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Institute
- Biowissenschaften (159) (remove)
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. ...
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.
Highlights
• PUR, PVC and PLA microplastics affect life-history parameters of Daphnia magna.
• Natural kaolin particles are less toxic than microplastics.
• Microplastic toxicity is material-specific, e.g. PVC is most toxic on reproduction.
• In case of PVC, plastic chemicals are the main driver of microplastic toxicity.
• PLA bioplastics are similarly toxic as conventional plastics.
Abstract
Given the ubiquitous presence of microplastics in aquatic environments, an evaluation of their toxicity is essential. Microplastics are a heterogeneous set of materials that differ not only in particle properties, like size and shape, but also in chemical composition, including polymers, additives and side products. Thus far, it remains unknown whether the plastic chemicals or the particle itself are the driving factor for microplastic toxicity. To address this question, we exposed Daphnia magna for 21 days to irregular polyvinyl chloride (PVC), polyurethane (PUR) and polylactic acid (PLA) microplastics as well as to natural kaolin particles in high concentrations (10, 50, 100, 500 mg/L, ≤ 59 μm) and different exposure scenarios, including microplastics and microplastics without extractable chemicals as well as the extracted and migrating chemicals alone. All three microplastic types negatively affected the life-history of D. magna. However, this toxicity depended on the endpoint and the material. While PVC had the largest effect on reproduction, PLA reduced survival most effectively. The latter indicates that bio-based and biodegradable plastics can be as toxic as their conventional counterparts. The natural particle kaolin was less toxic than microplastics when comparing numerical concentrations. Importantly, the contribution of plastic chemicals to the toxicity was also plastic type-specific. While we can attribute effects of PVC to the chemicals used in the material, effects of PUR and PLA plastics were induced by the mere particle. Our study demonstrates that plastic chemicals can drive microplastic toxicity. This highlights the importance of considering the individual chemical composition of plastics when assessing their environmental risks. Our results suggest that less studied polymer types, like PVC and PUR, as well as bioplastics are of particular toxicological relevance and should get a higher priority in ecotoxicological studies.
Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of intercellular communication, signaling via nucleotides. The signaling cascades involved in purinergic signaling include intracellular storage of nucleotides, nucleotide release, extracellular hydrolysis, and the effect of the released compounds or their hydrolysis products on target tissues via specific receptor systems. In this context ectonucleotidases play several roles. They inactivate released and physiologically active nucleotides, produce physiologically active hydrolysis products, and facilitate nucleoside recycling. This review briefly highlights the development of our knowledge of two types of enzymes involved in extracellular nucleotide hydrolysis and thus purinergic signaling, the ectonucleoside triphosphate diphosphohydrolases, and ecto-5′-nucleotidase.
The application of natural products (NPs) as drugs and lead compounds has greatly improved human health over the past few decades. Despite their success, we still need to find new NPs that can be used as drugs to combat increasing drug resistance via new modes of action and to develop safer treatments with less side effects.
Entomopathogenic bacteria of Xenorhabdus and Photorhabdus that live in mutualistic symbiosis with nematodes are considered as promising producers of NPs, since more than 6.5% of their genomes are assigned to biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites. The investigation on NPs from Xenorhabdus and Photorhabdus can not only provide new compounds for drug discovery but also help to understand the biochemical basis involved in mutualistic and pathogenic symbiosis of bacteria, nematode host and insect prey.
Nonribosomal peptides (NRPs) are a large class of NPs that are mainly found in bacteria and fungi. They are biosynthesized by nonribosomal peptide synthetases (NRPSs) and display diverse functions, representing more than 20 clinically used drugs. Although a large number of NRPs have been identified in Xenorhabdus and Photorhabdus, the advanced genome sequencing and bioinformatic analysis indicate that these bacteria still have many unknown NRPS-encoding gene clusters for NRP production that are worth to explore. Therefore, this thesis focuses on the discovery, biosynthesis, structure identification, and biological functions of new NRPs from Xenorhabdus and Photorhabdus.
The first publication describes the isolation and structure elucidation of seven new rhabdopeptide/xenortide-like peptides (RXPs) from X. innexi, incorporating putrescine or ammonia as the C-terminal amines. Bioactivity testing of these RXPs revealed potent antiprotozoal activity against the causative agents of sleeping sickness (Trypanosoma brucei rhodesiense) and malaria (Plasmodium falciparum), making them the most active RXP derivatives known to date. Biosynthetically, the initial NRPS module InxA might act iteratively with a flexible methyltransferase activity to catalyze the incorporation of the first five or six N-methylvaline/valine to these peptides.
The second publication focuses on the structure elucidation of seven unusual methionine-containing RXPs that were found as minor products in E. coli carrying the BGC kj12ABC from Xenorhabdus KJ12.1. To confirm the proposed structures from detailed HPLC-MS analysis, a solid-phase peptide synthesis (SPPS) method was developed for the synthesis of these partially methylated RXPs. These RXPs also exhibited good effects against T. brucei rhodesiense and P. falciparum, suggesting RXPs might play a role in protecting insect cadaver from soil-living protozoa to support the symbiosis with nematodes.
The third publication presents the identification of a new peptide library, named photohexapeptide library, which occurred after the biosynthetic gene phpS was activated in P. asymbiotica PB68.1 via promoter exchange. The chemical diversity of the photohexapeptides results from unusual promiscuous specificity of five out of six adenylation (A) domains being an excellent example of how to create compound libraries in nature. Furthermore, photohexapeptides enrich the family of the rare linear D-/L-peptide NPs.
The fourth publication concentrates on the structure elucidation of a new cyclohexapeptide, termed photoditritide, which was produced by P. temperata Meg1 after the biosynthetic gene pdtS was activated via promoter exchange. Photoditritide so far is the only example of a peptide from entomopathogenic bacteria that contains the uncommon amino acid homoarginine. The potent antimicrobial activity of photoditritide against Micrococcus luteus implies that photoditritide can protect the insect cadaver from food competitor bacteria in the complex life cycle of nematode and bacteria.
The last publication reports a new family of cyclic lipopeptides (CLPs), named phototemtides, which were obtained after the BGC pttABC from P. temperata Meg1 was heterologously expressed in E. coli. The gene pttA encodes an MbtH protein that was required for the biosynthesis of phototemtides in E. coli. To determine the absolute configurations of the hydroxy fatty acids, a total synthesis of the major compound phototemtide A was performed. Although the antimalarial activity of phototemtide A is only weak, it might be a starting point towards a selective P. falciparum compound, as it shows no activity against any other tested organisms.
Characterization of a dual BET/HDAC inhibitor for treatment of pancreatic ductal adenocarcinoma
(2020)
Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo‐ and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)‐JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)‐JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)‐JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1‐directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin‐targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.
The opportunistic human pathogen Acinetobacter baumannii is one of the leading causes of nosocomial infections. The high prevalence of multidrug‐resistant strains, a high adaptability to changing environments and an overall pronounced stress resistance contribute to persistence and spread of the bacteria in hospitals and thereby promote repeated outbreaks. Altogether, the success of A. baumannii is mainly built on adaptation and stress resistance mechanisms, rather than relying on ‘true’ virulence factors. One of the stress factors that pathogens must cope with is osmolarity, which can differ between the external environment and different body parts of the human host. A. baumannii ATCC 19606T accumulates the compatible solutes glutamate, mannitol and trehalose in response to high salinities. In this work, it was found that most of the solutes vanish immediately after reaching stationary phase, a very unusual phenomenon. While glutamate can be metabolized, mannitol produced by MtlD is excreted to the medium in high amounts. First results indicate that A. baumannii ATCC 19606T undergoes a rapid switch to a dormant state (viable but non‐culturable) after disappearance of the compatible solutes. Resuscitation from this state could easily be achieved in PBS or fresh medium.
Signal transduction and the regulation of gene expression are fundamental processes in every cell. RNA-binding proteins (RBPs) play a key role in the post-transcriptional modulation of gene expression in response to both internal and external stimuli. However, how signaling pathways regulate the assembly of RBPs with mRNAs remains largely unknown. Here, we summarize observations showing that the formation and composition of messenger ribonucleoprotein particles (mRNPs) is dynamically remodeled in space and time by specific signaling cascades and the resulting post-translational modifications. The integration of signaling events with gene expression is key to the rapid adaptation of cells to environmental changes and stress. Only a combined approach analyzing the signal transduction pathways and the changes in post-transcriptional gene expression they cause will unravel the mechanisms coordinating these important cellular processes.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
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.
One current goal in native mass spectrometry is the assignment of binding affinities to noncovalent complexes. Here we introduce a novel implementation of the existing laser-induced liquid bead ion desorption (LILBID) mass spectrometry method: this new method, LILBID laser dissociation curves, assesses binding strengths quantitatively. In all LILBID applications, aqueous sample droplets are irradiated by 3 µm laser pulses. Variation of the laser energy transferred to the droplet during desorption affects the degree of complex dissociation. In LILBID laser dissociation curves, laser energy transfer is purposely varied, and a binding affinity is calculated from the resulting complex dissociation. A series of dsDNAs with different binding affinities was assessed using LILBID laser dissociation curves. The binding affinity results from the LILBID laser dissociation curves strongly correlated with the melting temperatures from UV melting curves and with dissociation constants from isothermal titration calorimetry, standard solution phase methods. LILBID laser dissociation curve data also showed good reproducibility and successfully predicted the melting temperatures and dissociation constants of three DNA sequences. LILBID laser dissociation curves are a promising native mass spectrometry binding affinity method, with reduced time and sample consumption compared to melting curves or titrations.
The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.
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. ...
Background: Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood–Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study.
Results: We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+–NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis.
Discussion: We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme.
Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H2 + CO2 or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO2 reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy."
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).
The ability to vocalize is ubiquitous in vertebrates, but neural networks underlying vocal control remain poorly understood. Here, we performed simultaneous neuronal recordings in the frontal cortex and dorsal striatum (caudate nucleus, CN) during the production of echolocation pulses and communication calls in bats. This approach allowed us to assess the general aspects underlying vocal production in mammals and the unique evolutionary adaptations of bat echolocation. Our data indicate that before vocalization, a distinctive change in high-gamma and beta oscillations (50–80 Hz and 12–30 Hz, respectively) takes place in the bat frontal cortex and dorsal striatum. Such precise fine-tuning of neural oscillations could allow animals to selectively activate motor programs required for the production of either echolocation or communication vocalizations. Moreover, the functional coupling between frontal and striatal areas, occurring in the theta oscillatory band (4–8 Hz), differs markedly at the millisecond level, depending on whether the animals are in a navigational mode (that is, emitting echolocation pulses) or in a social communication mode (emitting communication calls). Overall, this study indicates that fronto-striatal oscillations could provide a neural correlate for vocal control in bats.
The interaction of microplastics with freshwater biota and their interaction with other stressors is still not very well understood. Therefore, we investigated the ingestion, excretion and toxicity of microplastics in the freshwater gastropod Lymnaea stagnalis.
MP ingestion was analyzed as tissues levels in L. stagnalis after 6–96 h of exposure to 5–90 μm spherical polystyrene (PS) microplastics. To understand the excretion, tissue levels were determined after 24 h of exposure followed by a 12 h–7 d depuration period. To assess the toxicity, snails were exposed for 28 d to irregular PS microplastics (<63 μm, 6.4–100,000 particles mL−1), both alone and in combination with copper as additional stressor. To compare the toxicity of natural and synthetic particles, we also included diatomite particles. Microplastics ingestion and excretion significantly depended on the particle size and the exposure/depuration duration. An exposure to irregular PS had no effect on survival, reproduction, energy reserves and oxidative stress. However, we observed slight effects on immune cell phagocytosis. Exposure to microplastics did not exacerbate the reproductive toxicity of copper. In addition, there was no pronounced difference between the effects of microplastics and diatomite. The tolerance towards microplastics may originate from an adaptation of L. stagnalis to particle-rich environments or a general stress resilience. In conclusion, despite high uptake rates, PS fragments do not appear to be a relevant stressor for stress tolerant freshwater gastropods considering current environmental levels of microplastics.
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.
The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
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.
What is in Umbilicaria pustulata? A metagenomic approach to reconstruct the holo-genome of a lichen
(2020)
Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis—the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)—were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ∼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.
Global landscapes are changing due to human activities with consequences for both biodiversity and ecosystems. For single species, terrestrial mammal population densities have shown mixed responses to human pressure, with both increasing and decreasing densities reported in the literature. How the impacts of human activities on mammal populations translates into altered global density patterns remains unclear. Here we aim to disentangle the effect of human impacts on large-scale patterns of mammal population densities using a global dataset of 6729 population density estimates for 468 mammal species (representing 59% and 44% of mammalian orders and families). We fitted a mixed effect model to explain the variation in density based on a 1-degree resolution as a function of the human footprint index (HFI), a global proxy of direct and indirect human disturbances, while accounting for body mass, trophic level and primary productivity (normalized vegetation index; NDVI). We found a significant positive relationship between population density and HFI, where population densities were higher in areas with a higher HFI (e.g. agricultural or suburban areas – no populations were located in very high HFI urban areas) compared to areas with a low HFI (e.g. wilderness areas). We also tested the effect of the individual components of the HFI and still found a consistent positive effect. The relationships remained positive even across populations of the same species, although variability among species was high. Our results indicate shifts in mammal population densities in human modified landscapes, which is due to the combined effect of species filtering, increased resources and a possible reduction in competition and predation. Our study provides further evidence that macroecological patterns are being altered by human activities, where some species will benefit from these activities, while others will be negatively impacted or even extirpated.
