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1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5′-UTR of SARS-CoV-2
(2022)
The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5′- and 3′-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5′-untranslated region (5′-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5′-UUUCGU-3′ hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.
The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here, we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in Arabidopsis thaliana, ribosomal cistrons are present in Fagus sylvatica only in four major regions, in line with FISH studies. On most assembled chromosomes, telomeric repeats were found at both ends, while centromeric repeats were found to be scattered throughout the genome apart from their main occurrence per chromosome. The genome-wide distribution of SNPs was evaluated using a second individual from Jamy Nature Reserve (Poland). SNPs, repeat elements and duplicated genes were unevenly distributed in the genomes, with one major anomaly on chromosome 4. The genome presented here adds to the available highly resolved plant genomes and we hope it will serve as a valuable basis for future research on genome architecture and for understanding the past and future of European Beech populations in a changing climate.
Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms.
Protein oxidation results from the reaction of amino-acid side chains with reactive oxygen species (ROS) and is partly irreversible. In non-photosynthetic tissues, mitochondria are a main source of ROS, whereas plastids are the major source in photosynthetic tissues. Oxidized proteins suffer from decreased structural integrity and even loss of function, and their accumulation leads to cytotoxic aggregates. In mammals, aggregate formation correlates with aging and is linked to several age-related pathologies. Mammalian proteolytic pathways for clearance of oxidized proteins are under intensive research, while mechanistic insights into this process in plants is scarce. Acylamino acid-releasing (AARE) enzymes are ATP-independent serine proteases, presumably acting on oxidized proteins and operating in a dual exo-/endopeptidase mode. They are found in all domains of life. Here, we investigated AARE enzymes in the moss Physcomitrella and the angiosperm Arabidopsis and identified three homologous nuclear genes in Physcomitrella (PpAARE1-3) and a single nuclear gene in Arabidopsis (AtAARE). Surprisingly, we observed triple localization of the proteins AtAARE and PpAARE1 to plastids, mitochondria and the cytosol in vivo, likely conserved across the plant lineage. This represents an ATP-independent possibility for degradation of oxidized proteins in the major source organelles of ROS in plants, which is distinct to mammals. Combinatorial knockout plants and protein interaction analysis revealed specific interactions of the moss AARE isoforms and functions in progressive aging. Analysis of an AtAARE T-DNA mutant further suggests the evolutionary conservation of AARE function in age-related development.
Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and support a unified concept of ageing.
Species of the genus Blautia are typical inhabitants of the human gut and considered as beneficial gut microbes. However, their role in the gut microbiome and their metabolic features are poorly understood. Blautia schinkii was described as an acetogenic bacterium, characterized by a functional Wood–Ljungdahl pathway (WLP) of acetogenesis from H2 + CO2. Here we report that two relatives, Blautia luti and Blautia wexlerae do not grow on H2 + CO2. Inspection of the genome sequence revealed all genes of the WLP except genes encoding a formate dehydrogenase and an electron-bifurcating hydrogenase. Enzyme assays confirmed this prediction. Accordingly, resting cells neither converted H2 + CO2 nor H2 + HCOOH + CO2 to acetate. Carbon monoxide is an intermediate of the WLP and substrate for many acetogens. Blautia luti and B. wexlerae had an active CO dehydrogenase and resting cells performed acetogenesis from HCOOH + CO2 + CO, demonstrating a functional WLP. Bioinformatic analyses revealed that many Blautia strains as well as other gut acetogens lack formate dehydrogenases and hydrogenases. Thus, the use of formate instead of H2 + CO2 as an interspecies hydrogen and electron carrier seems to be more common in the gut microbiome.
Although new advances in neuroscience allow the study of vocal communication in awake animals, substantial progress in the processing of vocalizations has been made from brains of anaesthetized preparations. Thus, understanding how anaesthetics affect neuronal responses is of paramount importance. Here, we used electrophysiological recordings and computational modelling to study how the auditory cortex of bats responds to vocalizations under anaesthesia and in wakefulness. We found that multifunctional neurons that process echolocation and communication sounds were affected by ketamine anaesthesia in a manner that could not be predicted by known anaesthetic effects. In wakefulness, acoustic contexts (preceding echolocation or communication sequences) led to stimulus-specific suppression of lagging sounds, accentuating neuronal responses to sound transitions. However, under anaesthesia, communication contexts (but not echolocation) led to a global suppression of responses to lagging sounds. Such asymmetric effect was dependent on the frequency composition of the contexts and not on their temporal patterns. We constructed a neuron model that could replicate the data obtained in vivo. In the model, anaesthesia modulates spiking activity in a channel-specific manner, decreasing responses of cortical inputs tuned to high-frequency sounds and increasing adaptation in the respective cortical synapses. Combined, our findings obtained in vivo and in silico reveal that ketamine anaesthesia does not reduce uniformly the neurons’ responsiveness to low and high frequency sounds. This effect depends on combined mechanisms that unbalance cortical inputs and ultimately affect how auditory cortex neurons respond to natural sounds in anaesthetized preparations.
Detailed information on species temperature preferences are needed to measure the effects of global warming on species and communities in European rivers. However, information currently available in the literature on taxon-specific temperature preferences or temperature tolerances is very heterogeneous and therefore not well suited for forecasting purposes. To close this gap, we derived so-called ’central temperature tendencies’ (CTTt values) for benthic invertebrate species. For this end, 547 species and temperature data from regional monitoring programmes in Germany collected at 4249 sites were analysed. Due to the vulnerability of species to high
temperatures, CTTt values were calculated for mean summer temperatures, following a robust approach of calculating a weighted average based on temperature classes. Derived CTTt values correspond well to species temperature preferences as reported in literature as long as the latter were homogeneous in terms of how they were derived and which temperature reference was at focus. Based on taxon-specific CTTt values, a community value, CTTCom, was calculated for each benthic invertebrate sample. CTTCom values were validated by correlation with mean summer water temperatures. As the slope a of the linear regression model between CTTCom values and measured summer temperatures was comparatively low (a = 0.49), a correction function was derived in order to optimise the relation between both. This was crucial, because it is assumed that although CTTt was derived solely from taxa abundances within summer temperature classes, CTTCom not only reflects the effect of (summer) water temperature itself, but also corresponds to a temperature equivalent value, which describes the overall quality of all respiration-relevant aquatic summer habitat conditions that determine the metabolism of respective benthic invertebrates. By comparing this equivalent value with water temperatures measured in the year previous of sampling, statements can be made about the influence of flow conditions and other factors determining oxygen availability.
Thus, CTTCom reflects the mean aerobic scope of the overall benthic invertebrate fauna: the better the respiration conditions for rheophilic species with high oxygen demand, the larger the aerobic scope and the lower CTTCom.
The approach taken in our study is promising and provides a tool to track and even project past, present, and future impacts of global warming on benthic invertebrates in rivers based on measured values of respiratory relevant environmental variables. We encourage all stakeholders in the field of freshwater ecology to test this
Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1-based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1-based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1-based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B-based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.
Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H2. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H2-dependent Fd reduction and Fd2--dependent H2 production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N′-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na+ for activity and did not translocate 22Na+ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H+-translocating, H+-reducing enzyme.
Orientation hypercolumns in the visual cortex are delimited by the repeating pinwheel patterns of orientation selective neurons. We design a generative model for visual cortex maps that reproduces such orientation hypercolumns as well as ocular dominance maps while preserving retinotopy. The model uses a neural placement method based on t–distributed stochastic neighbour embedding (t–SNE) to create maps that order common features in the connectivity matrix of the circuit. We find that, in our model, hypercolumns generally appear with fixed cell numbers independently of the overall network size. These results would suggest that existing differences in absolute pinwheel densities are a consequence of variations in neuronal density. Indeed, available measurements in the visual cortex indicate that pinwheels consist of a constant number of ∼30, 000 neurons. Our model is able to reproduce a large number of characteristic properties known for visual cortex maps. We provide the corresponding software in our MAPStoolbox for Matlab.
Bisphenols and phthalates, chemicals frequently used in plastic products, promote obesity in cell and animal models. However, these well-known metabolism-disrupting chemicals (MDCs) represent only a minute fraction of all compounds found in plastics. To gain a comprehensive understanding of plastics as a source of exposure to MDCs, we characterized the chemicals present in 34 everyday products using nontarget high-resolution mass spectrometry and analyzed their joint adipogenic activities by high-content imaging. We detected 55,300 chemical features and tentatively identified 629 unique compounds, including 11 known MDCs. Importantly, the chemicals extracted from one-third of the products caused murine 3T3-L1 preadipocytes to proliferate, and differentiate into adipocytes, which were larger and contained more triglycerides than those treated with the reference compound rosiglitazone. Because the majority of plastic extracts did not activate the peroxisome proliferator-activated receptor γ and the glucocorticoid receptor, the adipogenic effects are mediated via other mechanisms and, thus, likely to be caused by unknown MDCs. Our study demonstrates that daily-use plastics contain potent mixtures of MDCs and can, therefore, be a relevant yet underestimated environmental factor contributing to obesity.
Animals use the geomagnetic field and astronomical cues to obtain compass information. The magnetic compass is not a uniform mechanism, as several functional modes have been described in different animal groups. The Sun compass requires the internal clock to interpret the position of the Sun. For star compass orientation, night-migrating birds seem to use the star pattern as a whole, without involving the internal clock. Both the astronomical compass mechanisms are based on learning processes to adapt them to the geographic latitude where the animals live and, in long-living animals, to compensate for the seasonal changes. Several mechanisms are used to determine the compass course to a goal. Using information collected during the outward journey is mostly done by path integration: recording the direction with a compass and integrating its twists and turns. Migratory animals have innate programs to guide them to their still unknown goal. Highly mobile animals with large ranges develop a so-called navigational ‘map’, a mental representation of the spatial distribution of navigational factors within their home region and their migration route. The nature of the factors involved is not yet entirely clear; magnetic intensity and inclination are the ones best supported so far.
Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.
