables possibilities for selective manipulations of the regulatory cascades.
The overarching goal of this work was to examine the regulatory networks in Photorhabdus and Xenorhabdus strains. The first part of this work focused on the Hfq-dependent regulation of specialized metabolite production. In those genera, the RNA chaperone, Hfq, represses expression of hexA, which encodes for a global transcriptional regulator that acts as the master repressor for SM production. Multiple global approaches were used to identify the sRNA ArcZ, which targets a specific region in the 5’-untranslated region of the hexA mRNA and ultimately guides Hfq in order to repress its expression. It was shown that a deletion of arcZ led to a drastic reduction of SM production in Photorhabdus and Xenorhabdus, consistent with the phenotype of their respective hfq deletion mutants. Transcriptomic profiling revealed far-reaching effects on the transcriptome, with up to 735 coding sequences significantly affected in the arcZ deletion strain. Finally, it was shown that the resulting chemical background, devoid of SMs, in combination with targeted promotor exchange can be used to exclusively overproduce a desired natural product, representing an alternative route of genetic manipulation.
The second part of this work focused on the influence and identification of insect related compounds that affect SM production in P. laumondii, X. szentirmaii and X. nematophila. Insect homogenate was generated from G. mellonella larvae, a model host for these bacteria. Supplementation of the cultivation medium with homogenate induced considerable shifts in the SM profiles of those bacteria. A global effect on the transcriptional output was determined by transcriptomic profiling. The core response to the simulation of an insect environment consisted of ten CDS, eight of which are involved in the degradation of fatty acids or the import of maltose and maltodextrin into the cells. Two abundant components in the insect homogenate, trehalose and putrescin, were added to the cultivation medium of those strains and subsequent HPLC-MS analysis revealed a direct correlation of their concentration in the medium and the production titres of certain SMs. These results indicated that the bacteria sense the insect environment via different insect specific components in order to initiate a metabolic adjustment, which is probably required for adaptation to the insect host.
The last part of this work examined the influence of other, so far not directly related genes on SM production, based on the isolation of P. laumondii transposon-insertion mutants with clear phenotypic alterations. Re-sequencing and SM profiling of the mutant strains revealed that a transposon-insertion in the gene encoding for a putative DNA-adenine methyltransferase affected SM production. The phenotype was confirmed by deleting this gene. Based on Single-Molecule Real-Time sequencing, the complete methylome of the WT, deletion- and complementation mutant were analysed (experimental work performed by Sacha J. Pidot, Melbourne, Australia). No obvious alterations were detected in the methylation patterns of the strains, indicating that the dam gene product does not methylate the adenine in GATC-motifs, as it was described in literature for E. coli. This data raises the question what the function of the putative DNA-adenine methyltransferase is in P. laumondii and how it can influence the secondary metabolism. Even though there is currently no clear evidence, the potential role of epigenetic gene regulation mechanisms should be considered in further work.
Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete Saccharomyces cerevisiae and the basidiomycete Ustilago maydis. One extract that inhibited the growth of S. cerevisiae much stronger than that of U. maydis was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against S. cerevisiae but reduced activity against U. maydis were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether U. maydis could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in U. maydis.
Characterization of a dual BET/HDAC inhibitor for treatment of pancreatic ductal adenocarcinoma
(2020)
Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo‐ and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)‐JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)‐JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)‐JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1‐directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin‐targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.
The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD‐glucose and glucose‐6‐phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose‐6‐phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose‐6‐phosphate phosphatase activity accumulated trehalose‐6‐phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose‐6‐phosphate. Our results demonstrate that trehalose‐6‐phosphate affects multiple physiological activities in A. baumannii ATCC 19606.
Nonribosomal peptides produced by minimal and engineered synthetases with terminal reductase domains
(2020)
Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2‐electron reduction of the enzyme‐bound thioester releasing the generated peptides as C‐terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde‐generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.