Déterminants de l’utilisation de Acacia auriculiformis comme bois d’œuvre en Afrique de l’Ouest
(2020)
Acacia auriculiformis, un bois énergie, suscite de plus en plus des intérêts de bois d’œuvre au niveau des industriels de bois au Bénin. L’appréciation des performances de l’espèce dans les usines et en plantation est déterminante pour la vul- garisation de l’espèce comme alternative pour mitiger la déforestation en lien avec la demande en bois d’œuvre. L’objectif principal de ce travail est donc d’évaluer les conditions entourant l’adoption de Acacia auriculiformis comme espèce de bois d’œuvre au Bénin, Afrique de l’Ouest. Au total, 154 usines de bois et 25 plantations ont été enquêtées dans les zones abritant les plantations à A. auriculiformis. A. auriculiformis est l’espèce la plus fréquente dans les usines de bois (81%) suivie de Afzelia africana (55%), Tectona grandis (47%) et Khaya senegalensis (47%). Les superficies des plantations à A. auriculi- formis ont augmenté entre 1999 et 2019. Les connaissances sur l’utilisation de ce bois sont variables dans la zone d’étude. Le bois de A. auriculiformis est apprécié comme bois d’œuvre parce qu’il présente une couleur esthétique, un séchage rapide, une facilité de mise en œuvre, une imprégnabilité élevée, une densité moyenne à élevée et un bel aspect après mise en œuvre. Cependant, son bois fournit beaucoup de sciure, a beaucoup de nœuds et présente une déformabilité moyenne. Sa disponibili- té et son accessibilité sont les principaux facteurs justifiant la préférence de l’espèce par les industriels de bois d’œuvre. Cette forme d’utilisation de l’espèce est également remarquée au Togo, en Côte d’Ivoire. L’espèce présente une bonne perspective d’utilisation comme bois d’œuvre.
Trypanosoma cruzi, the causative agent of Chagas disease, colonizes the gut of triatomine insects, including Rhodnius prolixus. It is believed that this colonization upsets the microbiota that are normally present, presumably switching the environment to one more favorable for parasite survival. It was previously thought that one particular bacterium, Rhodococcus rhodnii, was essential for insect survival due to its ability to produce vital B-complex vitamins. However, these bacteria are not always identified in great abundance in studies on R. prolixus microbiota. Here we sequenced the microbiota of the insect anterior midgut using shotgun metagenomic sequencing in order to obtain a high-resolution snapshot of the microbes inside at two different time points and under two conditions; in the presence or absence of parasite and immediately following infection, or three days post-infection. We identify a total of 217 metagenomic bins, and recovered one metagenome-assembled genome, which we placed in the genus Dickeya. We show that, despite Rhodococcus being present, it is not the only microbe capable of synthesizing B-complex vitamins, with the genes required for biosynthesis present in a number of different microbes. This work helps to gain a new insight into the microbial ecology of R. prolixus.
Nonribosomal peptides produced by minimal and engineered synthetases with terminal reductase domains
(2020)
Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2‐electron reduction of the enzyme‐bound thioester releasing the generated peptides as C‐terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde‐generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.
The growing number of infections with multi-resistant bacteria or the current COVID-19 pandemic put compounds with therapeutic properties into the public focus. Non-ribosomal peptides (NRPs) are natural products that are already marketed as antibiotics, cytotoxic agents or immunosuppressants. Their biological activities rely on the structural diversity including non-proteinogenic amino acids (AAs), heterocycles or modifications like methylation or acylation.
The biosynthesis of NRPs is carried out by non-ribosomal peptide synthetases (NRPSs). These multifunctional megaenzymes show a modular architecture like in an assembly-line. Each module is thereby responsible for the incorporation and modification of one AA and therefore contains different catalytic domains. The adenylation (A) domain recognizes and activates its specific substrate in an ATP-dependent manner which is transferred to a 4’-phosphopantetheine cofactor post-translationally attached to the thiolation (T) domain. Peptide bond formation between two T domain bound substrates catalysed by the condensation (C) domain transfers the growing peptide chain to the following module. Such a C-A-T module can be extended with optional domains to integrate structural diversity and a terminal thioesterase (TE) domain usually releases the peptide via hydrolysis or intramolecular attack of nucleophiles. Inspired by the modular architecture, NRPS engineering deals with the modification of NRPs in order to increase biological activities, circumvent bacterial resistances or create de novo peptides. This can be achieved by mutasynthesis or modification of the substrate binding pocket as well as single and multiple domain substitution. However, the few successful approaches led to impaired enzymes and did not establish a general applicable guideline. In the first publication as part of this work, the development of such a guideline comprising three rules is addressed. First, the A-T-C tridomain named exchange unit (XU) is seen as a catalytic unit instead of a module. When using them as building blocks, the C domain’s specificity for the AA of the following XU has to be considered as second rule. Third, a conserved WNATE motif within the C-A linker depicts the fusion point of the XUs. Upon heterologous expression of the cloned plasmids in E. coli and high performance liquid chromatography coupled mass spectrometry-based analysis of the extracts, the ambactin-producing NRPS from Xenorhabdus was reprogrammed with one and two XUs. This only leads to a moderate loss of production titre or an even higher one when the AA configuration was changed by introducing a dual condensation/epimerization (C/E) domain. The pentamodular GameXPeptide-producing NRPS was reconstructed using up to five XUs of four different NRPSs and even completely de novo synthetases were created. The second publication describes the exchange unit condensation domain (XUC) concept and relies on a fusion point between the two subdomains (N-terminal CDsub and C-terminal CAsub) of the C domain’s V-shaped pseudodimeric structure which generates A-T didomains with flanking CAsub and CDsub. These hybrid C domain-forming building blocks depict an improvement to the XU concept by avoiding the drawback of C domain specificity. This allows a more flexible NRPS engineering that can e.g. enable peptide library design. Furthermore, beside a combination of both concepts within one NRPS and a transfer to Bacillus NRPSs, the use of XUC with relaxed A domain specificity allowed further peptide modifications by introducing non-natural AAs. The third publication deals with aldehyde and alcohol-generating reductase (R) domains which depict an alternative for peptide release in NRPSs. A promoter exchange in X. indica identified a pyrazine-producing NRPS with a minimal architecture of an A, T and R domain and was therefore termed ATRed. R domains were additionally used in engineered NRPSs to produce pyrazinones and derivatives thereof by XU substitution although most constructs failed to show production. Beyond that, an R domain has been shown to replace a TE domain in wild type synthetases leading to slightly modified NRPs and the postulated biosynthesis was incidentally revised. Furthermore, an NRPS with terminal R domain was engineered to produce a free peptide aldehyde, which are known to be potent proteasome inhibitors. For the above mentioned ATReds, the presence of up to three coding regions was further identified in 20 different Xenorhabdus strains but only six of them were verified to produce pyrazines. All ATReds share variable sequence similarities among each other and were subsequently divided into three subtypes. One subtype is supposed to perform the pyrazine biosynthesis via a non-canonical catalytic triad.
DnaK3, a highly conserved cyanobacterial chaperone of the Hsp70 family, binds to cyanobacterial thylakoid membranes, and an involvement of DnaK3 in the biogenesis of thylakoid membranes has been suggested. As shown here, light triggers synthesis of DnaK3 in the cyanobacterium Synechocystis sp. PCC 6803, which links DnaK3 to the biogenesis of thylakoid membranes and to photosynthetic processes. In a DnaK3 depleted strain, the photosystem content is reduced and the photosystem II activity is impaired, whereas photosystem I is regular active. An impact of DnaK3 on the activity of other thylakoid membrane complexes involved in electron transfer is indicated. In conclusion, DnaK3 is a versatile chaperone required for biogenesis and/or maintenance of thylakoid membrane-localized protein complexes involved in electron transfer reactions. As mentioned above, Hsp70 proteins are involved in photoprotection and repair of PS II in chloroplasts.
Peronospora belbahrii is one of the most destructive downy mildew diseases that has emerged throughout the past two decades. Due to the lack of quarantine regulations and its possible seed-borne nature, it has spread globally and is now present in most areas in which basil is produced. While most obligate biotrophic, plant parasitic oomycetes are highly host-specific, there are a few that have a wider host range, e.g. Albugo candida, Bremia tulasnei, and Pseudoperonospora cubensis. Recently, it was shown that Peronospora belbahrii is able to infect Rosmarinus, Nepetia, and Micromeria in Israel in cross-infection trials, hinting an extended host range for also this pathogen. In this study, a newly occurring downy mildew pathogen on lavender was investigated with respect to its morphology and phylogeny, and it is shown that it belongs to Peronospora belbahrii as well. Thus, it seems that Peronospora belbahrii is currently extending its host range to additional members of the tribe Mentheae and Ocimeae. Therefore, it seems advisable to scrutinise all commonly used members of these tribes in order to avoid further spread of virulent genotypes.
Peronospora aquilegiicola is a destructive pathogen of columbines and has wiped out most Aquilegia cultivars in several private and public gardens throughout Britain. The pathogen, which is native to East Asia was noticed in England and Wales in 2013 and quickly spread through the country, probably by infested plants or seeds. To our knowledge, the pathogen has so far not been reported from other parts of Europe. Here, we report the emergence of the pathogen in the northwest of Germany, based on morphological and phylogenetic evidence. As the pathogen was found in a garden in which no new columbines had been planted recently, we assume that the pathogen has already spread from its original point of introduction in Germany. This calls for an increased attention to the further spread of the pathogen and the eradication of infection spots to avoid the spread to naturally occurring columbines in Germany and to prevent another downy mildew from becoming a global threat, like Peronospora belbahrii and Plasmopara destructor, the downy mildews of basil and balsamines, respectively.
This study was performed to identify Peronosclerospora species found in Indonesia based on sequence analysis of the cox2 gene. In addition, sequence data in total, 26 isolates of Peronosclerospora were investigated in this study. They were obtained from 7 provinces in Indonesia, namely Lampung, Jawa Timur, Jawa Barat, Sumatera Utara, Jawa Tengah, Yogyakarta, and Sulawesi Selatan. Sequence analysis of cox2 and phylogenetic inference were performed on all the 26 isolates. A set of primers developed in this study, PCOX2F and PCOX2R, was used for PCR amplification. Phylogenetic analyses showed that all the Indonesian isolates were divided into two groups. Group I contained 13 isolates; 9 isolates obtained from Lampung, 3 isolates from Sumatera Utara, and 1 isolate from Jawa Barat. Group II consisted of 13 isolates; 7 isolates from Jawa Timur, 2 isolates from Jawa Tengah, 1 isolate from Yogyakarta, and 3 isolates from Sulawesi Selatan. All the members of group I clustered with the ex-type sequence of P. australiensis. Meanwhile, all members of Group II formed the sister clade of isolates obtained from Timor-Leste and may represent P. maydis.
Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification
Risk evaluations for agricultural chemicals are necessary to preserve healthy populations of honey bee colonies. Field studies on whole colonies are limited in behavioural research, while results from lab studies allow only restricted conclusions on whole colony impacts. Methods for automated long-term investigations of behaviours within comb cells, such as brood care, were hitherto missing. In the present study, we demonstrate an innovative video method that enables within-cell analysis in honey bee (Apis mellifera) observation hives to detect chronic sublethal neonicotinoid effects of clothianidin (1 and 10 ppb) and thiacloprid (200 ppb) on worker behaviour and development. In May and June, colonies which were fed 10 ppb clothianidin and 200 ppb thiacloprid in syrup over three weeks showed reduced feeding visits and duration throughout various larval development days (LDDs). On LDD 6 (capping day) total feeding duration did not differ between treatments. Behavioural adaptation was exhibited by nurses in the treatment groups in response to retarded larval development by increasing the overall feeding timespan. Using our machine learning algorithm, we demonstrate a novel method for detecting behaviours in an intact hive that can be applied in a versatile manner to conduct impact analyses of chemicals, pests and other stressors.
Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar‐based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2‐dependent CO2 reductase and furthermore belong to different classes of ‘Rnf’‐ and ‘Ech‐acetogens’. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood–Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g−1 h−1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO‐based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.
Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding.
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.
Competition over land is at the core of many sustainable development challenges in Myanmar: villagers, companies, governments, ethnic minority groups, civil society organisations and non-governmental organisations from local to the international level claim access to and decision-making power over the use of land. Therefore, this article investigates the actor interactions influencing land-use changes and their impacts on the supply of ecosystem services and human well-being. We utilise a transdisciplinary mixed-methods approach and the analytical lens of the social-ecological systems framework. Results reveal that the links between land-use changes, ecosystem services and human well-being are multifaceted; For example ecosystem services can decline, while human well-being increases. We explain this finding through three different pathways to impact (changes in the resource systems, the governance systems or the broader social, economic and political context). We conclude with implications of these results for future sustainable land governance.