Mining is one of the major pollution sources worldwide, causing huge disturbances to the environment. Industrial and artisanal mining activities are widespread in Mexico, a major global producer of various metals. This study aimed to assess the ecological impairments resulting from mining activities using aquatic macroinvertebrates assemblages (MA). A multiple co-inertia analysis was applied to determine the relationships between environmental factors, habitat quality, heavy metals, and aquatic macroinvertebrates in 15 study sites in two different seasons (dry and wet) along two rivers running across the Central Plateau of Mexico. The results revealed three contrasting environmental conditions associated with different MAs. High concentrations of heavy metals, nutrients, and salinity limit the presence of several families of seemingly sensitive macroinvertebrates. These factors were found to influence structural changes in MAs, showing that not only mining activities, but also agriculture and presence of villages in the basin, exert adverse effects on macroinvertebrate assemblages. Diversity indices showed that the lowest diversity matched both the most polluted and the most saline rivers. The rivers studied displayed high alkalinity and hardness levels, which can reduce the availability of metals and cause adverse effects on periphyton by inhibiting photosynthesis and damaging MAs. Aquatic biomonitoring in rivers, impacted by mining and other human activities, is critical for detecting the effect of metals and other pollutants to improve management and conservation strategies. This study supports the design of cost-effective and accurate water quality biomonitoring protocols in developing countries.
Reprogramming biosynthetic assembly-lines is a topic of intense interest. This is unsurprising as the scaffolds of most antibiotics in current clinical use are produced by such pathways. The modular nature of assembly-lines provides a direct relationship between the sequence of enzymatic domains and the chemical structure of the product, but rational reprogramming efforts have been met with limited success. To gain greater insight into the design process, we wanted to examine how Nature creates assembly-lines and searched for biosynthetic pathways that might represent evolutionary transitions. By examining the biosynthesis of the anti-tubercular wollamides, we uncover how whole gene duplication and neofunctionalization can result in pathway bifurcation. We show that, in the case of the wollamide biosynthesis, neofunctionalization is initiated by intragenomic recombination. This pathway bifurcation leads to redundancy, providing the genetic robustness required to enable large structural changes during the evolution of antibiotic structures. Should the new product be non-functional, gene loss can restore the original genotype. However, if the new product confers an advantage, depreciation and eventual loss of the original gene creates a new linear pathway. This provides the blind watchmaker equivalent to the design, build, test cycle of synthetic biology.
Hydrogen is a promising fuel in a carbon-neutral economy, and many efforts are currently undertaken to produce hydrogen. One of the challenges is to store and transport the highly explosive gas in a safe and easy way. One option that is intensively analyzed by chemists and biologists is the conversion of hydrogen and CO2 to formic acid, the liquid organic hydrogen carrier. Here, we demonstrate for the first time that a bio-based system, using Acetobacterium woodii as the biocatalyst, allows multiple cycles of bi-directional hydrogenation of CO2 to formic acid in one bioreactor. The process was kept running over 2 weeks producing and oxidizing 330 mM formic acid in total. Unwanted side-product formation of acetic acid was prevented through metabolic engineering of the organism. The demonstrated process design can be considered as a future “bio-battery” for the reversible storage of electrons in the form of H2 in formic acid, a versatile compound.
Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii
(2022)
Methanol is one of the most widely produced organic substrates from syngas and can serve as a bio-feedstock to cultivate acetogenic bacteria which allows a major contribution to reducing greenhouse gas. Acetobacterium woodii is one of the very few acetogens that can utilize methanol to produce acetate as sole product. Since A. woodii is genetically tractable, it is an interesting candidate to introduce recombinant pathways for production of bio-commodities from methanol. In this study, we introduced the butyrate production operon from a related acetogen, Eubacterium callanderi KIST612, into A. woodii and show a stable production of butyrate from methanol. This study also reveals how butyrate production by recombinant A. woodii strains can be enhanced with addition of electrons in the form of carbon monoxide. Our results not only show a stable expression system of non-native enzymes in A. woodii but also increase in the product spectrum of A. woodii to compounds with higher economic value.
This work characterizes the post-PKS modifications of AQ-256. Additionally, the second part describes the establishment of an AQ production platform for electrolyte generation that can be utilized in redox-flow-batteries. Lastly, a silent BGC that encodes the genes for terpenoid biosynthesis was described and characterized with regards to product formation and putative ecological function.
Only a few studies on the nocturnal behavior of African ungulates exist so far, with mostly small sample sizes. For a comprehensive understanding of nocturnal behavior, the data basis needs to be expanded. Results obtained by observing zoo animals can provide clues for the study of wild animals and furthermore contribute to a better understanding of animal welfare and better husbandry conditions in zoos. The current contribution reduces the lack of data in two ways. First, we present a stand-alone open-source software package based on deep learning techniques, named Behavioral Observations by Videos and Images using Deep-Learning Software (BOVIDS). It can be used to identify ungulates in their enclosure and to determine the three behavioral poses “Standing,” “Lying—head up,” and “Lying—head down” on 11,411 h of video material with an accuracy of 99.4%. Second, BOVIDS is used to conduct a case study on 25 common elands (Tragelaphus oryx) out of 5 EAZA zoos with a total of 822 nights, yielding the first detailed description of the nightly behavior of common elands. Our results indicate that age and sex are influencing factors on the nocturnal activity budget, the length of behavioral phases as well as the number of phases per behavioral state during the night while the keeping zoo has no significant influence. It is found that males spend more time in REM sleep posture than females while young animals spend more time in this position than adult ones. Finally, the results suggest a rhythm between the Standing and Lying phases among common elands that opens future research directions.
Carotenoids represent a class of pigmented terpenoids. They are distributed in all taxonomic groups of fungi. Most of the fungal carotenoids differ in their chemical structures to those from other organisms. The general function of carotenoids in heterotrophic organisms is protection as antioxidants against reactive oxygen species generated by photosensitized reactions. Furthermore, carotenoids are metabolized to apocarotenoids by oxidative cleavage. This review presents the current knowledge on fungal-specific carotenoids, their occurrence in different taxonomic groups, and their biosynthesis and conversion into trisporic acids. The outline of the different pathways was focused on the reactions and genes involved in not only the known pathways, but also suggested the possible mechanisms of reactions, which may occur in several non-characterized pathways in different fungi. Finally, efforts and strategies for genetic engineering to enhance or establish pathways for the production of various carotenoids in carotenogenic or non-carotenogenic yeasts were highlighted, addressing the most-advanced producers of each engineered yeast, which offered the highest biotechnological potentials as production systems.
In the framework of the PNRA (Italian National Antarctic Research Program) project CARBONANT focusing on biogenic carbonates and held in January–February 2002, several Ross Sea banks were sampled to obtain samples of biogenic carbonates. In the Mawson Bank, species belonging to the isopod genus Chaetarcturus Brandt, 1990 were recorded, including a specimen that did not match any described species. In this paper we describe Chaetarcturus cervicornis sp. n., which is characterized by supraocular spines and two pairs of tubercle-like protrusions on the cephalothorax. The new species is very similar to C. bovinus (Brandt & Wägele, 1988) and C. adareanus (Hodgson, 1902), but has a clearly different spine pattern. The study of the species of the genus Chaetarcturus in the Ross Sea contributes to increase our knowledge on the diversity of the Antarcturidae in the Southern Ocean. Ross Sea banks seem to hold an interesting and not-well-known fauna, deserving attention in future research.
Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). TAA-producing bacteria are the causative agent of many human diseases and TAA targeted anti-adhesive compounds might counteract such bacterial infections. The modularly structured Bartonella adhesin A (BadA) is one of the best characterised TAAs and serves as an attractive adhesin to study the domain-function relationship of TAAs during infection. BadA is a major virulence factor of B. henselae and is essential for the initial attachment to host cells via adhesion to extracellular matrix proteins. B. henselae is the causative agent of cat scratch disease and adheres to fibronectin using its long BadA fibres. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations.
Human, feline, and laboratory adapted B. henselae isolates display genomic and phenotypic differences. By analysing the genomes of eight B. henselae strains using long-read sequencing, a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes was identified. Moreover, numerous conserved flanking genes were characterised, however, their influence on the regulation of badA expression and modification remains to be explored. It seems that B. henselae G 5436 is the evolutionary ancestor of the other B. henselae strains analysed in this work. The diversity of the badA island among the B. henselae strains indicates that the downstream badA-like domain region might be used as a ‘toolbox’ for rearrangements in the badA gene. Overall, it is suggested that badA-domain duplications, insertions, and/or deletions are the result of active phase variation via site-specific recombination and contribute to rapid host adaptation in the scope of pathogenicity, immune evasion, and/or enhanced long-term colonisation.
The model strain B. henselae Marseille expresses a badA gene that includes 30 repetitive neck/stalk domains, each consisting of several predicted structural motifs. To further elucidate the motif sequences that mediate fibronectin binding, various modified badA constructs were generated. Their ability to bind fibronectin was assessed via whole-cell ELISA and fluorescence microscopy. In conclusion, it is suggested that BadA adheres to fibronectin in a cumulative fashion with quick saturation via unpaired β-strands appearing in structural motifs present in BadA neck/stalk domains 19, 27, and other homologous domains. Furthermore, antibodies targeting a 15-mer amino acid sequence in the DALL motif of BadA neck/stalk domain 27 were able to reduce fibronectin binding of the B. henselae mutant strain S27. Moreover, this DALL motif sequence is conserved in the genome of all analysed B. henselae strains. The identification of common binding motifs between BadA and fibronectin supports the development of new anti-adhesive compounds that might inhibit the initial adherence of B. henselae and other TAA-producing pathogens during infection.