Climate change forces many species to move their ranges to higher latitudes or elevations. Resulting immigration or emigration of species might lead to functional changes, e.g., in the trait distribution and composition of ecological assemblages. Here, we combined approaches from biogeography (species distribution models; SDMs) and community ecology (functional diversity) to investigate potential effects of climate-driven range changes on frugivorous bird assemblages along a 3000 m elevational gradient in the tropical Andes. We used SDMs to model current and projected future occurrence probabilities of frugivorous bird species from the lowlands to the tree line. SDM-derived probabilities of occurrence were combined with traits relevant for seed dispersal of fleshy-fruited plants to calculate functional dispersion (FDis; a measure of functional diversity) for current and future bird assemblages. Comparisons of FDis between current and projected future assemblages showed consistent results across four dispersal scenarios, five climate models and two representative concentration pathways. Projections indicated a decrease of FDis in the lowlands, an increase of FDis at lower mid-elevations and little changes at high elevations. This suggests that functional dispersion responds differently to global warming at different elevational levels, likely modifying avian seed dispersal functions and plant regeneration in forest ecosystems along tropical mountains.
The ongoing biodiversity crisis becomes evident in the widely observed decline in abundance and diversity of species, profound changes in community structure, and shifts in species’ phenology. Insects are among the most affected groups, with documented decreases in abundance up to 76% in the last 25–30 years in some terrestrial ecosystems. Identifying the underlying drivers is a major obstacle as most ecosystems are affected by multiple stressors simultaneously and in situ measurements of environmental variables are often missing. In our study, we investigated a headwater stream belonging to the most common stream type in Germany located in a nature reserve with no major anthropogenic impacts except climate change. We used the most comprehensive quantitative long‐term data set on aquatic insects available, which includes weekly measurements of species‐level insect abundance, daily water temperature and stream discharge as well as measurements of additional physicochemical variables for a 42‐year period (1969–2010). Overall, water temperature increased by 1.88 °C and discharge patterns changed significantly. These changes were accompanied by an 81.6% decline in insect abundance, but an increase in richness (+8.5%), Shannon diversity (+22.7%), evenness (+22.4%), and interannual turnover (+34%). Moreover, the community's trophic structure and phenology changed: the duration of emergence increased by 15.2 days, whereas the peak of emergence moved 13.4 days earlier. Additionally, we observed short‐term fluctuations (<5 years) in almost all metrics as well as complex and nonlinear responses of the community toward climate change that would have been missed by simply using snapshot data or shorter time series. Our results indicate that climate change has already altered biotic communities severely even in protected areas, where no other interacting stressors (pollution, habitat fragmentation, etc.) are present. This is a striking example of the scientific value of comprehensive long‐term data in capturing the complex responses of communities toward climate change.
DnaK3, a highly conserved cyanobacterial chaperone of the Hsp70 family, binds to cyanobacterial thylakoid membranes, and an involvement of DnaK3 in the biogenesis of thylakoid membranes has been suggested. As shown here, light triggers synthesis of DnaK3 in the cyanobacterium Synechocystis sp. PCC 6803, which links DnaK3 to the biogenesis of thylakoid membranes and to photosynthetic processes. In a DnaK3 depleted strain, the photosystem content is reduced and the photosystem II activity is impaired, whereas photosystem I is regular active. An impact of DnaK3 on the activity of other thylakoid membrane complexes involved in electron transfer is indicated. In conclusion, DnaK3 is a versatile chaperone required for biogenesis and/or maintenance of thylakoid membrane-localized protein complexes involved in electron transfer reactions. As mentioned above, Hsp70 proteins are involved in photoprotection and repair of PS II in chloroplasts.
Peronospora salviae‐officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae‐officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.
Active species reintroduction is an important conservation tool when aiming for the restoration of biological communities and ecosystems. The effective monitoring of reintroduction success is a crucial factor in this process. Here, we used a combination of environmental DNA (eDNA) techniques and species distribution models (SDMs) to evaluate the success of recent reintroductions of the freshwater fish Alburnoides bipunctatus in central Germany. We built SDMs without and with eDNA presence data to locate further suitable reintroduction sites and potentially overlooked populations of the species. We successfully detected eDNA of A. bipunctatus at all reintroduction sites, as well as several adjacent sites mostly in downstream direction, which supports the success of reintroduction efforts. eDNA‐based species detection considerably improved SDMs for A. bipunctatus, which allowed to identify species presence in previously unknown localities. Our results confirm the usefulness of eDNA techniques as standard tool to monitor reintroduced fish populations. We propose that combining eDNA with SDMs is a highly effective approach for long‐term monitoring of reintroduction success in aquatic species.