In the fish embryo toxicity (FET) test with zebrafish (Danio rerio) embryos, 3,4-dichloroaniline (3,4-DCA) is often employed as a positive control substance. Previous studies have characterized bioconcentration and transformation of 3,4-DCA in this test under flow-through conditions. However, the dynamic changes of chemical concentrations in exposure media and embryos were not studied systematically under the commonly used semi-static exposure conditions in multiwell plates. To overcome these limitations, we conducted semi-static exposures experiments where embryolarval zebrafish were exposed to 0.5, 2.0, and 4.0 mg L−1 of 3,4-DCA for up to 120 hpf, with 24-h renewal intervals. During each renewal interval, concentrations of 3,4-DCA were quantified in water samples at 0, 6, 18, and 24 h using high-performance liquid chromatography with diode array detection. Levels of 3,4-DCA in larvae were measured after 120 h exposure. Concentrations of 3,4-DCA in the test vessels decreased rapidly during exposure. Taking these dynamics into account, bioconcentration factors in the present study ranged from 12.9 to 29.8 L kg−1, depending on exposure concentration. In summary, this study contributed to our knowledge of chemical dynamics in the FET test with embryolarval zebrafish, which will aid in defining suitable exposure conditions for future studies.
Aquatic ecosystems are globally contaminated with microplastics (MP). However, comparative data on MP levels in freshwater systems is still scarce. Therefore, the aim of this study is to quantify MP abundance in water and sediment of the German river Elbe using visual, spectroscopic (Fourier-transform infrared spectroscopy) and thermo analytical (pyrolysis gas chromatography mass spectrometry) methods. Samples from eleven German sites along the German part of the Elbe were collected, both in the water and sediment phase, in order to better understand MP sinks and transport mechanisms. MP concentrations differed between the water and sediment phase. Sediment concentrations (mean: 3,350,000 particles m−3, 125–5000 μm MP) were in average 600,000-fold higher than water concentrations (mean: 5.57 particles m−3, 150–5000 μm MP). The abundance varied between the sampling sites: In sediments, the abundance decreased in the course of the river while in water samples no such clear trend was observed. This may be explained by a barrage retaining sediments and limiting tidal influence in the upstream parts of the river. Particle shape differed site-specifically with one site having exceptionally high quantities of spheres, most probably due to industrial emissions of PS-DVB resin beads. Suspended MP consisted predominantly of polyethylene and polypropylene whereas sediments contained a higher diversity of polymer types. Determined MP concentrations correspond well to previous results from other European rivers. In a global context, MP levels in the Elbe relate to the lower (water) to middle section (sediment) of the global range of MP concentrations determined for rivers worldwide. This highlights that elevated MP levels are not only found in single countries or continents, but that MP pollution is an issue of global concern.
Das Vorkommen von Kunststoffmaterialien <5 mm, sogenanntem Mikroplastik
(MP), in marinen Ökosystemen wurde bereits eingehend untersucht. Im Gegensatz dazu existieren erhebliche Wissenslücken hinsichtlich der Abundanz und der Auswirkung von MP in limnischen Ökosystemen. Vor diesem Hintergrund steht das Umweltvorkommen, mögliche Eintragspfade und die Auswirkungen von MP auf aquatische Invertebraten im Fokus dieser Arbeit. Zur Bestimmung der MP-Abundanz in Fließgewässern sind Sedimente der Elbe untersucht worden. Hierfür wurde zunächst eine Methode zur Extraktion und Identifizierung von MP aus Umweltproben entwickelt, optimiert und validiert. In der anschließenden Analyse konnten in elf Probenahmestellen 55–17400 MP kg-1 in den Sedimenten nachgewiesen werden. Der Einfluss der Gezeitenströmung wurde anhand der abnehmenden MP-Abundanz in der Tideelbe deutlich. Insgesamt weisen die Ergebnisse darauf hin, dass Sedimente von Fließgewässern eine Senke für MP darstellen. Für die Evaluation von Eintragspfaden von MP in Oberflächengewässer wurden die
Einleiter von fünf Kläranlagen beprobt und 240–897 MP m-3 in den Einleitern detektiert. Die Detailuntersuchung einer Kläranlage zeigte, dass >99% der MP-Fracht im Verlauf der Abwasseraufbereitung entfernt wird. Hierbei erfolgte die Hauptentfernung
bereits in der Vorklärung. Somit stellen Kläranlagen effektive Barrieren für den Eintrag von MP dar.
Insgesamt wird ersichtlich, dass die getesteten Arten C. riparius und G. pulex relativ insensitiv gegenüber einer MP-Exposition sind. So konnten bei G. pulex keine und bei C. riparius erst bei sehr hohen MP-Konzentrationen adverse Effekte detektiert werden. Hierbei ist die Autökologie der Spezies eine mögliche Erklärung für die Toleranz gegenüber partikulären Stressoren. Auf Basis dieser Daten sowie der ermittelten MPAbundanz kann das Umweltrisiko von MP in limnischen Ökosystemen vorläufig als
gering eingeschätzt werden. Hierbei gilt es jedoch zu beachten, dass eine abschließende
Bewertung aufgrund der nach wie vor existierenden Unsicherheiten nicht möglich ist. Diese Unsicherheiten betreffen die Umweltkonzentration von MP <80 μm, das Verhaltensowie das Wirkpotential dieser heterogenen und dynamischen Stressorenklasse
in umweltrelevanten Szenarien.
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.
Our knowledge of early evolution of snakes is improving, but all that we can infer about the evolution of modern clades of snakes such as boas (Booidea) is still based on isolated bones. Here, we resolve the phylogenetic relationships of Eoconstrictor fischeri comb. nov. and other booids from the early-middle Eocene of Messel (Germany), the best-known fossil snake assemblage yet discovered. Our combined analyses demonstrate an affinity of Eoconstrictor with Neotropical boas, thus entailing a South America-to-Europe dispersal event. Other booid species from Messel are related to different New World clades, reinforcing the cosmopolitan nature of the Messel booid fauna. Our analyses indicate that Eoconstrictor was a terrestrial, medium- to large-bodied snake that bore labial pit organs in the upper jaw, the earliest evidence that the visual system in snakes incorporated the infrared spectrum. Evaluation of the known palaeobiology of Eoconstrictor provides no evidence that pit organs played a role in the predator–prey relations of this stem boid. At the same time, the morphological diversity of Messel booids reflects the occupation of several terrestrial macrohabitats, and even in the earliest booid community the relation between pit organs and body size is similar to that seen in booids today.
Keystone mutualisms, such as corals, lichens or mycorrhizae, sustain fundamental ecosystem functions. Range dynamics of these symbioses are, however, inherently difficult to predict because host species may switch between different symbiont partners in different environments, thereby altering the range of the mutualism as a functional unit. Biogeographic models of mutualisms thus have to consider both the ecological amplitudes of various symbiont partners and the abiotic conditions that trigger symbiont replacement. To address this challenge, we here investigate 'symbiont turnover zones'--defined as demarcated regions where symbiont replacement is most likely to occur, as indicated by overlapping abundances of symbiont ecotypes. Mapping the distribution of algal symbionts from two species of lichen-forming fungi along four independent altitudinal gradients, we detected an abrupt and consistent β-diversity turnover suggesting parallel niche partitioning. Modelling contrasting environmental response functions obtained from latitudinal distributions of algal ecotypes consistently predicted a confined altitudinal turnover zone. In all gradients this symbiont turnover zone is characterized by approximately 12°C average annual temperature and approximately 5°C mean temperature of the coldest quarter, marking the transition from Mediterranean to cool temperate bioregions. Integrating the conditions of symbiont turnover into biogeographic models of mutualisms is an important step towards a comprehensive understanding of biodiversity dynamics under ongoing environmental change.
Das Gehirn weist in mehreren Bereichen anatomische Asymmetrien zwischen beiden Hemisphären auf, so auch in Bereichen der Hörrinde. Zudem ist bereits langjährig bekannt, dass menschliche Sprache vorrangig in der linken Gehirnhälfte, d.h. linksseitig lateralisiert, verarbeitet wird. Daraus folgend stellt sich die Frage, ob dies eine besondere Spezialisierung ist, oder ob es noch weitere lateralisierte Hirnfunktionen gibt. Viele akustische Signale haben dabei frequenzmodulierte (FM) Komponenten, die im Hörsystem für die Erkennung nach Parametern wie Richtung und Dauer der Modulation analysiert werden müssen. Ob die Analyse von FM-Komponenten oder einzelner Reizparameter im Gehirn lateralisiert stattfindet, wurde in der Literatur meist mit bildgebenden Verfahren untersucht.
Für das Erkennen und Unterscheiden der Modulationsrichtung weist eine Vielzahl von Studien auf eine erhöhte Aktivität in der rechten Hörrinde hin. Für die Analyse von Stimulusdauern ist es bisher allerdings noch unklar bzw. umstritten, ob diese lateralisiert erfolgt. Für die Untersuchung der Lateralisierung einfacher Sprachkomponenten werden häufig Konsonant-Vokal-Silben (CV-Silben) verwendet. In einer Vielzahl von Studien konnte eine linkslastige Lateralisierung, wie bei der Spracherkennung, gezeigt werden.
In der vorliegenden Arbeit wurde nun untersucht, ob ein eindeutigeres Muster von Lateralisierung zu finden ist, wenn diese in Wahrnehmungsexperimenten, untersucht wird. Dabei wurde ein zu untersuchender Teststimulus (FM-/CV-Stimulus) auf einem Ohr mit einem kontralateralen breitbandigen Rauschen auf dem anderen Ohr gleichzeitig präsentiert. Durch die Struktur der Hörbahn kann dabei davon ausgegangen werden, dass in einer Hemisphäre des Vorderhirns vorrangig Informationen aus dem kontralateralen Ohr verarbeitet und Informationen aus dem ipsilateralen Ohr unterdrückt werden und sich somit Rückschlüsse auf die Funktion/Beteiligung einer Hemishpäre ziehen lassen. Das Rauschen diente dabei zur unspezifischen Aktivierung der gegenüberliegenden Hemisphäre.
Die Lateralisierung wurde systematisch für unterschiedlich komplexe Reize untersucht. Dazu wurden in zwei Versuchsreihen Unterscheidungsexperimente durchgeführt, die sich in mehrere Messungen (mit mehreren Durchläufen) mit unterschiedlichen Parametereinstellungen gliederten. Pro Durchlauf musste sich die Versuchsperson immer zwischen zwei Antwortmöglichkeiten entscheiden (2-AFC-Verfahren). Der Schalldruckpegel des Rauschens war dabei für alle Messungen konstant. Der Schalldruckpegel der Teststimuli blieb zwar während einer Messung konstant, wurde jedoch innerhalb eines Experimentes von Messung zu Messung reduziert.
In einer gemeinsamen Analyse wurden jeweils die Fehlerraten und Reaktionszeiten beider Ohren, getrennt nach Seite und FM-/ CV-Stimulus, miteinander verglichen, um so auf eine mögliche Lateralisierung schließen zu können. Damit die Daten der Versuchspersonen bei vergleichbarer Schwierigkeit analysiert werden konnten, wurde als Vergleichswert zwischen allen Versuchspersonen der Schalldruckpegel der ersten Messung mit einer Fehlerrate von mindestens 15,0 % gewählt (15 %-Kriterium). Um auszuschließen, dass das Hörvermögen der Versuchspersonen Unterschiede zwischen beiden Ohren aufweist, wurde vor jeder Messung der „Punkt subjektiver Gleichheit“ für die Lautstärke-wahrnehmung zwischen linkem und rechten Ohr bestimmt.
In der ersten Versuchsreihe wurde dabei die Verarbeitung der Modulationsrichtung und der Stimulusdauer von FM-Stimuli untersucht. Es zeigte sich für beide Experimente, dass ein sinkender Schalldruckpegel des FM-Stimulus zu einer steigenden Fehlerrate führte. Unter Anwendung des 15 %-Kriteriums waren die Fehlerraten für die Unterscheidung der Modulationsrichtung signifikant geringer, wenn der FM-Stimulus auf dem linken Ohr präsentiert wurde. Dies ist ein deutlicher Hinweis für eine rechtslastige Lateralisierung.
Für die Unterscheidung der Stimulusdauer gab es dagegen keinen signifikanten Unterschied zwischen den Fehlerraten beider Ohren. Somit muss davon ausgegangen werden, dass beide Hemisphären für diese Aufgabe benötigt werden und eine bilaterale Verarbeitung stattfindet. In den Reaktionszeiten konnten in beiden Experimente keine signifikanten Unterschiede gezeigt werden. Die Unterscheidung der Modulationsrichtung wurde dabei von allen Versuchspersonen als einfacher eingestuft als die Unterscheidung der Stimulusdauer, was sich auch in niedrigeren Antwortschnelligkeit und Fehlerraten bei vergleichbaren Schalldruckpegeln zeigte.