Lipopolysaccharide (LPS) is a major glycolipid component in the outer leaflet of the outer membrane of Gram-negative bacteria and known as endotoxin exhibited by the lipid A moiety, which serves as a membrane anchor. The effective permeability barrier properties of the outer membrane contributed by the presence of LPS in the extracellular layer of the outer membrane confer Gram-negative bacteria a high resistance against hydrophobic compounds such as antibiotics, bile salts and detergents to survive in harsh environments. The biogenesis of LPS is well studied in Escherichia coli (herewith E. coli) and the LPS transport (Lpt) is carried out by a transenvelope complex composed of seven essential proteins (LptABCDEFG), which are located in the three compartments of the cell such as the outer membrane, the inner membrane and the periplasm. The Lpt system also exists in Anabaena sp. PCC 7120 (herewith Anabaena sp.), however, homologues of LptC and LptE are still missing. BLAST search failed to identify a homologue of LptC, in contrast, the secondary structure analysis using the Pfam database based on the existing ecLptC secondary structure identified one open reading frame All0231 as the putative Anabaena sp. homologue of LptC, which is designated anaLptC. Despite the low sequence similarity, the secondary structure alignment between anaLptC and ecLptC using the HHpred server showed that both proteins share high secondary structural similarities. The genotypic analysis of the insertion mutant anaLptC did not identify a fully segregated genome and its phenotypic analysis revealed that it was sensitive against chemicals, suggesting that the analptC gene is essential for the growth of Anabaena sp. and involved in the outer membrane biogenesis. This is further supported by the observation of the small cell phenotype in the anaLptC mutant via transmission electron microscopy. Moreover, physical interactions between the anaLptC periplasmic domain with anaLptA as well as with anaLptF were established, indicating that the anaLptC periplasmic domain is correctly folded and alone functional and that the transmembrane helix is not required for the interaction with anaLptA and anaLptF. Furthermore, the reduction of the O-antigen containing LPS was observed in the insertion mutant anaLptC and the dissociation constant Kd of the anaLptC periplasmic domain for ecLPS was determined.The three-dimensional structure of the periplasmic domain of anaLptC was solved by X-ray crystallography with a resolution of 2.8 Å. The structural superposition between the ecLptC crystal structure (PDB number 3my2) and the crystal structure of anaLptC periplasmic domain obtained by this study showed the similarity in the folding of the two proteins with a Cα r.m.s.d value of about 1 Å and confirmed that the length of anaLptC is more than two times longer than that of ecLptC. The structural comparison also revealed that both structures share the typical β-jellyroll fold and conserved amino acids, which were shown in ecLptC to bind to LPS in vivo and found in anaLptC. Overall, these data strongly suggest that anaLptC is involved in the transport of LPS and support the model whereby the bridge spanning the inner membrane and the outer membrane would be assembled via interactions of the structurally conserved β-jellyroll domains shared by five (LptACDFG) out of seven Lpt proteins.
Die oxygene Photosynthese bildet den Grundpfeiler des heutigen Ökosystems unseres Planeten. Neben den gut untersuchten Landpflanzen bilden Mikroalgen eine äußerst bedeutende Organismengruppe der phototrophen Lebewesen. Zu den Mikroalgen zählen die Diatomeen, welche sich beispielsweise durch eine Silikatschale und spezielle Lichtsammelkomplexe auszeichnen und für einen Großteil der marinen Primärproduktion verantwortlich sind. Die stoffwechselphysiologischen Grundlagen des ökologischen Erfolgs der Kieselalgen sind bislang noch unzureichend erforscht. Ein Vertreter der zentrischen Diatomeen, Cyclotella, wurde bereits zur Jahrtausendwende zur biochemischen Charakterisierung der Diatomeen Photosynthese verwendet (Eppard und Rhiel, 1998; Eppard und Rhiel, 2000), das Genom des Organismus aber erst vor kurzem sequenziert (Traller et al., 2016). Die Sequenzierung des Genoms konnte einige Gene für Lichtsammelproteine identifizieren, die Homologie zu den LhcSR-Proteinen aus C. reinhardtii aufweisen, welche nachweislich eine photoprotektive Funktion besitzen (Peers et al., 2009). Diese sogenannten Lhcx-Proteine der Diatomeen sind in den zwei Gruppen der Kieselalgen, den zentrischen und pennaten Diatomeen zu finden, unterscheiden sich aber in ihren jeweiligen Lhcx-Kandidaten. So können in der pennaten Diatomee P. tricornutum vier lhcx-Gene ausgemacht werden, während die zentrische Kieselalge T. pseudonana sechs lhcx-Gene besitzt und C. cryptica vier verschiedene lhcx-Kandidaten genomisch aufweist (Armbrust et al., 2004; Bowler et al., 2008; Traller et al., 2016). Die beschriebenen Diatomeen weisen alle eine Homologie im Lhcx1 auf, während sich die übrigen Lhcx-Kandidaten zwischen pennaten und zentrischen Diatomeen unterscheiden. Ein zwischen T. pseudonana und C. cryptica konserviertes Lhcx ist das Lhcx6_1, welches 2011 das erste Mal massenspektrometrisch an Photosystemen von T. pseudonana nachgewiesen wurde (Grouneva et al., 2011) und in weiteren Massenspektrometrie-gestützten Untersuchungen in beiden zentrischen Diatomeen an Photosynthese-Komplexen gefunden werden konnte (Gundermann et al., 2019; Calvaruso et al., 2020). Die Funktion des Lhcx6_1 ist bislang unklar.
Diese Arbeit konnte das Lhcx6_1 aus C. meneghiniana charakterisieren und Antikörper-gestützt genauer lokalisieren, eine nicht dynamische Phosphorylierung der Thylakoidmembran-Proteine der zentrischen Diatomee nachweisen und die molekularbiologische Zugänglichkeit des Organismus optimieren. qRT-PCR gestützte Expressions-Analysen konnten eine unerwartete Expression des lhcx6_1-Gens aufdecken. Dieses weist, im Vergleich zum Lhcx1, keine Starklicht induzierte Expression auf. Die Expression des Gens konnte nach wenigen Stunden Schwachlicht als maximal bestimmt werden, während sie im Starklicht abnimmt. Das Muster der Genexpression glich im Schwachlicht eher der des lhcf1-Gens. Die Sequenzierung des lhcx6_1 aus C. meneghiniana identifizierte eine verlängerte N-terminale Sequenz des Proteins, welche Homologie zu den minoren Antennen aus A. thaliana besitzt und Teil des reifen Proteins ist. Mittels eines C-terminalen Epitops wurde ein Antikörper gegen das Lhcx6_1 entworfen, welcher das Protein in C. meneghiniana spezifisch nachweisen kann. Die Isolation von Thylakoidmembranen der zentrischen Diatomee und weitergehende Aufreinigung mittels Saccharosedichtegradienten und lpBN-PAGE konnten die Lokalisation des Lhcx6_1 eingrenzen. Das Protein zeigt dabei keine Unterschiede in seiner Lokalisation nach Inkubation in Schwach-, Stark- und Fernrot-Licht und ist vorrangig mit Photosystem I assoziiert. In geringerer Menge konnte es zudem an Photosystem II nachgewiesen werden, während der immunologische Nachweis in Lichtsammelkomplexen (FCPs) minimale Mengen erbrachte. Ferner konnte eine Phosphorylierung des Lhcx6_1 an Threonin-Resten nachgewiesen werden, während die meisten anderen Thylakoidmembran-Proteine mittels Phospho-Serin Antikörper detektiert werden konnten. Weder die Phosphorylierung des Lhcx6_1, noch der anderen Thylakoidmembran-Proteine, zeigt eine dynamische Regulation, im Stile einer state-transition ähnlichen Kinase auf. Die Qualität des Umgebungslichts führte zu keinerlei Unterschieden in Phosphorylierungsmustern. Weiterführende Untersuchungen der Lhcx6_1-Phosphorylierung mittels Phos-tag PAGE identifizieren eine unphosphorylierte und eine einfach phosphorylierte Form des Proteins. Dabei kann an PSI ausschließlich die phosphorylierte Version des Lhcx6_1 gefunden werden. Im Zuge der Arbeit konnte zudem erstmalig die Elektroporation und Konjugation für C. meneghiniana als Transformations-Methoden etabliert werden, während das Protokoll für die biolistische Transformation optimiert wurde. Die Elektroporation erbrachte die höchste Transformationseffizienz. Molekularbiologische Unterfangen eines Lhcx6_1-Knockdowns mittels Antisense-RNA erzielten zunächst, aufgrund der starken Gegenregulation der Diatomee, keinen Erfolg...
he most basic behavioural states of animals can be described as active or passive. While high-resolution observations of activity patterns can provide insights into the ecology of animal species, few methods are able to measure the activity of individuals of small taxa in their natural environment. We present a novel approach in which a combination of automatic radiotracking and machine learning is used to distinguish between active and passive behaviour in small vertebrates fitted with lightweight transmitters (<0.4 g).
We used a dataset containing >3 million signals from very-high-frequency (VHF) telemetry from two forest-dwelling bat species (Myotis bechsteinii [n = 52] and Nyctalus leisleri [n = 20]) to train and test a random forest model in assigning either active or passive behaviour to VHF-tagged individuals. The generalisability of the model was demonstrated by recording and classifying the behaviour of tagged birds and by simulating the effect of different activity levels with the help of humans carrying transmitters. The model successfully classified the activity states of bats as well as those of birds and humans, although the latter were not included in model training (F1 0.96–0.98).
We provide an ecological case-study demonstrating the potential of this automated monitoring tool. We used the trained models to compare differences in the daily activity patterns of two bat species. The analysis showed a pronounced bimodal activity distribution of N. leisleri over the course of the night while the night-time activity of M. bechsteinii was relatively constant. These results show that subtle differences in the timing of species' activity can be distinguished using our method.
Our approach can classify VHF-signal patterns into fundamental behavioural states with high precision and is applicable to different terrestrial and flying vertebrates. To encourage the broader use of our radiotracking method, we provide the trained random forest models together with an R package that includes all necessary data processing functionalities. In combination with state-of-the-art open-source automated radiotracking, this toolset can be used by the scientific community to investigate the activity patterns of small vertebrates with high temporal resolution, even in dense vegetation.
