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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.