In der zweiten Versuchsreihe wurde als Referenzmessung nochmals die Unterscheidung der Modulationsrichtungen von FM-Stimuli durchgeführt. Anschließend wurde die Unterscheidung von „da“ und „ga“ untersucht. Diese CV-Silben differieren ausschließlich in der FM-Komponente. Die Untercheidung von CV-Silben ohne Unterschied in der FM-Komponente wurde mittels „ta“ und „ka“ getestet. Für alle drei Experimente zeigte sich, dass ein geringerer Schalldruckpegel des FM- oder CV-Stimulus zu einer steigenden Fehlerrate führte. Unter Anwendung des 15 %-Kriteriums zeigte sich für die Unterscheidung der Modulationsrichtung ein Trend zu niedrigeren Fehlerraten bei der Präsentation des FM-Stimulus auf dem linken im Vergleich mit dem rechten Ohr. In den Reaktionszeiten konnten keine signifikanten Unterschiede gezeigt werden.
Für die Unterscheidung von „da“ und „ga“ ließ sich unter Anwendung des 15 %-Kriteriums in den Fehlerraten und Reaktionszeiten kein Vorteil eines Ohres nachweisen. Dagegen zeigten sich klare Unterschiede bei einzelnen Versuchspersonen. So waren die Fehlerraten für Versuchspersonen, die vorwiegend „da“ erkannt bzw. gehört hatten signifikant höher, wenn der CV-Stimulus auf dem rechten Ohr präsentiert wurde, für „ga“-Hörer war das Gegenteil der Fall. In den Reaktionszeiten konnte kein signifikanter Zusammenhang nachgewiesen werden. Somit ließ sich zeigen, dass je nach Strategie der Versuchsperson bzw. deren individueller Wahrnehmung der CV-Silben, Unterschiede in der Lateralisierung erreicht werden können.
Für die Unterscheidung von „ta“ und „ka“ zeigten sich unter Anwendung des 15 %-Kriteriums signifikant niedrigere Fehlerraten und Reaktionszeiten, wenn der CV-Stimulus auf dem linken Ohr präsentiert wurde. Dies weist deutlich auf eine rechtslastige Lateralisierung hin. Vergleicht man alle drei Experimente ließ sich zudem zeigen, dass die Unterscheidung der Modulationsrichtung einfacher war als die Unterscheidung verschiedener CV-Stimuli. Dabei war die Unterscheidung von „da“ und „ga“ für die Versuchspersonen schwieriger als die Unterscheidung von „ta“ und „ka“. Allerdings konnte in den Lateralisierungsdaten kein direkter Zusammenhang zwischen den FM- und „da“-/„ga“-Stimuli gezeigt werden.
Zusammenfassend konnte in allen fünf Experimenten eine verschieden stark lateralisierte Verarbeitung von akustischen Stimuli bei gleichzeitigem kontralateralen Rauschen gezeigt werden. Der Vorteil eines Ohres (bzw. einer Hemisphäre) war sowohl von der Aufgabe als auch vom Stimulustyp abhängig. Dabei gab es zum Teil starke Unterschiede in der Effektstärke und dem Grad der Lateralisierung zwischen den einzelnen Versuchspersonen. Insgesamt konnte gezeigt werden, dass sich die hier angewendete psychophysische Methode gut eignet, um Ergebnisse zur Lateralisierung von akustischen Stimuli zu gewinnen und somit die Verhaltensrelevanz von Ergebnissen aus Studien mit bildgebenden Verfahren zu überprüfen.
Derzeit breiten sich gebietsfremde Stechmücken (Diptera: Culicidae) aufgrund von Globalisierung und Klimawandel auf der ganzen Welt aus und bilden neue, stabile Populationen. Wegen ihrer hämatophagen Ernährungsweise sind sie Überträger von Pathogenen, die teilweise schwere bis tödliche Krankheiten beim Menschen, seinen Haustieren oder auch Wildtieren auslösen können. Mit den Stechmücken treten daher auch Infektionskrankheiten vermehrt in Gebieten auf, in denen sie vorher nicht vorkamen oder als bereits ausgerottet galten. Da die meisten im Menschen wirksamen Pathogene nicht durch Impfungen kontrolliert werden können, bleibt als eine der wenigen Möglichkeit der Krankheitsprävention die Dezimierung der Stechmückenpopulation. Daher sind Stechmücken momentan im Fokus von biologischer und epidemiologischer Forschung. Diese hat zum Ziel epidemische Krankheitsausbrüche vektorübertragener Krankheiten in der menschlichen Population zu verhindern. Eine Verringerung der lokalen Stechmückenpopulation bis hin zum Aussterben kann durch die Verwendung von Insektiziden, die Vernichtung von Bruthabitaten oder anderen Kontrollmaßnahmen erreicht werden. Jedoch sind diese Maßnahmen unterschiedlich effektiv, haben zum Teil unerwünsch-te ökologische und gesundheitsschädigende Folgen und sind unterschiedlich aufwendig und kostenintensiv in der Anwendung. Für die Entwicklung eines integrierten, effektiven, zielgerichteten und kostengünstigen Vektormanagements fehlen bislang jedoch die populationsbiologischen Grundlagen.
Ziel dieser Arbeit ist daher die Schaffung der Datengrundlage eines Integrierten Stechmückenmanagements für die Asiatische Buschmücke (Aedes japonicus japonicus THEOBALD 1901), die am weitesten verbreitete exotische Stechmücke in Deutschland. Schwerpunkte dafür wurden auf das zeitliche und räumliche Vorkommen, die Temperaturabhängigkeit des Lebenszyklus, sowie die Wirksamkeit von Kontrollmethoden gelegt.
Die Kenntnis der räumlichen Verbreitung und saisonalen Häufigkeit der Stechmücken ist notwendig, um befallene Standorte und Zeitpunkte des größten Populationszuwachses definieren zu können. Die Verbreitung und die Häufigkeit der endothermen Stechmücken sind stark von der Umgebungstemperatur abhängig, die beispielsweise deren Entwicklungsdauer und Sterblichkeit beeinflusst. Dabei entwickeln sich die verschiedenen Stadien (Ei, Larven, Puppe, Imago), die eine Stechmücke während ihres Lebens durchläuft, in Abhängigkeit von der Umgebungstemperatur unterschiedlich und haben jeweils andere Temperaturpräferenzen. Lebenszyklustabellen geben die Entwicklungsdauer und Mortalität pro Stadium in Abhängigkeit von der Temperatur an. Mit ihrer Hilfe können somit die räumlichen und zeitlichen Vorkommen und Häufigkeiten einer Stechmückenart berechnet werden. Dies ist insbesondere für Stechmücken in Gebieten mit jahreszeitlichen Temperaturveränderungen wichtig. Um Daten für eine solche Lebenszyklustabelle aufnehmen zu können, ist es notwendig Laborexperimente bei festgelegten Temperaturen durchzuführen. Die Voraussetzung dafür ist, dass die Stechmückenart im Labor optimale Bedingungen erhält, um ihren Lebenszyklus abschließen zu können. In dieser Arbeit wurde daher ein Laborprotokoll entwickelt, mithilfe dessen der Lebenszyklus der Asiatischen Buschmücke im Labor untersucht werden kann. Dazu wurden systematisch die Fütterung, die innerartliche Konkurrenz und das Wasservolumen des Brutge-fäßes für die aquatischen Stadien erprobt. Auf Basis dieses Protokolls wurden anschließend die Temperatureinflüsse auf die Entwicklung aller Stadien aufgenommen. Diese Daten dienten der Parametrisierung eines populationsdynamischen Modells. Dieses wurde verwendet, um Standorte mehrjähriger Populationen zu definieren, saisonale Häufigkeiten für Deutschland zu berechnen, durch Temperaturveränderungen hervorgerufene zukünftige Verbreitungsgebiete vorherzusagen, sowie Effekte von Kontrollmaßnahmen auf die Häufigkeit der Asiatischen Buschmücke zu modellieren.
Um eine dauerhafte Kontrolle der Stechmückenvektoren zu gewährleisten, ist weiterhin die permanente Neuentwicklung von wirksamen Kontrollmethoden notwendig. Dazu gehört die präventive Vermeidung von Bruthabitaten der aquatischen Stadien von Stechmücken. Die exotischen Stechmücken, die in Deutschland etabliert sind, gehören mehrheitlich der Gattung Aedes an und sind sogenannte Gefäßbrüter. Ihre bevorzugten Bruthabitate sind kleine Was-seransammlungen wie sie in Baumhöhlen, Gesteinsauswaschungen, Gießkannen, Regentonnen und Blumenuntersetzern vorkommen. In dieser Arbeit wurde untersucht, welche Farben und Volumina von Plastikbechern die Asiatische Buschmücke zur Eiablage bevorzugt, um präferierte Bruthabitate gezielt zu identifizieren und verringern zu können. Auch die Bereitstellung von Insektiziden wird durch in Stechmücken auftretende Insektizidresistenzen erschwert. Insektizide sollen dabei umweltfreundlich, spezifisch für den Zielorganismus und nicht gesundheitsschädlich für den Menschen sein. Weiterhin sind eine gute Anwendbarkeit, geringe Kosten und eine hohe Effizienz wünschenswert. Eine Quelle für potentielle Insektizide sind pflanzliche Stoffe, zum Beispiel ätherische Öle. Diese sind leicht erhältlich, natürlichen Ursprungs und wirksame Vergrämungsmittel gegen stechbereite Stechmückenweibchen. In dieser Arbeit wurde nach einer Literaturrecherche Nelkenöl ausgewählt und als Insektizid gegen Larven der Asiatischen Buschmücke getestet. Dafür wurden die akute toxische Wirkung von Nelkenöl bei drei Temperaturen untersucht und zusätzlich die Wirkung von Nelkenöl auf die Eiablage im Freiland. Nelkenöl zeigte dabei sowohl eine larvizide als auch eine eiablagehemmende Wirkung. Weiterhin wurde Kupfer in Form von kupferhaltigen Euromünzen als Larvizid untersucht. Kupfer ist ein wirksamer Stoff gegen die aquatischen Stadien von Stechmücken. Allerdings wurde der Stoff noch nicht in Form der einfach zu handhabenden, leicht erhältlichen Kupfermünzen getestet. Dazu wurden Vorexperimente durchgeführt, um herauszufinden, wieviel Kupferionen sich aus den Münzen lösen lassen. Anschließend wurde der akut toxische Effekt auf Larven der Asiatischen Buschmücke untersucht.
Ein Integriertes Stechmückenmanagement hat zum Ziel, die lokale Stechmückenpopulation zu kontrollieren, um so Stichen und daraus resultierender Krankheitsübertragung vorzubeugen. Dies erfolgt über die Aufklärung von Betroffenen, der Überwachung der Stechmückenpopulation, dem Testen auf Pathogenbefall und der direkten Kontrolle von Stechmücken. Diese Arbeit leistet einen Beitrag zu den Kenntnissen über die Laborhaltung einer exotischen Stechmückenart, zur Identifizierung von Bruthabitaten, zur zeitlichen und räumlichen Festlegung von Kontrollmaßnahmen und zur Anwendung von Larviziden und eines Vergrämungsmittels. Mit dieser Arbeit wurde die Grundlage eines faktenbasierten Integrativen Stechmückenmanagements für die Asiatische Buschmücke entwickelt, das eventuell auch auf weitere Aedes-Arten übertragbar ist, und als Handlungsempfehlung für politische Entscheidungstragende dienen kann.
Brain aging is one of the major risk factors for the development of several neurodegenerative diseases. Therefore, mitochondrial dysfunction plays an important role in processes of both, brain aging and neurodegeneration. Aged mice including NMRI mice are established model organisms to study physiological and molecular mechanisms of brain aging. However, longitudinal data evaluated in one cohort are rare but are important to understand the aging process of the brain throughout life, especially since pathological changes early in life might pave the way to neurodegeneration in advanced age. To assess the longitudinal course of brain aging, we used a cohort of female NMRI mice and measured brain mitochondrial function, cognitive performance, and molecular markers every 6 months until mice reached the age of 24 months. Furthermore, we measured citrate synthase activity and respiration of isolated brain mitochondria. Mice at the age of three months served as young controls. At six months of age, mitochondria-related genes (complex IV, creb-1, β-AMPK, and Tfam) were significantly elevated. Brain ATP levels were significantly reduced at an age of 18 months while mitochondria respiration was already reduced in middle-aged mice which is in accordance with the monitored impairments in cognitive tests. mRNA expression of genes involved in mitochondrial biogenesis (cAMP response element-binding protein 1 (creb-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), nuclear respiratory factor-1 (Nrf-1), mitochondrial transcription factor A (Tfam), growth-associated protein 43 (GAP43), and synaptophysin 1 (SYP1)) and the antioxidative defense system (catalase (Cat) and superoxide dismutase 2 (SOD2)) was measured and showed significantly decreased expression patterns in the brain starting at an age of 18 months. BDNF expression reached, a maximum after 6 months. On the basis of longitudinal data, our results demonstrate a close connection between the age-related decline of cognitive performance, energy metabolism, and mitochondrial biogenesis during the physiological brain aging process.