The most basic behavioural states of animals can be described as active or passive. However, while high-resolution observations of activity patterns can provide insights into the ecology of animal species, few methods are able to measure the activity of individuals of small taxa in their natural environment. We present a novel approach in which the automated VHF radio-tracking of small vertebrates fitted with lightweight transmitters (< 0.2 g) is used to distinguish between active and passive behavioural states.
A dataset containing > 3 million VHF signals was used to train and test a random forest model in the assignment of either active or passive behaviour to individuals from two forest-dwelling bat species (Myotis bechsteinii (n = 50) and Nyctalus leisleri (n = 20)). The applicability of the model to other taxonomic groups was demonstrated by recording and classifying the behaviour of a tagged bird and by simulating the effect of different types of vertebrate activity with the help of humans carrying transmitters. The random forest model successfully classified the activity states of bats as well as those of birds and humans, although the latter were not included in model training (F-score 0.96–0.98).
The utility of the model in tackling ecologically relevant questions was demonstrated in a study of the differences in the daily activity patterns of the two bat species. The analysis showed a pronounced bimodal activity distribution of N. leisleri over the course of the night while the night-time activity of M. bechsteinii was relatively constant. These results show that significant differences in the timing of species activity according to ecological preferences or seasonality can be distinguished using our method.
Our approach enables the assignment of VHF signal patterns to fundamental behavioural states with high precision and is applicable to different terrestrial and flying vertebrates. To encourage the broader use of our radio-tracking method, we provide the trained random forest models together with an R-package that includes all necessary data-processing functionalities. In combination with state-of-the-art open-source automated radio-tracking, this toolset can be used by the scientific community to investigate the activity patterns of small vertebrates with high temporal resolution, even in dense vegetation.
Cardiolipin, the mitochondria marker lipid, is crucially involved in stabilizing the inner mitochondrial membrane and is vital for the activity of mitochondrial proteins and protein complexes. Directly targeting cardiolipin by a chemical-biology approach and thereby altering the cellular concentration of “available” cardiolipin eventually allows to systematically study the dependence of cellular processes on cardiolipin availability. In the present study, physics-based coarse-grained free energy calculations allowed us to identify the physical and chemical properties indicative of cardiolipin selectivity and to apply these to screen a compound database for putative cardiolipin-binders. The membrane binding properties of the 22 most promising molecules identified in the in silico approach were screened in vitro, using model membrane systems finally resulting in the identification of a single molecule, CLiB (CardioLipin-Binder). CLiB clearly affects respiration of cardiolipin-containing intact bacterial cells as well as of isolated mitochondria. Thus, the structure and function of mitochondrial membranes and membrane proteins might be (indirectly) targeted and controlled by CLiB for basic research and, potentially, also for therapeutic purposes.
Tilletia caries and T. laevis, which are the causal agents of common bunt, as well as T. controversa, which causes dwarf bunt of wheat, threaten especially organic wheat farming. The three closely related fungal species differ in their teliospore morphology and partially in their physiology and infection biology. The gene content as well as intraspecies variation in these species and the genetic basis of their separation is unknown. We sequenced the genome of four T. caries, five T. controversa, and two T. laevis and extended this dataset with five publicly available ones. The genomes of the three species displayed microsynteny with up to 94.3% pairwise aligned regions excluding repetitive regions. The majority of functionally characterized genes involved in pathogenicity, life cycle, and infection of corn smut, Ustilago maydis, were found to be absent or poorly conserved in the draft genomes and the biosynthetic pathway for trimethylamine in Tilletia spp. could be different from bacteria. Overall, 75% of the identified protein-coding genes comprising 84% of the total predicted carbohydrate utilizing enzymes, 72.5% putatively secreted proteins, and 47.4% of effector-like proteins were conserved and shared across all 16 isolates. We predicted nine highly identical secondary metabolite biosynthesis gene clusters comprising in total 62 genes in all species and none were species-specific. Less than 0.1% of the protein-coding genes were species-specific and their function remained mostly unknown. Tilletia controversa had the highest intraspecies genetic variation, followed by T. caries and the lowest in T. laevis. Although the genomes of the three species are very similar, employing 241 single copy genes T. controversa was phylogenetically distinct from T. caries and T. laevis, however these two could not be resolved as individual monophyletic groups. This was in line with the genome-wide number of single nucleotide polymorphisms and small insertions and deletions. Despite the conspicuously different teliospore ornamentation of T. caries and T. laevis, a high degree of genomic identity and scarcity of species-specific genes indicate that the two species could be conspecific.
Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties. As a result, many peptides have entered the clinics for various applications. Two main routes for the biosynthesis of complex peptides have evolved in nature: ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic pathways and non-ribosomal peptide synthetases (NRPSs). Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes. These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides. In this review, we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications. In addition, the influence of the fundamentally different biosynthetic principles on past, current and future engineering approaches is illustrated. Despite the different biosynthetic principles of both peptide biosynthetic routes, the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping. The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.
Three of the four species of giraffe are threatened, particularly the northern giraffe (Giraffa camelopardalis), which collectively have the smallest known wild population estimates. Among the three subspecies of the northern giraffe, the West African giraffe (Giraffa camelopardalis peralta) had declined to 49 individuals by 1996 and only recovered due to conservation efforts undertaken in the past 25 years, while the Kordofan giraffe (Giraffa camelopardalis antiquorum) remains at <2300 individuals distributed in small, isolated populations over a large geographical range in Central Africa. These combined factors could lead to genetically depauperated populations. We analyzed 119 mitochondrial sequences and 26 whole genomes of northern giraffe individuals to investigate their population structure and assess the recent demographic history and current genomic diversity of West African and Kordofan giraffe. Phylogenetic and population structure analyses separate the three subspecies of northern giraffe and suggest genetic differentiation between populations from eastern and western areas of the Kordofan giraffe’s range. Both West African and Kordofan giraffe show a gradual decline in effective population size over the last 10 ka and have moderate genome-wide heterozygosity compared to other giraffe species. Recent inbreeding levels are higher in the West African giraffe and in Kordofan giraffe from Garamba National Park, Democratic Republic of Congo. Although numbers for both West African and some populations of Kordofan giraffe have increased in recent years, the threat of habitat loss, climate change impacts, and illegal hunting persists. Thus, future conservation actions should consider close genetic monitoring of populations to detect and, where practical, counteract negative trends that might develop.
Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade.
Biodiversity patterns of marine crustaceans are still unknown in many locations or might have been overlooked due to our knowledge gaps, despite increasing sampling and data sharing efforts during the last decades. By analysing big data extracted from open portals such as Ocean Biodiversity Information System (OBIS) and Global Biodiversity Information System (GBIF), we aim to revisit the distribution and biodiversity patterns of the highly speciose and abundant Crustacea in the Northwest Pacific (NWP) from shallowest depths to the deep sea. This study focussed on selected benthic and pelagic crustacean (sub) classes and their species richness, sampling effort, and expected species richness (ES50) using equal/sized hexagonal cells, 5° latitudinal bands, 500 m depth intervals were analyzed. Crustacean species richness was highest in the tropical Philippines as well as around the Japanese islands. Pelagic crustacean species richness peaked at 30° latitude and declined beyond that. Benthic taxa; however, depicted high levels of species richness across most of the latitudinal gradient, reaching its highest point at 45° latitude. Due to the prevalence of certain crustacean orders in the deep sea, benthic species richness showed a distribution pattern with two distinct peaks across bathymetric gradients; with highest species richness recorded at shallow-water depths and also at abyssal depths. The most important environmental drivers of benthic and pelagic crustacean species richness were primary productivity (positive correlation) and salinity (negative correlation). Our study provides first insights into biodiversity patterns of the highly diverse Crustacea in the NWP and highlights strong differences between benthic and pelagic taxa. The results presented here could help us to better understand whether benthic or pelagic taxa might respond differently to climate changes in the NWP based on their distinct physiological and biological characteristics. This information is crucial in establishing species management strategies and ecosystem restorations in both shallow water and deep-sea environments.
mRNA localization to subcellular compartments has been reported across all kingdoms of life and it is generally believed to promote asymmetric protein synthesis and localization. In striking contrast to previous observations, we show that in S. cerevisiae the B-type cyclin CLB2 mRNA is localized and translated in the yeast bud, while the Clb2 protein, a key regulator of mitosis progression, is concentrated in the mother nucleus. Using single-molecule RNA imaging in fixed (smFISH) and living cells (MS2 system), we show that the CLB2 mRNA is transported to the yeast bud by the She2-She3 complex, via an mRNA ZIP-code situated in the coding sequence. In CLB2 mRNA localization mutants, Clb2 protein synthesis in the bud is decreased resulting in changes in cell cycle distribution and genetic instability. Altogether, we propose that CLB2 mRNA localization acts as a sensor for bud development to couple cell growth and cell cycle progression, revealing a novel function for mRNA localization.
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
Size and shape variation of molar crowns in primates plays an important role in understanding how species adapted to their environment. Gorillas are commonly considered to be folivorous primates because they possess sharp cusped molars which are adapted to process fibrous leafy foods. However, the proportion of fruit in their diet can vary significantly depending on their habitats. While tooth morphology can tell us what a tooth is capable of processing, tooth wear can help us to understand how teeth have been used during mastication. The objective of this study is to explore if differences in diet at the subspecies level can be detected by the analysis of molar macrowear. We analysed a large sample of second lower molars of Grauer’s, mountain and western lowland gorilla by combining the Occlusal Fingerprint Analysis method with other dental measurements. We found that Grauer’s and western lowland gorillas are characterised by a macrowear pattern indicating a larger intake of fruit in their diet, while mountain gorilla’s macrowear is associated with the consumption of more folivorous foods. We also found that the consumption of herbaceous foods is generally associated with an increase in dentine and enamel wear, confirming the results of previous studies.
Background: Long sequencing reads allow increasing contiguity and completeness of fragmented, short-read–based genome assemblies by closing assembly gaps, ideally at high accuracy. While several gap-closing methods have been developed, these methods often close an assembly gap with sequence that does not accurately represent the true sequence.