The Brachybasidiaceae are a family of 22 known species of plant-parasitic microfungi belonging to Exobasidiales, Basidiomycota. Within this family, species of the largest genus Kordyana develop balls of basidia on top of stomatal openings. Basidial cells originate from fungal stroma filling substomatal chambers. Species of Kordyana typically infect species of Commelinaceae. During fieldwork in the neotropics, fungi morphologically similar to Kordyana spp. were found on Goeppertia spp. (syn. Calathea spp., Marantaceae), namely on G. panamensis in Panama and on G. propinqua in Bolivia. These specimens are proposed as representatives of a genus new to science, Marantokordyana, based on the distinct host family and molecular sequence data of ITS and LSU rDNA regions. The specimens on the two host species represent two species new to science, M. oberwinkleriana on G. panamensis and M. boliviana on G. propinqua. They differ by the size and shape of their basidia, molecular sequence data of ITS and LSU rDNA regions, and host plant species. In the past, the understanding of Brachybasidiaceae at order and family level was significantly improved by investigation realized by Franz Oberwinkler and his collaborators at the University of Tübingen, Germany. On species level, however, our knowledge is still very poor due to incomplete species descriptions of several existing names in literature, scarceness of specimens, as well as sequence data lacking for many taxa and for further barcode regions. Especially species of Kordyana and species of Dicellomyces are in need of revision.
Acinetobacter baumannii is an opportunistic pathogen, which has become a rising threat in healthcare facilities worldwide due to increasing antibiotic resistances and optimal adaptation to clinical environments and the human host. We reported in a former publication on the identification of three phopholipases of the phospholipase D (PLD) superfamily in A. baumannii ATCC 19606T acting in concerted manner as virulence factors in Galleria mellonella infection and lung epithelial cell invasion. This study focussed on the function of the three PLDs. A Δpld1-3 mutant was defect in biosynthesis of the phospholipids cardiolipin (CL) and monolysocardiolipin (MLCL), whereas the deletion of pld2 and pld3 abolished the production of MLCL. Complementation of the Δpld1-3 mutant with pld1 restored CL biosynthesis demonstrating that the PLD1 is implicated in CL biosynthesis. Complementation of the Δpld1-3 mutant with either pld2 or pld3 restored MLCL and CL production leading to the conclusion that PLD2 and PLD3 are implicated in CL and MLCL production. Mutant studies revealed that two catalytic motifs are essential for the PLD3-mediated biosynthesis of CL and MLCL. The Δpld1-3 mutant exhibited a decreased colistin and polymyxin B resistance indicating a role of CL in cationic antimicrobial peptides (CAMPs) resistance.
Reprogramming of tomato leaf metabolome by the activity of heat stress transcription factor HsfB1
(2020)
Plants respond to high temperatures with global changes of the transcriptome, proteome, and metabolome. Heat stress transcription factors (Hsfs) are the core regulators of transcriptome responses as they control the reprogramming of expression of hundreds of genes. The thermotolerance-related function of Hsfs is mainly based on the regulation of many heat shock proteins (HSPs). Instead, the Hsf-dependent reprogramming of metabolic pathways and their contribution to thermotolerance are not well described. In tomato (Solanum lycopersicum), manipulation of HsfB1, either by suppression or overexpression (OE) leads to enhanced thermotolerance and coincides with distinct profile of metabolic routes based on a metabolome profiling of wild-type (WT) and HsfB1 transgenic plants. Leaves of HsfB1 knock-down plants show an accumulation of metabolites with a positive effect on thermotolerance such as the sugars sucrose and glucose and the polyamine putrescine. OE of HsfB1 leads to the accumulation of products of the phenylpropanoid and flavonoid pathways, including several caffeoyl quinic acid isomers. The latter is due to the enhanced transcription of genes coding key enzymes in both pathways, in some cases in both non-stressed and stressed plants. Our results show that beyond the control of the expression of Hsfs and HSPs, HsfB1 has a wider activity range by regulating important metabolic pathways providing an important link between stress response and physiological tomato development.
Correction to: Scientific Reports https://doi.org/10.1038/srep21848, published online 22 February 2016
This Article contains an error. Among the studied species, the orangutan was erroneously specified as Bornean orangutan Pongo pygmaeus. In fact, the studied individual was a Sumatran orangutan Pongo abelii.
Iron is part of many redox and other enzymes and, thus, it is essential for all living beings. Many oxic environments have extremely low concentrations of free iron. Therefore, many prokaryotic species evolved siderophores, i.e., small organic molecules that complex Fe3+ with very high affinity. Siderophores of bacteria are intensely studied, in contrast to those of archaea. The haloarchaeon Haloferax volcanii contains a gene cluster that putatively encodes siderophore biosynthesis genes, including four iron uptake chelate (iuc) genes. Underscoring this hypothesis, Northern blot analyses revealed that a hexacistronic transcript is generated that is highly induced under iron starvation. A quadruple iuc deletion mutant was generated, which had a growth defect solely at very low concentrations of Fe3+, not Fe2+. Two experimental approaches showed that the wild type produced and exported an Fe3+-specific siderophore under low iron concentrations, in contrast to the iuc deletion mutant. Bioinformatic analyses revealed that haloarchaea obtained the gene cluster by lateral transfer from bacteria and enabled the prediction of enzymatic functions of all six gene products. Notably, a biosynthetic pathway is proposed that starts with aspartic acid, uses several group donors and citrate, and leads to the hydroxamate siderophore Schizokinen.
Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ("wide" and "narrow"), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.
Photorhabdus and Xenorhabdus bacteria live in a highly specific symbiosis with nematodes that belong to the genus of Heterorhabditis and Steinernema, respectively. These cruiser type nematodes actively search for soil-dwelling insects and infect them via natural openings. Inside of the insect, the bacteria are released into the hemocoel where they start producing an array of secondary metabolites to bypass the insect immune system and kill the prey within 48 hours. Many of those natural products possess bioactivities against other bacteria, fungi, protozoa or insects, which makes them interesting candidates for pharmaceutical applications. Even though advanced molecular biological methods in combination with bioinformatics tools can now be used to predict biosynthetic gene clusters (BGCs) and their products, there are still many BGCs with unknown products. Even for the plethora of natural products that were successfully identified in the last couple of years, the exact ecological function often remains elusive, as laboratory conditions can vary considerably from the natural environment of the bacteria. Knowledge about the natural conditions that stimulate, or repress production of certain natural products and their underlying regulatory mechanisms yield new approaches for natural product research and enables possibilities for selective manipulations of the regulatory cascades.
The overarching goal of this work was to examine the regulatory networks in Photorhabdus and Xenorhabdus strains. The first part of this work focused on the Hfq-dependent regulation of specialized metabolite production. In those genera, the RNA chaperone, Hfq, represses expression of hexA, which encodes for a global transcriptional regulator that acts as the master repressor for SM production. Multiple global approaches were used to identify the sRNA ArcZ, which targets a specific region in the 5’-untranslated region of the hexA mRNA and ultimately guides Hfq in order to repress its expression. It was shown that a deletion of arcZ led to a drastic reduction of SM production in Photorhabdus and Xenorhabdus, consistent with the phenotype of their respective hfq deletion mutants. Transcriptomic profiling revealed far-reaching effects on the transcriptome, with up to 735 coding sequences significantly affected in the arcZ deletion strain. Finally, it was shown that the resulting chemical background, devoid of SMs, in combination with targeted promotor exchange can be used to exclusively overproduce a desired natural product, representing an alternative route of genetic manipulation.
The second part of this work focused on the influence and identification of insect related compounds that affect SM production in P. laumondii, X. szentirmaii and X. nematophila. Insect homogenate was generated from G. mellonella larvae, a model host for these bacteria. Supplementation of the cultivation medium with homogenate induced considerable shifts in the SM profiles of those bacteria. A global effect on the transcriptional output was determined by transcriptomic profiling. The core response to the simulation of an insect environment consisted of ten CDS, eight of which are involved in the degradation of fatty acids or the import of maltose and maltodextrin into the cells. Two abundant components in the insect homogenate, trehalose and putrescin, were added to the cultivation medium of those strains and subsequent HPLC-MS analysis revealed a direct correlation of their concentration in the medium and the production titres of certain SMs. These results indicated that the bacteria sense the insect environment via different insect specific components in order to initiate a metabolic adjustment, which is probably required for adaptation to the insect host.
The last part of this work examined the influence of other, so far not directly related genes on SM production, based on the isolation of P. laumondii transposon-insertion mutants with clear phenotypic alterations. Re-sequencing and SM profiling of the mutant strains revealed that a transposon-insertion in the gene encoding for a putative DNA-adenine methyltransferase affected SM production. The phenotype was confirmed by deleting this gene. Based on Single-Molecule Real-Time sequencing, the complete methylome of the WT, deletion- and complementation mutant were analysed (experimental work performed by Sacha J. Pidot, Melbourne, Australia). No obvious alterations were detected in the methylation patterns of the strains, indicating that the dam gene product does not methylate the adenine in GATC-motifs, as it was described in literature for E. coli. This data raises the question what the function of the putative DNA-adenine methyltransferase is in P. laumondii and how it can influence the secondary metabolism. Even though there is currently no clear evidence, the potential role of epigenetic gene regulation mechanisms should be considered in further work.
Background: The gut microbiome can influence life history traits associated with host fitness such as fecundity and longevity. In most organisms, these two life history traits are traded-off, while they are positively linked in social insects. In ants, highly fecund queens can live for decades, while their non-reproducing workers exhibit much shorter lifespans. Yet, when fertility is induced in workers by death or removal of the queen, worker lifespan can increase. It is unclear how this positive link between fecundity and longevity is achieved and what role the gut microbiome and the immune system play in this. To gain insights into the molecular regulation of lifespan in social insects, we investigated fat body gene expression and gut microbiome composition in workers of the ant Temnothorax rugatulus in response to an experimental induction of fertility and an immune challenge.
Results: Fertile workers upregulated several molecular repair mechanisms, which could explain their extended lifespan. The immune challenge altered the expression of several thousand genes in the fat body, including many immune genes, and, interestingly, this transcriptomic response depended on worker fertility. For example, only fertile, immune-challenged workers upregulated genes involved in the synthesis of alpha-ketoglutarate, an immune system regulator, which extends the lifespan in Caenorhabditis elegans by down-regulating the TOR pathway and reducing oxidant production. Additionally, we observed a dramatic loss in bacterial diversity in the guts of the ants within a day of the immune challenge. Yet, bacterial density did not change, so that the gut microbiomes of many immune challenged workers consisted of only a single or a few bacterial strains. Moreover, the expression of immune genes was linked to the gut microbiome composition, suggesting that the ant host can regulate the microbiome in its gut.
Conclusions: Immune system flare-ups can have negative consequence on gut microbiome diversity, pointing to a previously underrated cost of immunity. Moreover, our results provide important insights into shifts in the molecular regulation of fertility and longevity associated with insect sociality.
A large number of chemicals are constantly introduced to surface water from anthropogenic and natural sources. Although substantial efforts have been made to identify these chemicals (e.g potentially anthropogenic contaminants) in surface waters using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), a large number of LC-HRMS chemical signals often with high peak intensity are left unidentified. In addition to synthetic chemicals and transformation products, these signals may also represent plant secondary metabolites (PSMs) released from vegetation through various pathways such as leaching, surface run-off and rain sewers or input of litter from vegetation. While this may be considered as a confounding factor in screening of water contaminants, it could also contribute to the cumulative toxic risk of water contamination. However, it is hardly known to what extent these metabolites contribute to the chemical mixture of surface waters. Thus, reducing the number of unknowns in water samples by identifying also PSMs in significant concentrations in surface waters will help to improve monitoring and assessment of water quality potentially impacted by complex mixtures of natural and synthetic compounds. Therefore, the main focus of the present study was to identify the occurrence of PSMs in river waters and explore the link between the presence of vegetation along rivers and detection of their corresponding PSMs in river
water.
In order to achieve the goals of the present thesis, two chemical screening approaches, namely, non-target and target screening using LC-HRMS were implemented. (1) Non-target analysis involving a novel approach has been applied to associate unknown peaks of high intensity in LC-HRMS to PSMs from surrounding vegetation by focusing on peaks overlapping between river water and aqueous plant extracts (Annex A1). (2) LC–HRMS target screening in river waters were performed for about 160 PSMs, which were selected from a large phytotoxin database (Annex A2 and A3) considering their expected abundance in the vegetation, their potential mobility, persistence and toxicity in the water cycle and commercial availability of standards.
In non-target screening (Annex A1), a high number of overlapping peaks has been found in between aqueous plant extracts and water from adjacent location, suggesting a significant impact of vegetation on chemical mixtures detectable in river waters. The chemical structures were assigned for 12 pairs of peaks while several pairs of peaks
whose MS/MS spectra matched but no structure suggestion were made by the implemented software tools for retrieving possible chemical structure. Nevertheless, the pairs of peaks with matching spectra represented the same chemical structure. The identified compound belonged to different compound classes such as coumarins, flavonoids besides others. For the identified PSMs individual concentration up to 5 µg/L were measured. The concentration and the number of detected PSMs per sample were correlated with the rain event and vegetation coverage.
Target screening unraveled the occurrence of 33 out of 160 target compounds in river waters (Annex A2 and A3). The identified compounds belonged to different classes such as alkaloids, coumarins, flavonoids, and other compounds. Individual compound concentrations were up to several thousand ng/L with the toxic alkaloids narciclasine and
lycorine recording highest maximum concentrations. The neurotoxic alkaloid coniine from poison hemlock was detected at concentrations up to 0.4 µg/L while simple coumarins
esculetin and fraxidin occurred at concentrations above 1 µg/L. The occurrence of some PSMs in river water were correlated to the specific vegetation growing along the rivers while the others were linked to a wide range of vegetation. As an example, narciclasine and lycorine was emitted by the dominant plant species from Amaryllidaceae family (e.g. Galanthus nivalis (snow drop), Leucojum vernum and Anemone nemorosa) while intermedine and echimidine were from Symphytum officinale. The ubiquitous occurrence of simple coumarins fraxidin, scopoletin and aesculetin could be linked to their presence in a wide range of vegetation.
Due to lack of aquatic toxicity data for the identified PSMs (in both target and non-target) and extremely scarce exposure data, no reliable risk assessment was possible.
Alternatively, risk estimation was performed using the threshold for toxicological concern (TTC) concept developed for drinking water contaminants. Many of the identified PSMs
exceeded the TTC value (0.1 µg/L) thus caution should be taken when using such surface waters for drinking water abstraction or recreational use.
This thesis provides an overview of the occurrence of PSMs in river water impacted by the massive presence of vegetation. Concentration for many of the identified PSMs are well within the range of those of synthetic environmental contaminants. Thus, this study adds to a series of recent results suggesting that possibly toxic PSMs occur in relevant concentrations in European surface waters and should be considered in monitoring and risk assessment of water resources. Aquatic toxicity data for PSMs are extensively lacking but are required to include these compounds in the assessment of risks to aquatic organisms and for eliminating risks to human health during drinking water production.
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.
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.
Enhanced LTP of population spikes in the dentate gyrus of mice haploinsufficient for neurobeachin
(2020)
Deletion of the autism candidate molecule neurobeachin (Nbea), a large PH-BEACH-domain containing neuronal protein, has been shown to affect synaptic function by interfering with neurotransmitter receptor targeting and dendritic spine formation. Previous analysis of mice lacking one allele of the Nbea gene identified impaired spatial learning and memory in addition to altered autism-related behaviours. However, no functional data from living heterozygous Nbea mice (Nbea+/−) are available to corroborate the behavioural phenotype. Here, we explored the consequences of Nbea haploinsufficiency on excitation/inhibition balance and synaptic plasticity in the intact hippocampal dentate gyrus of Nbea+/− animals in vivo by electrophysiological recordings. Based on field potential recordings, we show that Nbea+/− mice display enhanced LTP of the granule cell population spike, but no differences in basal synaptic transmission, synapse numbers, short-term plasticity, or network inhibition. These data indicate that Nbea haploinsufficiency causes remarkably specific alterations to granule cell excitability in vivo, which may contribute to the behavioural abnormalities in Nbea+/− mice and to related symptoms in patients.
Following severe population decline and local extinction due to massive habitat destruction and persecution, wildcats have recently reappeared in several parts of Germany’s low mountain region. It remains unknown how this reemergence occurred, specifically if local populations have been overlooked at low densities or if the species has successfully spread across the highly fragmented anthropogenic landscape. In the central German Rhön Mountains, for instance, wildcats were believed to be extinct during most of the twentieth century, however, the species was recently detected and subsequent genetic monitoring found the presence of a sizeable population. In this study, we used microsatellite and SNP genotypes from 146 wildcat individuals from 2008 to 2017 across a ~ 15,000 km2 area in the central German low mountain region to understand the population re-establishment of wildcats in the region. Bayesian clustering and subsequent analyses revealed that animals in the Rhön Mountains appear to be a mix from the two adjacent populations in the North and South of the area, suggesting a recent range expansion from two different directions. Both populations meet in the Rhön Biosphere Reserve, leading to an admixture of the northern, autochthonous, and the southern reintroduced wildcat population. While we cannot completely exclude the possibility of undetected population persistence, the high genetic homogeneity in the central German wildcat population and the lack of any signatures of past population decline in the Rhön favor a scenario of natural expansion. Our findings thus suggest that wildcats are well capable of rapid range expansion across richly structured landscape mosaics consisting of open land, settlements, and forest patches and document the potential of massive non-invasive genetic sampling when aiming to reconstruct the complex population and range dynamics of wildlife.
Nature affects human well-being in multiple ways. However, the association between species diversity and human well-being at larger spatial scales remains largely unexplored. Here, we examine the relationship between species diversity and human well-being at the continental scale, while controlling for other known drivers of well-being. We related socio-economic data from more than 26,000 European citizens across 26 countries with macroecological data on species diversity and nature characteristics for Europe. Human well-being was measured as self-reported life-satisfaction and species diversity as the species richness of several taxonomic groups (e.g. birds, mammals and trees). Our results show that bird species richness is positively associated with life-satisfaction across Europe. We found a relatively strong relationship, indicating that the effect of bird species richness on life-satisfaction may be of similar magnitude to that of income. We discuss two, non-exclusive pathways for this relationship: the direct multisensory experience of birds, and beneficial landscape properties which promote both bird diversity and people's well-being. Based on these results, this study argues that management actions for the protection of birds and the landscapes that support them would benefit humans. We suggest that political and societal decision-making should consider the critical role of species diversity for human well-being.
Nature and its constituents are known to affect human well-being in positive and negative ways. Nature can be beneficial for humans by providing, for instance, food, recreation or inspiration. Natural disasters or transmitted diseases are, on the other hand, examples of nature’s detrimental or harmful contributions to human well-being. Such positive as well as negative effects have been termed Nature’s Contributions to People (NCP) by the Intergovernmental Science-Policy Platform for Biodiversity and Ecosystem Services (IPBES) and can be categorized into three different types of contributions: regulating, material and non-material NCP. While regulating and material NCP have been studied extensively, research on the non-material NCP is less common in comparison, especially regarding non-material NCP of biodiversity and wildlife. This dissertation therefore aims at shedding light on the non-material links between biodiversity, wildlife and human well-being. The thesis presents the results of three individual research studies in three separate chapters (CH1, 2 & 3).
In the first chapter (CH1) I conduct a systematic literature review on the non-material contributions of wildlife. Several previous reviews have published overviews on the non-material contributions of wildlife. However, only a few of these reviews examine both the positive and negative effects of wildlife in combination. These reviews usually cover few aspects of human well-being (e.g. recreation, health, psychological well-being) or just focus on a specific group of wildlife species (e.g. carnivores, scavengers). In addition, the pathways determining how wildlife affects human well-being are yet little understood. The aim of this review is therefore to create a holistic overview of the current knowledge on non-material contributions of wildlife (WCP), by summarising research on positive and negative effects and disentangling potential channels of human-wildlife experiences.
My results show that most studies in scientific literature report negative WCP. However, over the last decade the number of publications on positive WCP has increased, mainly in the Global North. This change in research focus, at the turn of the century, may be related to the newly emerging ideas and perspectives on nature during that time (e.g. Ecosystem Services and NCP). The results may also indicate different research interests across global regions and a focus on positive WCP (especially in the Global North). Surprisingly, the review identifies a lack of joint systematic assessments of positive and negative WCP across taxa, human well-being dimensions and ways (channels) of wildlife experiences. Studies show taxon-specific differences, with predominantly positive WCP reported for birds and predominantly negative WCP published for mammals and reptiles. Physical health was the most examined human well-being dimension, while many others, such as subjective well-being, social well-being, learning, identity or sense of place were rarely studied in comparison. The two channels of wildlife experiences that have been mainly studied or reported are Interaction and Knowing. While Interaction describes multisensory experiences in which people physically interact with wildlife. Knowing describes the metaphysical connection between humans and wildlife that arises through thinking or remembering experiences from wildlife encounters (including knowledge about wildlife).
To date, only few published studies examine the relationship between biodiversity and human well-being across larger spatial scales. For instance, little is known about how biodiversity is related to human well-being on the national or continental level. The second and third chapter (CH2 & 3) are thus comprised of two empirical case studies which examine the relationship between biodiversity and human well-being across Germany and Europe, respectively. As indicator for biodiversity, I use different species diversity measures including species richness and abundance. In the second chapter (CH2) I analyse the association between species richness and human health across Germany. The results demonstrate a significant positive relationship between plant and bird species richness and mental health while controlling for a multitude of socio-economic and demographic factors as well as other nature characteristics. In the third chapter (CH3) I conduct the first study on the relationship between species diversity and subjective well-being on the continental level. The results show that bird species richness (unlike mammal, megafauna and tree richness) is positively associated with life-satisfaction, a measure for subjective well-being across Europe. These results are robust while accounting for socio-economic and macro-economic factors. The results of both empirical studies are in correspondence with previous research, conducted on the local and national level.
Overall, my dissertation shows that wildlife and biodiversity greatly affect human well-being and provide substantial non-material NCP.
...
Bioaerosols are considered to play a relevant role in atmospheric processes, but their sources, properties, and spatiotemporal distribution in the atmosphere are not yet well characterized. In the Amazon Basin, primary biological aerosol particles (PBAPs) account for a large fraction of coarse particulate matter, and fungal spores are among the most abundant PBAPs in this area as well as in other vegetated continental regions. Furthermore, PBAPs could also be important ice nuclei in Amazonia. Measurement data on the release of fungal spores under natural conditions, however, are sparse. Here we present an experimental approach to analyze and quantify the spore release from fungi and other spore-producing organisms under natural and laboratory conditions. For measurements under natural conditions, the samples were kept in their natural environment and a setup was developed to estimate the spore release numbers and sizes as well as the microclimatic factors temperature and air humidity in parallel to the mesoclimatic parameters net radiation, rain, and fog occurrence. For experiments in the laboratory, we developed a cuvette to assess the particle size and number of newly released fungal spores under controlled conditions, simultaneously measuring temperature and relative humidity inside the cuvette. Both approaches were combined with bioaerosol sampling techniques to characterize the released particles using microscopic methods. For fruiting bodies of the basidiomycetous species, Rigidoporus microporus, the model species for which these techniques were tested, the highest frequency of spore release occurred in the range from 62 % to 96 % relative humidity. The results obtained for this model species reveal characteristic spore release patterns linked to environmental or experimental conditions, indicating that the moisture status of the sample may be a regulating factor, whereas temperature and light seem to play a minor role for this species. The presented approach enables systematic studies aimed at the quantification and validation of spore emission rates and inventories, which can be applied to a regional mapping of cryptogamic organisms under given environmental conditions.
Cell fate clusters in ICM organoids arise from cell fate heredity and division: a modelling approach
(2020)
During the mammalian preimplantation phase, cells undergo two subsequent cell fate decisions. During the first decision, the trophectoderm and the inner cell mass are formed. Subsequently, the inner cell mass segregates into the epiblast and the primitive endoderm. Inner cell mass organoids represent an experimental model system, mimicking the second cell fate decision. It has been shown that cells of the same fate tend to cluster stronger than expected for random cell fate decisions. Three major processes are hypothesised to contribute to the cell fate arrangements: (1) chemical signalling; (2) cell sorting; and (3) cell proliferation. In order to quantify the influence of cell proliferation on the observed cell lineage type clustering, we developed an agent-based model accounting for mechanical cell–cell interaction, i.e. adhesion and repulsion, cell division, stochastic cell fate decision and cell fate heredity. The model supports the hypothesis that initial cell fate acquisition is a stochastically driven process, taking place in the early development of inner cell mass organoids. Further, we show that the observed neighbourhood structures can emerge solely due to cell fate heredity during cell division.
Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete Saccharomyces cerevisiae and the basidiomycete Ustilago maydis. One extract that inhibited the growth of S. cerevisiae much stronger than that of U. maydis was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against S. cerevisiae but reduced activity against U. maydis were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether U. maydis could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in U. maydis.
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. ...
The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K+, in osmostress response. Growth of A. baumannii was K+-dependent and the K+-dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K+ and K+ accumulation increased with the salinity of the medium. K+ uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K+ concentration: mannitol and glutamate concentrations increased with increasing K+ concentrations whereas trehalose was highest at low K+. After osmotic upshock, cells first accumulated K+ followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K+ and glutamate. These experiments demonstrate K+ uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K+ and different organic solutes.