Findings: Here, we present DENTIST, a sensitive, highly accurate, and automated pipeline method to close gaps in short-read assemblies with long error-prone reads. DENTIST comprehensively determines repetitive assembly regions to identify reliable and unambiguous alignments of long reads to the correct loci, integrates a consensus sequence computation step to obtain a high base accuracy for the inserted sequence, and validates the accuracy of closed gaps. Unlike previous benchmarks, we generated test assemblies that have gaps at the exact positions where real short-read assemblies have gaps. Generating such realistic benchmarks for Drosophila (134 Mb genome), Arabidopsis (119 Mb), hummingbird (1 Gb), and human (3 Gb) and using simulated or real PacBio continuous long reads, we show that DENTIST consistently achieves a substantially higher accuracy compared to previous methods, while having a similar sensitivity.
Conclusion: DENTIST provides an accurate approach to improve the contiguity and completeness of fragmented assemblies with long reads. DENTIST's source code including a Snakemake workflow, conda package, and Docker container is available at https://github.com/a-ludi/dentist. All test assemblies as a resource for future benchmarking are at https://bds.mpi-cbg.de/hillerlab/DENTIST/.
Highlights
• Determination of styrene-butadiene rubber as tire constituent using TED-GC/MS.
• Determination of zinc content as tire constituent using ICP-OES.
• Representative sampling strategy with large-volume mixed samples.
• Tire wear content is decreasing with increasing sampling depth and distance to road.
• Deposited tire wear particles are mainly present in soil fraction <100 μm.
Abstract
Tire wear (TW) constitutes a significant source of microplastic in terrestrial ecosystems. It is known that particles emitted by roads can have an effect up to 100 m into adjacent areas. Here, we apply for the first-time thermal extraction desorption gas chromatography-mass spectrometry (TED-GC/MS) to determine TW in soil samples by detection of thermal decomposition products of styrene-butadiene rubber (SBR), without additional enrichment. Additionally, zinc contents were determined as an elemental marker for TW. Mixed soil samples were taken along three transects along a German motorway in 0.3, 2.0, and 5.0 m distance from the road. Sampling depths were 0–2, 2–5, 5–10, and 10–20 cm. Four fine fractions, 1 000–500, 500–100, 100–50, and <50 μm, were analyzed.
TW contents based on SBR ranged from 155 to 15 898 mg kg−1. TW contents based on zinc were between 413 and 44 812 mg kg−1. Comparison of individual values of SBR and zinc reveals SBR as a more specific marker. Results confirm that most TW ends up in the topsoil within a 2 m distance.
The sampling strategy resulted in representative data for a larger area. Standard deviations of quadruple TED-GC/MS determination of SBR were <10% for all grain size fractions. TED-GC/MS is a suitable analytical tool for determining TW in soil samples without the use of toxic chemicals, enrichment, or special sample preparation.
Mitglieder der ubiquitär verbreiteten Cryptochrom-Photolyase-Familie sind Blaulicht-absorbierende Flavoproteine mit hoher Sequenzhomologie aber diversen Funktionen. Photolyasen katalysieren die Reparatur UV-Licht-induzierter DNA-Schäden. Cryptochrome (CRYs) wirken als lichtunabhängige Transkriptionsrepressoren innerhalb des Kern-Oszillators der circadianen Uhr oder als primäre Photorezeptoren zur Synchronisation dieser mit dem äußeren Tag-Nacht-Rhythmus und steuern durch Regulation der Genexpression Wachstum und Entwicklung. Gemeinsames Strukturmerkmal aller CPF-Vertreter ist die Photolyase- homologe Region (PHR), die das Chromophor Flavinadenindinukleotid (FAD) bindet, das lichtabhängig zwischen den Redoxformen oxidiert (FADox), semireduziert (FAD●- bzw. FADH●) und vollreduziert (FADH-) wechseln kann und damit die CRY-Konformation und -Aktivität beeinflusst. Unterscheidungsmerkmale sind die spezifische C-terminale Erweiterung (CTE) sowie die Komposition der FAD-Bindetasche, die unterschiedliche FAD-Redoxformen stabilisiert. Die Mechanismen der CRY-Photosignaltransduktion sind nicht völlig erforscht.
CryP ist eines von vier CRYs in der Diatomee Phaeodactylum tricornutum und gehört zur bislang nicht charakterisierten Gruppe pflanzenähnlicher CRYs. In vorhergehenden Untersuchungen wurde für CryP eine nukleare Lokalisation und damit verbunden eine blaulicht- sowie dunkelabhängige Regulation der Transkription unterschiedlichster Gene gezeigt. Zudem reguliert CryP das Proteinlevel photosynthetischer Lichtsammelkomplexe. CryP interagiert mit bisher nicht charakterisierten Proteinen aus dem Bereich DNA und Regulation sowie Ribosomen und Translation. Heterolog exprimiertes und isoliertes CryP stabilisiert das Neutralradikal FADH● und das Antennenchromophor Methenyltetrahydrofolat (MTHF).
In vorliegender Dissertation wurde die Bedeutung des FAD-Redoxzustands und der C-terminalen Proteindomäne für Strukturänderungen hinsichtlich der Oligomerisierung und Konformation sowie für das CryP-Interaktionsverhalten untersucht. Hierzu wurden rekombinante CryP-Varianten heterolog isoliert, die Mutationen in für die FAD-Reduzierbarkeit entscheidenden Aminosäuren oder eine Deletion der CTE tragen.
Die Analyse der CryP-Oligomerisierungsstufe und Konformation erfolgte mittels Ko-Präzipitation, nativen und zweidimensionalen PAGEs sowie partieller Proteolyse. Dabei wurde heterolog isoliertes CryP in seinen drei Redoxformen oxidiert (mit FADox), semireduziert (mit FADH●) und vollreduziert (mit FADH-) sowie das um die CTE-verkürzte CryP-PHR verglichen. Für CryP wurde eine redoxunabhängige, PHR-vermittelte Di- und Tetramerisierung über elektrostatische Wechselwirkung der Monomere beobachtet. Die CTE bindet spezifisch und redoxunabhängig an die PHR in einem Bereich um die FAD-Bindetasche. Dies schließt eine großräumige Konformationsänderung zwischen PHR und CTE infolge einer FAD-Photoreduktion wie für pflanzliche und viele tierische CRYs als Aktivierungsmechanismus für CryP aus.
Interaktionsstudien mittels zweidimensionaler PAGE gaben Aufschluss über unterschiedliche Bindeverhalten der beiden betrachteten Interaktionspartner an CryP. Sowohl BolA, ein potentieller redoxregulierter Transkriptionsfaktor, als auch ID42612 mit unbekannter Funktion interagieren mit CryP unabhängig von der FAD-Redoxform. Dabei bindet BolA an die CTE des CryP-Dimers und -Monomers, während ID42612 einen Komplex mit dem CryP-Dimer bildet.
Mittels in vitro Absorptions- und Fluoreszenzspektroskopie wurde die FAD-Redoxchemie von CryP und CryP-PHR verglichen. Die beiden Varianten unterscheiden sich in der FAD-Photoreduzierbarkeit und -Oxidationskinetik. Das Volllängenprotein CryP kann ohne externes Reduktionsmittel zum semireduzierten FADH● phototreduziert werden, das im Gegensatz zu bekannten CRYs über Tage im Dunkeln stabil gegen aerobe Oxidation ist. Eine Belichtung mit Reduktionsmittel führt zur Bildung des vollreduzierten FADH-, das innerhalb von Minuten zu FADH● rückoxidiert. Das um die CTE verkürzte CryP-PHR kann nur mit externem Reduktionsmittel zu FADH● photoreduziert werden, der vollreduzierte Zustand wird nie erreicht. Die Stabilisierung von FADH● gegen aerobe Oxidation im CryP-Holoprotein ist vergleichbar zur FAD-Redoxchemie von Photolyasen. Verglichen mit sonstigen charakterisierten CRYs ist die Wichtigkeit der CTE für eine effiziente FAD-Photoreduktion und FADH●-Stabilisierung eine CryP-spezifische Charakteristik.
Neben der CTE trägt die zu FAD-N5 proximal gelegene Position zur FADH●-Stabilisierung bei, wie Absorptionsmessungen an CryP_N417C zeigten. CryP weist mit Asparagin die gleiche Konservierung an dieser Position wie Photolyasen auf und unterscheidet sich damit ebenfalls von klassischen CRYs.
Analysen zur cryp-Transkription mittels qRT-PCR zeigten eine rhythmische Expression mit maximalen Transkriptmengen in der Nacht und eine rasche photoinduzierte Herunterregulation der Transkription...
Mitochondria are dynamic eukaryotic organelles involved in a variety of essential cellular processes including the generation of adenosine triphosphate (ATP) and reactive oxygen species as well as in the control of apoptosis and autophagy. Impairments of mitochondrial functions lead to aging and disease. Previous work with the ascomycete Podospora anserina demonstrated that mitochondrial morphotype as well as mitochondrial ultrastructure change during aging. The latter goes along with an age-dependent reorganization of the inner mitochondrial membrane leading to a change from lamellar cristae to vesicular structures. Particularly from studies with yeast, it is known that besides the F1Fo-ATP-synthase and the phospholipid cardiolipin also the “mitochondrial contact site and cristae organizing system” (MICOS) complex, existing of the Mic60- and Mic10-subcomplex, is essential for proper cristae formation. In the present study, we aimed to understand the mechanistic basis of age-related changes in the mitochondrial ultrastructure. We observed that MICOS subunits are coregulated at the posttranscriptional level. This regulation partially depends on the mitochondrial iAAA-protease PaIAP. Most surprisingly, we made the counterintuitive observation that, despite the loss of lamellar cristae and of mitochondrial impairments, the ablation of MICOS subunits (except for PaMIC12) leads to a pronounced lifespan extension. Moreover, simultaneous ablation of subunits of both MICOS subcomplexes synergistically increases lifespan, providing formal genetic evidence that both subcomplexes affect lifespan by different and at least partially independent pathways. At the molecular level, we found that ablation of Mic10-subcomplex components leads to a mitohormesis-induced lifespan extension, while lifespan extension of Mic60-subcomplex mutants seems to be controlled by pathways involved in the control of phospholipid homeostasis. Overall, our data demonstrate that both MICOS subcomplexes have different functions and play distinct roles in the aging process of P. anserina.
Diurnal and nocturnal behaviour of cheetahs (Acinonyx jubatus) and lions (Panthera leo) in zoos
(2022)
Mammals are constantly exposed to exogenous and endogenous influences that affect their behaviour and daily activity. Light and temperature, as well as anthropogenic factors such as husbandry routines, visitors, and feeding schedules are potential influences on animals in zoological gardens. In order to investigate the effects of some of these factors on animal behaviour, observational studies based on the analyses of activity budgets can be used. In this study, the daily and nightly activity budgets of six lions (Panthera leo) and five cheetahs (Acinonyx jubatus) from four EAZA institutions were investigated. Focused on the influencing factor light and feeding, we analysed these activity budgets descriptively. Behaviour was recorded and analysed during the winter months over an observation period of 14 days and 14 nights using infrared-sensitive cameras. Our results show that lions and cheetahs exhibit activity peaks at crepuscular and feeding times, regardless of husbandry. Thus, lions in captivity shift nocturnal behaviour familiar from the wild to crepuscular and diurnal times. In cheetahs, in contrast, captive and wild individuals show similar 24 h behavioural rhythms. The resting behaviour of both species is more pronounced at night, with cheetahs having a shorter overall sleep duration than lions. This study describes the results of the examined animals and is not predictive. Nevertheless, the results of this study make an important contribution to gaining knowledge about possible factors influencing the behaviour of lions and cheetahs in zoos and offer implications that could be useful for improving husbandry and management.
Oaks may contribute to the stabilization of European forests under climate change. We utilized two common gardens established in contrasting growth regimes, in Greece (Olympiada) and Germany (Schwanheim), to compare the diurnal photosynthetic performance of a Greek and an Italian provenance of two Mediterranean oaks (Quercus pubescens and Q. frainetto) during the 2019 growing season. Although the higher radiation in the southern common garden led to a strong midday depression of chlorophyll a fluorescence parameters (maximum quantum efficiency of PSII, performance index on absorption basis), comparable light-saturated net photosynthetic rates were achieved in both study areas. Moreover, both species and provenances exhibited analogous responses. Q. pubescens had enhanced chlorophyll a fluorescence traits but similar photosynthetic rates compared to Q. frainetto, whereas the provenances did not differ. These findings indicate the high photosynthetic efficiency of both oaks under the current climate in Central Europe and their suitability for assisted migration schemes.
Many metabolic pathways of eukaryotes are carried out in form of interconnected pathways, which take place in organelles. The organelle membrane separates the reaction compartments from each other, making it a key feature of organelle existence in the cell. To maintain cellular homeostasis, organelle positioning in and transport through the cell as well as organelle interaction are important for the organisms. In plants, organellar movement of peroxisomes, Golgi stacks and mitochondria was shown to be mediated by the actin-myosin machinery. The molecular mechanisms are not elucidated, but working models comprise classical movement mechanisms of motor proteins pulling their cargo on cytoskeletal filaments. In contrast, many mechanisms of chloroplasts movement, which are regulated by blue and red light, are deciphered but follow a different molecular mechanism. Plastidal relatives of the chloroplast have long been disregarded by scientific research but carry out important metabolic reactions to maintain cellular homeostasis. The cellular transport and movement mechanisms of root plastids have not been described in detail until now. Additionally, all plastid subspecies can form tubular structures, called stromules. Those are thought to be involved in the organelle communication and metabolite exchange. Since they are very mobile structures, they influence the organellar dynamic of plastids. This work aimed for an in-detail description of the cellular movements of root plastids in the plant Arabidopsis thaliana to elucidate underlying mechanisms of their movement. Additionally, the dynamics of root plastid stromules were investigated, led by the questions, if and how stromules are involved in the mediation of plastidal movement and their overall dynamics. Plastidal movement in Arabidopsis thaliana was captured using light sheet-based fluorescence microscopy. 4D image data was automatically analyzed using the program Arivis Vision 4D with subsequent manual correction. Additionally to the 4D approach, a manual 3D analysis of plastid and stromule dynamics was performed. The results of the semiautomated analysis displayed heterologous distribution of the plastidal movement. Using a combination of the vector length of each motion event and the angle in relation to previous motion vectors, the proportions of different movement patterns were determined. Main fractions of the data showed undirected motion of plastids, whereas small proportions displayed directed movement with speed up to 8.5 µm/sec. Directed motion was shown to be carried out on defined routes in the cell. Salt stress did not affect plastidal motion, whereas drought stress lead to its reduction. Sucrose depletion led to a drastic decrease of plastidal movement. Additionally, stromule dynamics were investigated using the acquired image data. Stromules were observed in high frequency mainly at stationary plastids giving them the opportunity of dynamic interaction in their cellular surrounding. Stromules reached lengths of up to 60 µm. Additionally, they displayed a variety of movement patterns that contributed greatly to the overall plastid dynamics. Stromule related motion events were captured reaching up to 3.2 µm/sec. Similar to determined plastid dynamics, stromule motions were reduced during drought stress and sucrose depletion, but also were negatively influenced by salt stress. Those results strongly favor an actin-myosin mediated movement machinery mediating the plastidal and stromule movement. This stands in contrast to previous results describing the movement mechanisms of light induced chloroplast movement.
In an additional approach, the molecular mechanisms underlying stromule formation were analyzed. Previous results describe that stromule formation can be induced at isolated chloroplasts of the plant Nicotiana benthamiana by mixing it with concentrated cell extract. During this work, a variation of the described assay was established using the plant Pisum sativum. It was shown that an unknown protein factor presumably undergoing protein-lipid interaction is responsible for in vitro stromule formation. Using a combination of sucrose gradient centrifugation and anion exchange chromatography, the desired factor could be enriched, while the majority of unwanted proteins could be reduced drastically. A following LC-MS analysis revealed a selection of proteins with membrane interaction- and unknown functions that might be involved in in vitro stromule formation.
The mammalian frontal and auditory cortices are important for vocal behaviour. Here, using local field potential recordings, we demonstrate for the first time that the timing and spatial pattern of oscillations in the fronto-auditory cortical network of vocalizing bats (Carollia perspicillata) predict the purpose of vocalization: echolocation or communication. Transfer entropy analyses revealed predominantly top-down (frontal-to-auditory cortex) information flow during spontaneous activity and pre-vocal periods. The dynamics of information flow depended on the behavioural role of the vocalization and on the timing relative to vocal onset. Remarkably, we observed the emergence of predominantly bottom-up (auditory-to-frontal cortex) information transfer patterns specific echolocation production, leading to self-directed acoustic feedback. Electrical stimulation of frontal areas selectively enhanced responses to echolocation sounds in auditory cortex. These results reveal unique changes in information flow across sensory and frontal cortices, potentially driven by the purpose of the vocalization in a highly vocal mammalian model.
The mammalian frontal and auditory cortices are important for vocal behavior. Here, using local-field potential recordings, we demonstrate that the timing and spatial patterns of oscillations in the fronto-auditory network of vocalizing bats (Carollia perspicillata) predict the purpose of vocalization: echolocation or communication. Transfer entropy analyses revealed predominant top-down (frontal-to-auditory cortex) information flow during spontaneous activity and pre-vocal periods. The dynamics of information flow depend on the behavioral role of the vocalization and on the timing relative to vocal onset. We observed the emergence of predominant bottom-up (auditory-to-frontal) information transfer during the post-vocal period specific to echolocation pulse emission, leading to self-directed acoustic feedback. Electrical stimulation of frontal areas selectively enhanced responses to sounds in auditory cortex. These results reveal unique changes in information flow across sensory and frontal cortices, potentially driven by the purpose of the vocalization in a highly vocal mammalian model.
Plastic pollution is a pervasive problem. In the environment, both the physical and chemical aspects of the material contribute to pollution. For instance, discarded plastic is useless waste that is fragmented upon degradation and so-called microplastics <5 mm are formed. Besides, the chemicals added into plastics are usually customized for specific functions, but these can easily transfer from the polymer into an ambient medium. This work examined both of these aspects. Moreover, the question of whether ecotoxicological effects are more likely to appear because of the microparticle properties or the chemicals transferring from the microplastics was addressed. A special focus was laid on the UV-weathering-induced chemical release.
First, conventional and biodegradable plastics made from fossil and bio-based resources were chosen. The different materials (pre-production and recycled pellets as well as final products)were weathered and their leachates evaluated in vitro. The leachates were analyzed with nontarget screening in order to measure the number of transferred chemicals. Plastics identified as toxic were subjected to further investigations in vivo. A biodegradable shampoo bottle was processed to microplastics and the particles’ physical and chemical properties were assessed with the freshwater worm Lumbriculus variegatus. Here, commonly used endpoints such as mortality, reproduction and weight were tested via different exposure routes. Moreover, the freshwater shrimp Neocaridina palmata was exposed to microplastic beads and fragments to clarify if the shape of the particles affects the ingestion and egestion, respectively. Thereafter, two materials that displayed the strongest toxic responses in vitro within the first study were weathered and leached. Finally, the shrimps were exposed to the leachates and the locomotor behavior was used as an ecologically relevant but less frequently studied endpoint.
The results of the studies highlight that plastics are chemically complex mixtures, containing a wide range of chemicals in terms of the number and functionality. These chemicals induced oxidative stress, baseline toxicity and endocrine activities. This shows that pellets represent a processing state that comprises chemically heterogenous materials. Moreover, it was shown that a degradation initiator is not necessarily relevant to trigger inherent substances to leach out from plastics. Despite this, the UV-weathering resulted in increasingly released chemicals and exacerbated the in vitro toxicities. Even plastics assessed as toxicologically harmless prior to weathering released toxic chemical mixtures once they were weathered. One recycled and all of the biodegradable plastics were toxicologically most concerning. This means that such materials are currently not better than conventional, virgin plastics in terms of their toxicity.