Communication with the hematopoietic system is a vital component of regulating brain function in health and disease. Traditionally, the major routes considered for this neuroimmune communication are by individual molecules such as cytokines carried by blood, by neural transmission, or, in more severe pathologies, by the entry of peripheral immune cells into the brain. In addition, functional mRNA from peripheral blood can be directly transferred to neurons via extracellular vesicles (EVs), but the parameters that determine their uptake are unknown. Using varied animal models that stimulate neuronal activity by peripheral inflammation, optogenetics, and selective proteasome inhibition of dopaminergic (DA) neurons, we show that the transfer of EVs from blood is triggered by neuronal activity in vivo. Importantly, this transfer occurs not only in pathological stimulation but also by neuronal activation caused by the physiological stimulus of novel object placement. This discovery suggests a continuous role of EVs under pathological conditions as well as during routine cognitive tasks in the healthy brain.
The Rnf complex is a Na+ coupled respiratory enzyme in a fermenting bacterium, Thermotoga maritima
(2020)
rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na+ pump. These studies provide the proof that the Rnf complex is indeed an ion (Na+) translocating, respiratory enzyme. Together with a Na+-F1FO ATP synthase it builds a simple, two-limb respiratory chain in T. maritima. The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.
Flavin-based electron bifurcation is a long hidden mechanism of energetic coupling present mainly in anaerobic bacteria and archaea that suffer from energy limitations in their environment. Electron bifurcation saves precious cellular ATP and enables lithotrophic life of acetate-forming (acetogenic) bacteria that grow on H2 + CO2 by the only pathway that combines CO2 fixation with ATP synthesis, the Wood–Ljungdahl pathway. The energy barrier for the endergonic reduction of NADP+, an electron carrier in the Wood–Ljungdahl pathway, with NADH as reductant is overcome by an electron-bifurcating, ferredoxin-dependent transhydrogenase (Nfn) but many acetogens lack nfn genes. We have purified a ferredoxin-dependent NADH:NADP+ oxidoreductase from Sporomusa ovata, characterized the enzyme biochemically and identified the encoding genes. These studies led to the identification of a novel, Sporomusa type Nfn (Stn), built from existing modules of enzymes such as the soluble [Fe–Fe] hydrogenase, that is widespread in acetogens and other anaerobic bacteria.
The production of bulk chemicals mostly depends on exhausting petroleum sources and leads to emission of greenhouse gases. Within the last decades the urgent need for alternative sources has increased and the development of bio-based processes received new attention. To avoid the competition between the use of sugars as food or fuel, other feedstocks with high availability and low cost are needed, which brought acetogenic bacteria into focus. This group of anaerobic organisms uses mixtures of CO2, CO and H2 for the production of mostly acetate and ethanol. Also methanol, a cheap and abundant bulk chemical produced from methane, is a suitable substrate for acetogenic bacteria. In methylotrophic acetogens the methyl group is transferred to the Wood-Ljungdahl pathway, a pathway to reduce CO2 to acetate via a series of C1-intermediates bound to tetrahydrofolic acid. Here we describe the biochemistry and bioenergetics of methanol conversion in the biotechnologically interesting group of anaerobic, acetogenic bacteria. Further, the bioenergetics of biochemical production from methanol is discussed.
Droughts impair plant growth, limit global net primary production and are predicted to increase in the course of climate change. Knowledge of the plant drought response on a molecular level can facilitate the selection of drought resistant genotypes and genetic engineering and thereby can help to implement strategies, such as assisted migration projects or crop improvement, in order to preserve natural and agricultural vegetation against droughts.
Studies on gene expression under drought stress were conducted in three species each of the genera Quercus and Panicum, to shed light on the molecular drought response in these species and identify drought responsive genes as a basis for technical applications.
In the genus Quercus, gene expression studies were conducted in the three major European forest trees Q. ilex, Q. pubescens and Q. robur, for which a distributional shift caused by climate change is predicted for the 21st century. RNA-Seq experiments were conducted in the three Quercus species for the first time, ortholog groups were assigned and unregulated genes, as well as drought responsive genes, were identified (Madritsch et al. 2019). For a set of the unregulated genes, a stable expression over the course of long-term drought periods was evaluated in order to enable an application as reference genes for normalizing qRT-PCR experiments (Kotrade 2019a). The reference genes were used in subsequent experiments to generate gene expression profiles over the course of a two-year drought experiment with consecutive drought periods for a set of twelve drought responsive genes and revealed a highly variable gene regulation under long-term drought stress in the Quercus species (Kotrade et al. 2019b).
In the genus Panicum, the gene expression in response to drought was examined in the two wild crop species, P. laetum and P. turgidum, and in the less drought tolerant species P. bisulcatum via RNA-Seq experiments (Kotrade et al. 2020 (in revision). The transcriptomes of the species were sequenced for the first time, ortholog groups were assigned and the gene regulation was compared across the species. The common grounds of the drought response in Panicum were determined by identifying similarities across the species, while the identification of differences between the species led to genes that might contribute to the higher drought tolerance of P. laetum and P. turgidum
A comparison across the two genera showed large differences in the gene regulation upon drought. This might be largely explained by different experimental setups that resulted in different drought conditions in the genera, such as drought intensity, drought duration and velocity of drought development.
The sequence information and the drought responsive genes identified in the Quercus and Panicum species can be used to develop marker assays for marker-assisted selection. The genes that putatively contribute to the higher drought tolerance of the two wild crop Panicum species should be considered as candidate targets in genetic engineering studies. Marker-assisted selection and genetic engineering can be applied, for example, in assisted migration projects to support natural vegetation in the course of climate change or to breed more drought tolerant crop strains to mitigate crop failure rates caused by droughts.
The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.
The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus’ proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.
The genus Ebolavirus comprises some of the deadliest viruses for primates and humans and associated disease outbreaks are increasing in Africa. Different evidence suggests that bats are putative reservoir hosts and play a major role in the transmission cycle of these filoviruses. Thus, detailed knowledge about their distribution might improve risk estimations of where future disease outbreaks might occur. A MaxEnt niche modelling approach based on climatic variables and land cover was used to investigate the potential distribution of 9 bat species associated to the Zaire ebolavirus. This viral species has led to major Ebola outbreaks in Africa and is known for causing high mortalities. Modelling results suggest suitable areas mainly in the areas near the coasts of West Africa with extensions into Central Africa, where almost all of the 9 species studied find suitable habitat conditions. Previous spillover events and outbreak sites of the virus are covered by the modelled distribution of 3 bat species that have been tested positive for the virus not only using serology tests but also PCR methods. Modelling the habitat suitability of the bats is an important step that can benefit public information campaigns and may ultimately help control future outbreaks of the disease.
The facile synthesis and detailed investigation of a class of highly potent protease inhibitors based on 1,4-naphthoquinones with a dipeptidic recognition motif (HN-l-Phe-l-Leu-OR) in the 2-position and an electron-withdrawing group (EWG) in the 3-position is presented. One of the compound representatives, namely the acid with EWG = CN and with R = H proved to be a highly potent rhodesain inhibitor with nanomolar affinity. The respective benzyl ester (R = Bn) was found to be hydrolyzed by the target enzyme itself yielding the free acid. Detailed kinetic and mass spectrometry studies revealed a reversible covalent binding mode. Theoretical calculations with different density functionals (DFT) as well as wavefunction-based approaches were performed to elucidate the mode of action.
Zoos attract millions of visitors every year, many of whom are schoolchildren. For this reason, zoos are important institutions for the environmental education of future generations. Empirical studies on the educational impact of environmental education programs in zoos are still rare. To address this issue, we conducted two studies: In study 1, we investigated students’ interests in different biological topics, including zoos (n = 1,587). Data analysis of individual topics revealed large differences of interest, with advanced students showing less interest in zoos. In study 2, we invited school classes of this age group to visit different guided tours at the zoo and tested connection to nature before and after each educational intervention (n = 608). The results showed that the guided tours are an effective tool to raise students’ connection to nature. Add-on components have the potential to further promote connection to nature. The education programs are most effective with students with a low initial nature connection.
In recent environmental research, relational values (RVs) have emerged as a new group of values to explain environmental behavior. Although this new concept is attracting attention, empirical studies on the subject are still rare. On this basis, we have conducted three studies to analyze an existing measurement tool for RVs and compared the construct with the concept of connection to nature. In study 1, we confirmed convergent and discriminant validity of the RV scale by comparing it with the Two Factor Model of Environmental Values (2-MEV) model using a sample of n = 350 university students. Additionally, study 1 verified reliability using test–retest reliability on three different groups of students (n1 = 53; n2 = 37; n3 = 48). In study 2, principal component analyses were performed to examine the structure of RVs and to compare it to the concept of connection to nature by reusing the sample 350 university students from study 1. The results show that RVs and connection to nature are not fundamentally distinct constructs, but overlap. However, if the structure of the RV measurement is forced to a single factor, no perfect fit is found, making a multidimensional solution more likely. A third study was conducted to review the results from study 2 using confirmatory factor analysis on a new sample of 878 university and high school students. Study 3 confirmed RVs as a multidimensional construct with three factors: care, community, and connection. It also proved the overlap of the connection to nature and RV concepts to some extent.
Impact of biological education and gender on students' connection to nature and relational values
(2020)
The new concept of relational values (RVs) is gaining more and more attention in environmental research, but empirical analyses are still rare. However, this type of research is necessary because the RVs have an influence on environmental behavior. To evaluate the impact of biological education on attributing higher importance to RVs and connectedness to nature, we compared the connection to nature scores (using the inclusion of nature scale (INS) and connectedness to nature scale (CNS)) and RV scores of biologically interested high school students (n = 417) with first year (n = 593) and advanced biology (n = 223) students. While high school students showed significant lower connection to nature scores than university students, there was no significant difference in RVs between the test groups. These results suggest that there is a lack of factors in the university study of biology that can change RVs. The gender comparison of RVs and connection to nature showed a significant higher RV score for females while INS and CNS did not show a gender difference. Thus, the study makes an important contribution to the research, as it was able to prove that gender has an influence on a person's RVs but not on their connection to nature.
The UN's sustainable development goals (SDGs), which aim to solve important economic, social, and environmental problems of humanity, are to be supported by education for sustainable development (ESD). Empirical studies on the success of the implementation of the SDGs in the field of education are still pending. For this reason, using the loss of global biodiversity as an example, this study examined the extent to which high school students, teacher trainees in biology, and biology bachelor students can identify the causes of the global biodiversity loss. A new questioning tool was developed and tested on 889 participants. In addition, the relationship between connection to nature and the personal assessment about biodiversity threats was examined. The factor analysis of the scale used showed that 11 out of 16 items were assigned to the intended factor. The comparison between high school students, teacher trainees in biology, and biology bachelor students showed no significant difference in overall assessment of the reasons for global biodiversity loss. When comparing the three risk levels in which the risk factors for biodiversity could be divided, across the three student groups, only minor differences were found. Therefore, a specific education of prospective teachers is necessary, as they have to pass on the competence as multipliers to their students. No significant difference could be found when examining the relationship between connection to nature and the overall scores of the assessment scale for the reasons of biodiversity loss. However, it was found that people who felt more connected to nature were more capable of assessing the main causes of risk for global biodiversity, while people who felt less connected to nature achieved better scores for the medium factors
In biological systems (cell culture media, cells, body fluids), drugs/toxicants are usually not freely dissolved but partially bound to biomolecules; only a fraction of the chemical is free/unbound (fu). To predict pharmacological effects and toxicity, it is important that the fu of the drug is known. As the differences between free and nominal concentrations are determined by test system parameters (e.g., the protein and lipid content, and the type of surface material), comparison of nominal concentrations between two different new approach methods (NAM) may lead to faulty conclusions. The same problem exists when in vitro concentrations are compared to those in human subjects. Therefore, the respective fu of a chemical in a test system needs to be determined for in vitro-to-in vivo extrapolations (IVIVE). Besides direct measurements, prediction models can help to obtain fu. Here we describe a simplified approach to approximate fu and provide background information on the underlying assumptions. Comparative predictions and measurements of fu of various drugs are shown to exemplify the approach. Basic input data, like protein and lipid concentrations, are also provided. Beyond such test systems data, the only required chemical-specific inputs are the lipophilicity of the candidate drug and its ionization state, as determined by the dissociation constants of its acidic or basic groups. This overview is intended to be used by any lab scientist without specific toxicokinetics training to obtain an estimate of fu in a given cell culture medium.
Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO2-based bioeconomy and a H2-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H2 cycle and thus prime candidates as driving forces in a H2- and CO2-bioeconomy. Naturally, they convert two molecules of CO2 via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO2, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.
Connectomic analysis of apical dendrite innervation in pyramidal neurons of mouse cerebral cortex
(2020)
The central goal of this study was to generate synapse-resolution maps of local and long-range innervation on apical dendrites (AD) in mouse cerebral cortex. We used three-dimensional electron microscopy (3D-EM) to first measure the cell-type specific balance in the excitatory and inhibitory input on ADs. Further, we found two inhibitory axon populations with preference for apical dendrites originating from layer 2 and 3/5. Additionally, we used a combination of large-scale volumetric light and electron microscopy to investigate the innervation preference of long-range cortical projections onto ADs. To generate such large-scale 3D-EM datasets, we also developed a software package to automate aberration adjustment.
The balance of excitation and inhibition defines the computational properties of neurons. We, therefore, generated 6 datasets and annotated 26,548 excitatory and inhibitory synapses to map the relative inhibitory strength on the AD of pyramidal neurons in layers 1 and 2 (L1 and 2) of the cortex. We found consistent and cell-type specific patterns of inhibitory strength along the apical dendrite of L2-5 pyramidal neurons in primary somatosensory (S1), secondary visual (V2), posterior parietal (PPC) and anterior cingulate (ACC) cortices. L2 and L5 pyramidal neurons had inhibitory hot-zones at their main bifurcation and distal apical dendrite tuft, respectively. In contrast, L3 neurons had a baseline (~10%) level of inhibition along their apical dendrite. As controls, we quantified the effect of synapse strength (size), dendrite diameter, AD classification and synapse identification methods on the cell-type specific synapse densities. To classify L5 pyramidal subtypes, we performed hierarchical clustering using morphological properties that were described to differentiate slender- and thick-tufted L5 neurons.
We also investigated the distance to soma as a predictor of fractional inhibition around the main bifurcation of apical dendrites. Interestingly, we found a strong exponential relationship that was absent in density of either synapse type. This suggests a distance dependent control mechanism designed specifically for the balance (in synapse numbers) of excitation and inhibition.
Next, we focused on the inhibitory innervation preference for apical dendrite of pyramidal neuron. We, therefore, annotated 5,448 output synapses of AD-targeting inhibitory axons and found two populations specific for either L2 or L3/5 apical dendrites. Together with previous findings on preferential innervation of sub-cellular structures by inhibitory axons, this suggests two distinct inhibitory circuits for control of AD activity in L2 vs. deep-layer pyramidal neurons. This innervation preference was surprisingly consistent across S1, V2, PPC and ACC cortices.
3D-EM data acquisition is a laborious process that is made easier and more popular everyday by technical progress in the laboratory and industrial settings. To make data acquisition robust using our custom-built 3D-EM microscopes, an automatic aberration software was implemented to adjust the objective lens and the stigmators of the electron microscope. This method was used in multiple month-long experiments across 2 microscopes and 10 datasets. The aberration adjustment used the reduction in image details (high-frequency elements) to estimate the level of deviation from optimal focus and stigmator parameters. However, large objects in EM micrographs such as blood vessel and nuclei cross-sections generated anomalous results. We, therefore, added image processing routines based on edge detection combined with morphological operations to exclude such large objects.
Finally, we performed a correlative three-dimensional (3D) light (LM) and electron (EM) microscopy experiment to map the long-range primary visual (V1) and secondary motor (M2) cortical input to ADs in layer 1 of PPC using the “FluoEM” approach. This method allows for identification of the long-range source of projection axons in EM volumes without the need for EM-dense label conversion or heat-induced markings. The long-range source of an axon in EM is identified based on the fluorescent protein that is expressed in its LM counterpart. In comparison to M2 input, Long-range axons from V1 had a higher tendency to target L3 pyramidal neurons in PPC according to our preliminary analysis. In combination with the difference observed in the synapse composition of L2 and L3 apical dendrites, this suggests the need for separate functional and structural analysis of L2 and 3 pyramidal neurons.
Au Tchad, à cause de ses retombées financières une attention particulière est prêtée aux arbres à karité (Vitella- ria paradoxa C.F.Gaertn.). Cependant, cette culture est menacée par les plantes vasculaires parasites de la famille des Lo- ranthaceae. La présente étude a été effectuée dans 3 sites dans la région du Mandoul pour évaluer l’ampleur des attaques de Loranthaceae (gui africain) sur des arbres en fonction des classes de circonférence du tronc à 1,5 cm du sol. Elle a consisté à dénombrer sur une de surface, les arbres à karité infestés et les touffes de parasites rencontrées sur ces arbres, afin de dé- terminer leur taux et leur intensité d’infestation. Les résultats obtenus montrent que Tapinanthus dodoneifolus (DC) Danser a été trouvée comme la seule espèce de Loranthaceae qui parasite les arbres karité étudiés dans la zone d’étude. Le taux moyen d’infestation estimé à 73% augmente avec l’âge des arbres karité. La moyenne d’intensité de l’infestation/arbre (2,75 touffes à Békôh, 2,27 à Yomi and 2,04 à Bébopen) montre que Tapinanthus dodoneifolus constitue une réelle menace pour les peu- plements de karité dans la zone d’étude. Il reste à rechercher le seuil d’infestation qui provoque une réduction significative de la fructification. Pour l’instant, bien que pénible à cause de la hauteur des arbres adultes, la lutte mécanique contre les Ta- pinanthus par la coupe systématique des branches infestées est urgente dans les parcs à karité dans cette zone d’étude.
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.
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.
In situ burning (ISB) is discussed to be one of the most suitable response strategies to combat oil spills in extreme conditions. After burning, a highly viscous and sticky residue is left and may over time pose a risk of exposing aquatic biota to toxic oil compounds. Scientific information about the impact of burn residues on the environment is scarce. In this context, a comprehensive ISB field experiment with approx. 1000L IFO 180 was conducted in a fjord in Greenland. The present study investigated the toxicity of collected ISB residues to early life stages of zebrafish (Danio rerio) as a model for potentially exposed pelagic organisms. The toxicity of ISB residues on zebrafish embryos was compared with the toxicity of the initial (unweathered) IFO 180 and chemically dispersed IFO 180. Morphological malformations, hatching success, swimming behavior, and biomarkers for exposure (CYP1A activity, AChE inhibition) were evaluated in order to cover the toxic response on different biological organization levels. Across all endpoints, ISB residues did not induce greater toxicity in zebrafish embryos compared with the initial oil. The application of a chemical dispersant increased the acute toxicity most likely due to a higher bioavailability of dissolved and particulate oil components. The results provide insight into the adverse effects of ISB residues on sensitive life stages of fish in comparison with chemical dispersant application.
The hydrogen-dependent carbon dioxide reductase is a soluble enzyme complex that directly utilizes hydrogen (H2) for the reduction of carbon dioxide (CO2) to formate in the first step of the acetyl-coenzyme A- or Wood-Ljungdahl pathway (WLP). HDCR consists of 2 catalytic subunits, a hydrogenase and a formate dehydrogenase (FDH) and two small subunits carrying iron-sulfur clusters. The enzyme complex has been purified and characterized from two acetogenic bacteria, from the mesophile Acetobacterium woodii and, recently, from the thermophile Thermoanaerobacter kivui. Physiological studies toward the importance of the HDCR for growth and formate metabolism in acetogens have not been carried out yet, due to the lack of genetic tools. Here, we deleted the genes encoding HDCR in T. kivui taking advantage of the recently developed genetic system. As expected, the deletion mutant (strain TKV_MB013) did not grow with formate as single substrate or under autotrophic conditions with H2 + CO2. Surprisingly, the strain did also not grow on any other substrate (sugars, mannitol or pyruvate), except for when formate was added. Concentrated cell suspensions quickly consumed formate in the presence of glucose only. In conclusion, HDCR provides formate which was essential for growth of the T. kivui mutant. Alternatively, extracellularly added formate served as terminal electron acceptor in addition to CO2, complementing the growth deficiency. The results show a tight coupling of multi-carbon substrate oxidation to the WLP. The metabolism in the mutant can be viewed as a coupled formate + CO2 respiration, which may be an ancient metabolic trait.
To support future research based on natural sciences collection data, DiSSCo (Distributed System of Scientific Collections) – the European Research Infrastructure for Natural Science Collections – adopts Digital Object Architecture as the basis for its planned data infrastructure. Using the outputs of one Research Data Alliance (RDA) interest group (IG) and five working groups (WGs) we show how RDA recommendations and supporting documents have been applied to the various stages of the DiSSCo data lifecycle.
The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD‐glucose and glucose‐6‐phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose‐6‐phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose‐6‐phosphate phosphatase activity accumulated trehalose‐6‐phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose‐6‐phosphate. Our results demonstrate that trehalose‐6‐phosphate affects multiple physiological activities in A. baumannii ATCC 19606.
Transcriptional basis for differential thermosensitivity of seedlings of various tomato genotypes
(2020)
Transcriptional reprograming after the exposure of plants to elevated temperatures is a hallmark of stress response which is required for the manifestation of thermotolerance. Central transcription factors regulate the stress survival and recovery mechanisms and many of the core responses controlled by these factors are well described. In turn, pathways and specific genes contributing to variations in the thermotolerance capacity even among closely related plant genotypes are not well defined. A seedling-based assay was developed to directly compare the growth and transcriptome response to heat stress in four tomato genotypes with contrasting thermotolerance. The conserved and the genotype-specific alterations of mRNA abundance in response to heat stress were monitored after exposure to three different temperatures. The transcripts of the majority of genes behave similarly in all genotypes, including the majority of heat stress transcription factors and heat shock proteins, but also genes involved in photosynthesis and mitochondrial ATP production. In turn, genes involved in hormone and RNA-based regulation, such as auxin- and ethylene-related genes, or transcription factors like HsfA6b, show a differential regulation that associates with the thermotolerance pattern. Our results provide an inventory of genes likely involved in core and genotype-dependent heat stress response mechanisms with putative role in thermotolerance in tomato seedlings.
Peronospora salviae‐officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae‐officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.
Downy mildew of common sage (Salvia officinalis), caused by Peronospora salviae-officinalis, has become a serious problem in sage production worldwide. The causal agent of the disease belongs to the Pe. belbahrii species complex and was described as a species of its own in 2009. Nevertheless, very little is known about its infection biology and epidemiology. The aims of the current study were therefore to unravel the life cycle of this downy mildew and gain deeper insights into the epidemiology of the disease, as well as to clarify the species boundaries in the Pe. belbahrii species complex.
Infection studies showed that temperatures between 15 and 20 °C were most favourable for infection and disease progress. At 5 °C Pe. salviae-officinalis is still able to infect sage plants, but sporulation was only observed at higher temperatures. Furthermore, Pe. salviae-officinalis needs two events of leaf wetness or high humidity, a first one of at least three hours for conidial germination and penetration of the host, and a second one for sporulation. Additionally, contamination of sage seeds by Pe. salviae-officinalis was proven by seed washing and by PCR and DNA sequence comparisons, suggesting that infested seeds might play a major role in the fast spread of sage downy mildew, which is an important finding for phytosanitary or quarantine measures.
A protocol for fluorescence staining and confocal laser scanning microscopy was established and the whole life cycle of Pe. salviae-officinalis was tracked including oospore formation. The method was also used to examine samples of Pe. lamii on Lamium purpureum and Pe. belbahrii on Ocimum basilicum demonstrating the usefulness of this method for studying the infection process of downy mildews in general.
Peronospora species parasitizing S. sclarea, S. pratensis, O. basilicum, and Plectranthus scutellarioides were studied using light microscopy and molecular phylogenetic analyses based on six loci (ITS rDNA, cox1, cox2, ef1a, hsp90 and β-tubulin). The downy mildew on S. pratensis was shown to be distinct from Pe. salviae-officinalis and closely related to Pe. glechomae, and is herein described as a new taxon, Peronospora salviae-pratensis. The downy mildew on S. sclarea was found to be caused by Peronospora salviae-officinalis. The multi-gene phylogeny revealed that the causal agent of downy mildew on coleus is distinct from Pe. belbahrii on basil, and is herein described as a new taxon, Pe. choii.
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.
Tsetse flies are the transmitting vector of trypanosomes causing human sleeping sickness and animal trypanosomiasis in sub-saharan Africa. 3-alkylphenols are used as attractants in tsetse fly traps to reduce the spread of the disease. Here we present an inexpensive production method for 3-ethylphenol (3-EP) and 3-propylphenol (3-PP) by microbial fermentation of sugars. Heterologous expression in the yeast Saccharomyces cerevisiae of phosphopantetheinyltransferase-activated 6-methylsalicylic acid (6-MSA) synthase (MSAS) and 6-MSA decarboxylase converted acetyl-CoA as a priming unit via 6-MSA into 3-methylphenol (3-MP). We exploited the substrate promiscuity of MSAS to utilize propionyl-CoA and butyryl-CoA as alternative priming units and the substrate promiscuity of 6-MSA decarboxylase to produce 3-EP and 3-PP in yeast fermentations. Increasing the formation of propionyl-CoA by expression of a bacterial propionyl-CoA synthetase, feeding of propionate and blocking propionyl-CoA degradation led to the production of up to 12.5 mg/L 3-EP. Introduction of a heterologous ‘reverse ß-oxidation’ pathway provided enough butyryl-CoA for the production of 3-PP, reaching titers of up to 2.6 mg/L. As the concentrations of 3-alkylphenols are close to the range of the concentrations deployed in tsetse fly traps, the yeast broths might become promising and inexpensive sources for attractants, producible on site by rural communities in Africa.