To clarify the source of the microplastic toxicity, L. variegatus was exposed to biodegradable microplastics. The particles were ingested by the worms and adversely affected the examined endpoints. In comparison, microplastics that were depleted from their chemicals via a solvent treatment were less toxic. Kaolin as a natural particle control was evaluated alongside and positively affected the weight of the worms. This emphasizes the ecological relevance of fine-sized matter for the test species. The chemicals extracted from the microplastics induced a 100% mortality. A chemical analysis of the material revealed two ecotoxicologically relevant biocides. The physically-mediated effects of the microplastics seemed to be less of a concern for the worms, which is probably linked to their adaptation to high concentrations of naturally occurring particles in the environment. However, the effects related to the chemicals of plastic cannot be ignored, especially for materials that are claimed to be environmentally friendly.
In the third study, the role of the particle shape in the gut passaging of N. palmata was studied. While the particle size was a determinant factor for the ingestion, the ingestion and egestion of the beads and fragments did not differ, respectively. The shrimps ingested less fragments when food was provided than in the absence of food. As for the worms, the shrimps are known to ingest many naturally occurring particles. Their unselective feeding behavior towards the particle shape could indicate that microplastics as a physical pollutant are negligible for the shrimps. That is why the chemicals of the two most toxic in vitro materials were tested with N. palmata. However, no trend towards elevated or reduced movements of the shrimps was observed, even though the leachates contained baseline toxicants. This shows that the in vitro toxicities of plastics are not necessarily indicative for effects to occur at the in vivo level...
The European Community has set a milestone in the European water policy in 2000: all water directives and policies were united into one comprehensive document – the European Water Framework Directive (EU WFD). The EU WFD requires the monitoring of 45 priority substances, primarily in the water phase, which is not related to a substantial amount of chemicals available on the market worldwide (about 50,000). About 60% of these are human and environmentally toxic. Hence, the currently monitored 45 priority substances are not even close to being sufficient to provide a comprehensive picture of the actual chemical pollution in the aquatic environment.
Furthermore, the EU WFD in its original shape paid less attention to sediments as an important source and sink for chemical contamination. Under stable hydrological conditions, polluted old sediments are covered by less polluted younger sediments preventing erosion of deeper sediment layers and, therefore, the release of particle-bound contaminants. However, urbanization, deforestation, flooding, dredging, riverbed renaturation, and stormwater overflow basin releases can lead to an unpredictable release of particle-bound pollutants. Therefore, in 2008, sediments were added to the EU WFD as a monitoring matrix for substances that tend to accumulate there. As a result, after 18 years of the EU WFD, less than half of all European waterbodies reached a good ecological (40%) and chemical (38%) status.
One of the primary pollution sources in aquatic ecosystems are wastewater treatment plants (WWTPs). Advanced wastewater treatment by ozonation is promising to remove most micropollutants. However, the knowledge about the possible improvement of the receiving waterbody is rare. The latter aspects were the main reasons for the start of the DemO3AC project in 2014. The study area was located in the federal state of North Rhine-Westphalia (Germany). The study area included the Wurm River and its tributary, the Haarbach River. Both waterbodies act as receiving waterbodies for WWTPs. One of them is the Aachen-Soers WWTP (receiving waterbody: Wurm River), upgraded by full stream ozonation as an advanced effluent treatment. Therefore, the extensive investigation program within the DemO3AC project included an investigation of the ecological and chemical status of both receiving waterbodies and the investigation of a possible improvement of the Wurm River after implementing advanced effluent treatment.
The current study was a part of the DemO3AC project and covered the sediment toxicity and a possible impact of the ozonation on aquatic organisms in the receiving waterbody. Time-resolved sampling campaigns allowed investigations under different hydrological conditions, mainly determined by the weather. The first sampling campaign took place in June 2017 during a prolonged dry period with low water flow in the receiving waterbodies. The second sampling campaign was performed exactly one year later (June 2018) after a long rainy period and corresponding high-water levels. Full-stream ozonation at the Aachen-Soers WWTP had been in operation for half a year. Furthermore, a wide range of organic micropollutants was investigated in the effluent of the studied WWTPs to assess a possible hazard emerging from contaminants released into the receiving waterbody.
The study design was developed based on the holistic approach to assessing the ecotoxicological pollution of surface waterbodies. It included the detection of chemical compounds combined with effect-based methods to identify possible drivers of toxicity. The sediment's ecotoxicological assessment included studies on endocrine-disrupting activity, genotoxic and embryotoxic potentials. These endpoints were evaluated using in vitro and in vivo bioassays. In addition, sediments’ chemical profiling was performed using modern analytical chemistry techniques.
The genotoxic potential was investigated using the Ames fluctuation assay with Salmonella typhimurium bacterial strains TA98, TA100, YG1041, and YG1042, sensitive to different classes of compounds, and the Micronucleus assay as a eukaryotic assay with mammalian cells. A unique feature of the present study was the implementation of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay. Moreover, a comprehensive genotoxicity ranking of chemical compounds identified in sediments was used and combined with statistical analysis to identify the drivers of genotoxicity. The results of this study were published in Shuliakevich et al. (2022a) (see also Annex 1), describing the mutagenic potential of all sampling sites, which was primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. In addition, the rainwater overflow basin was identified as a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay to assess the different classes of mutagenic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.
The sediments' endocrine-disruptive activity was investigated using the cell-based reporter gene CALUX® assay. A simultaneous launch of the full-scale effluent ozonation at the Aachen-Soers WWTP was used for investigation of the entrance of the ozonated effluent into the Wurm River and the endocrine-disrupting activity in the water phase. A particular focus of the present study was the unique investigation of PAHs as possible drivers of the endocrine-disrupting activity in sediments of the Wurm River. The results of this study were laid down in the publication by Shuliakevich et al. (2022b) (see also Annex 2), describing variations in endocrine-disrupting activity in the Wurm River under different weather conditions. Briefly, under stable hydrological conditions in June 2017, the estrogenic and the antiandrogenic activities in sediments of the Wurm River were within the range of 0.03-0.1 ng E2 equivalents (eq.)/g dry weight sediment equivalents (dw SEQ) and 3.0-13.9 µg Flu eq./g dw SEQ, respectively. After extensive rain events in June 2018, the sediments' estrogenic and antiandrogenic activities were detected within the range of 0.06-0.2 ng E2 eq./g dw SEQ and 1.7-39.2 µg Flu eq./g de SEQ, respectively. Increased endocrine-disruptive activity (up to 0.2 ng E2 eq./g dw SEQ in ERα- and 39.2 µg Flu eq./g dw SEQ in anti-AR-CALUX® assays) in sediments downstream of the rainwater overflow basin suggested it as a possible source of pollution. A unique result of the second study was finding a positive correlation between measured particle-bound antiandrogenic activity and detected polyaromatic hydrocarbons (PAHs) ...
The toxicity of microplastics on Daphnia magna as key model for freshwater zooplankton is well described. While several studies predict population-level effects based on short-term, individual-level responses, only very few have validated these predictions experimentally. Thus, we exposed D. magna populations to irregular polystyrene microplastics and diatomite as natural particle (both ≤63 µm) over 50 days. We used mixtures of both particle types at fixed particle concentrations (50,000 mL-1) and recorded the overall population density, the size of the individual animals, and resting egg production. Particle exposure adversely affected the population density and structure and induced resting egg production. The terminal population size was 31–42% lower in exposed compared to control populations. Interestingly, mixtures containing diatomite induced stronger effects than microplastics alone highlighting that natural particles are not per se less toxic than microplastics. Our results demonstrate that an exposure to synthetic and natural particles has negative population-level effects on zooplankton. Understanding the mixture toxicity of microplastics and natural particles is important given that aquatic organisms will experience exposure to both. Just as for chemical pollutants, better knowledge of such joint effects is essential to fully understand the environmental risks of complex particle mixtures.
Environmental Implications While microplastics are commonly considered hazardous based on individual-level effects, there is a dearth of information on how they affect populations. Since the latter is key for understanding the environmental impacts of microplastics, we investigated how particle exposures affect the population size and structure of Daphnia magna. In addition, we used mixtures of microplastics and natural particles because neither occurs alone in nature and joint effects can expected in an environmentally realistic scenario. We show that such mixtures adversely affect daphnid populations and highlight that population-level and mixture-toxicity designs are one important step towards more environmental realism in microplastics research.
Patients harboring mutations in the gene DEPDC5 often display variations of neurological diseases including epilepsy, autism spectrum disorders (ASD) and other neuro-architectural alterations. DEPDC5 protein has been identified as an amino acid sensor responsible for negatively regulating the mechanistic target of rapamycin (mTOR), a central regulator in cell growth and cell homeostasis. Often, mutations of the DEPDC5 protein result in mTOR hyperactivity leading to abnormal neuronal phenotypes and the generation of excitatory/inhibitory imbalances in animal models. Complete knockout (KO) of DEPDC5 results in death shortly after birth, while inhibition of mTOR activity recovers postnatal death (Marsan et al. 2016). However, heterozygous DEPDC5-KOs in animals have been variable in their disease phenotypes during adulthood indicating developmental differences between subspecies and early development mechanisms which could be impactful on the outcome of the diseases.
To understand the mechanisms underlying DEPDC5 mutations during early development, a novel primary human neural progenitor cell line extracted from fetal tissue was characterized during proliferation and differentiation. CRISPR-Cas9 induced mutations of the DEPDC5 gene resulted in hyperphosphorylation of mTOR signaling processes and rapid expansion of the neuronal population during differentiation. Analysis of transcriptome data identified deregulation amongst p53 signaling, ribosome biogenesis, nucleotide and lipid synthesis as well as protein degradation pathways due to loss of DEPDC5. Disease gene datasets identified a correlation between Tuberous Sclerosis mutations as being more closely associated with DEPDC5 mutations while also finding overlap with some ASD and epilepsy genes. By using the mTOR inhibitor rapamycin, a substantial amount of the deregulated gene network was recovered while also reversing rapid neuronal differentiation caused by loss of DEPDC5. Though we saw increased dendritic arborization and subsequent decreases in dendrite lengths and soma sizes, rapamycin failed to recover these effects suggesting mTOR independent processes produced by DEPDC5-KO. This study provides new insights on the relationship between mutations in DEPDC5 and the functional, genomic and deregulatory networks it intertwines in humans and highlights that the DEPDC5 associated pathomechanisms are not fully related to mTOR hyperactivation, but include independent processes. This also sheds light on the question why rapamycin treatment only partially restores DEPDC5 related phenotypes and gives insight on treatments for DEPDC5 patients.
Energy-conserving dimethyl sulfoxide reduction in the acetogenic bacterium Moorella thermoacetica
(2022)
Moorella thermoacetica is one of the well-studied thermophilic acetogenic bacteria. It grows by oxidation of organic substrates, CO or H2 coupled to CO2 reduction to acetate. Here, we describe that M. thermoacetica can also use dimethyl sulfoxide as terminal electron acceptor. Growth of M. thermoacetica on glucose or H2 + CO2 was stimulated by dimethyl sulfoxide (DMSO). Membranes showed a DMSO reductase activity, that was induced by growing cells in presence of DMSO. The enzyme used reduced anthraquinone-2,6-disulfonate, benzyl- and methyl viologen as electron donor, but not NAD(P)H. Activity was highest at pH 5 and 60°C, the Km for DMSO was 2.4 mM. Potential DMSO reductase subunits were identified by peptide mass fingerprinting; they are encoded in a genomic region that contains three potential dmsA genes, three dmsB genes and one dmsC gene. Transcriptome analysis revealed that two different dmsAB gene clusters were induced in the presence of DMSO. The function of these two and their predicted biochemical features are discussed. In addition, the data are in line with the hypothesis that M. thermoacetica can use DMSO alongside CO2 as electron acceptor and DMSO reduction is catalysed by an energy-conserving, membrane-bound electron transport chain with DMSO as final electron acceptor.
Specialized surveillance mechanisms are essential to maintain the genetic integrity of germ cells, which are not only the source of all somatic cells but also of the germ cells of the next generation. DNA damage and chromosomal aberrations are, therefore, not only detrimental for the individual but affect the entire species. In oocytes, the surveillance of the structural integrity of the DNA is maintained by the p53 family member TAp63α. The TAp63α protein is highly expressed in a closed and inactive state and gets activated to the open conformation upon the detection of DNA damage, in particular DNA double-strand breaks. To understand the cellular response to DNA damage that leads to the TAp63α triggered oocyte death we have investigated the RNA transcriptome of oocytes following irradiation at different time points. The analysis shows enhanced expression of pro-apoptotic and typical p53 target genes such as CDKn1a or Mdm2, concomitant with the activation of TAp63α. While DNA repair genes are not upregulated, inflammation-related genes become transcribed when apoptosis is initiated by activation of STAT transcription factors. Furthermore, comparison with the transcriptional profile of the ΔNp63α isoform from other studies shows only a minimal overlap, suggesting distinct regulatory programs of different p63 isoforms.
In recent decades, zoos have been increasingly transformed into education centers with the goal of raising awareness about environmental issues and providing environmental education. Probably the simplest and most widespread environmental education program in the zoo is the guided tour. This study therefore aims to test whether a one hour zoo tour has an influence on the participants’ connection to nature and attitude towards species conservation. For this purpose, 269 people who had voluntarily registered for a zoo tour were surveyed before and after the tour. In addition to the regular zoo tour, special themed tours and tours with animal feedings were included. The results show a positive increase in connection to nature and a strengthening of positive attitudes towards species conservation for all tour types. For nature connectedness, in particular, people with an initial high connection to nature benefitted from the special themed tours and the tours, including animal feedings. For attitudes towards species conservation, no difference was found between the tour types. The results prove the positive influence of a very simple environmental education program, even for people with a preexisting high level of connection to nature and positive attitude towards species conservation.
Several clinically used drugs are derived from microorganisms that often produce them via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and connect individual amino acids in an assembly line fashion. Since NRPS are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of several different peptides including linear, cyclic and further modified derivatives. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain important in natural NRPS evolution. From this an evolutionary-inspired eXchange Unit between T domains (XUT) approach was developed, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as was shown for the specific production of a proteasome inhibitor, designed and assembled from five different NRPS fragments.
Many clinically used drugs are derived from or inspired by bacterial natural products that often are biosynthesised via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and join individual amino acids in an assembly line fashion. Since NRPS are not limited to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of complex peptides including linear, cyclic and further modified natural product analogues, e.g. to optimise natural product leads. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain that is important for natural NRPS evolution. From this, an evolution-inspired eXchange Unit between T domains (XUT) approach was developed which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.
Highlights
• Enables immunostaining and visualization of epitopes deep within brain slices
• Utilizes expansion microscopy to increase imaging resolution
• Optimized for brain organotypic slice cultures and tested in acute brain slices
• Analysis workflow for protein distribution (surface vs. intracellular pool) using Imaris
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Summary
Assessing protein distribution with super-resolution in tissue is often complicated and restrictive. Here, we describe a protocol for immunostaining and expansion microscopy imaging of mouse brain organotypic slice cultures. We detail an Imaris analysis workflow to analyze the surface vs intracellular distribution of AMPA receptors at super-resolution during homeostatic plasticity. We have optimized the protocol for brain organotypic slice culture and tested in acute brain slices. This protocol is suitable to study protein distribution under multiple plasticity paradigms.
Exploring the power of moth samples to reveal community patterns along shallow ecological gradients
(2022)
1. Analysing the effects of environmental variation on species assemblages is a key topic in community ecology. However, the outcome may strongly depend on the focal species group. Moths have often been used as the target in ecological studies due to their fast response to environmental change. Yet, some moth subgroups might be more sensitive than others to reflect environmental differences, depending on their functional and physiological characteristics.
2. We investigated which moth subsets are especially suitable to mirror responses to subtle variation in vegetation. We analysed the susceptibility of different subsets to local weather conditions and inter-annual fluctuations. Finally, we checked for the importance of including abundance information. We analysed moth communities (392 species, 23.870 individuals) at 60 sites within two Mediterranean forest reserves and investigated relationships between community composition and environment of (1) all moths (with and without taking abundances into account), and of subsets comprising only (2) small-sized species, (3) host-plant specialists, (4) moss, lichen and detritus feeding species, (5) ‘microlepidoptera’, (6) ‘macro-moths’ and (7) random subsets of 50, 100 and 200 species.
3. Incidence data performed similarly to abundance data in matrix regression models. Host plant specialists responded especially sensitive to small-scaled variation in vegetation composition. Macro-moth samples in contrast were highly prone to local weather conditions and to inter-annual abundance fluctuations. Accordingly, a focus on host-specialists and micro-moths is the best way to analyse relationships between shallow environmental gradients and insect communities.
Background: The causative agent of Chagas disease, Trypanosoma cruzi, and its nonpathogenic relative, Trypanosoma rangeli, are transmitted by haematophagous triatomines and undergo a crucial ontogenetic phase in the insect’s intestine. In the process, the parasites interfere with the host immune system as well as the microbiome present in the digestive tract potentially establishing an environment advantageous for development. However, the coherent interactions between host, pathogen and microbiota have not yet been elucidated in detail. We applied a metagenome shotgun sequencing approach to study the alterations in the microbiota of Rhodnius prolixus, a major vector of Chagas disease, after exposure to T. cruzi and T. rangeli focusing also on the functional capacities present in the intestinal microbiome of the insect.
Results: The intestinal microbiota of R. prolixus was dominated by the bacterial orders Enterobacterales, Corynebacteriales, Lactobacillales, Clostridiales and Chlamydiales, whereas the latter conceivably originated from the blood used for pathogen exposure. The anterior and posterior midgut samples of the exposed insects showed a reduced overall number of organisms compared to the control group. However, we also found enriched bacterial groups after exposure to T. cruzi as well as T rangeli. While the relative abundance of Enterobacterales and Corynebacteriales decreased considerably, the Lactobacillales, mainly composed of the genus Enterococcus, developed as the most abundant taxonomic group. This applies in particular to vectors challenged with T. rangeli and at early timepoints after exposure to vectors challenged with T. cruzi. Furthermore, we were able to reconstruct four metagenome-assembled genomes from the intestinal samples and elucidate their unique metabolic functionalities within the triatomine microbiome, including the genome of a recently described insect symbiont, Candidatus Symbiopectobacterium, and the secondary metabolites producing bacteria Kocuria spp.
Conclusions: Our results facilitate a deeper understanding of the processes that take place in the intestinal tract of triatomine vectors during colonisation by trypanosomal parasites and highlight the influential aspects of pathogen-microbiota interactions. In particular, the mostly unexplored metabolic capacities of the insect vector’s microbiome are clearer, underlining its role in the transmission of Chagas disease.
Motivation Expert curation to differentiate between functionally diverged homologs and those that may still share a similar function routinely relies on the visual interpretation of domain architecture changes. However, the size of contemporary data sets integrating homologs from hundreds to thousands of species calls for alternate solutions. Scoring schemes to evaluate domain architecture similarities can help to automatize this procedure, in principle. But existing schemes are often too simplistic in the similarity assessment, many require an a-priori resolution of overlapping domain annotations, and those that allow overlaps to extend the set of annotations sources cannot account for redundant annotations. As a consequence, the gap between the automated similarity scoring and the similarity assessment based on visual architecture comparison is still too wide to make the integration of both approaches meaningful.
Results Here, we present FAS, a scoring system for the comparison of multi-layered feature architectures integrating information from a broad spectrum of annotation sources. Feature architectures are represented as directed acyclic graphs, and redundancies are resolved in the course of comparison using a score maximization algorithm. A benchmark using more than 10,000 human-yeast ortholog pairs reveals that FAS consistently outperforms existing scoring schemes. Using three examples, we show how automated architecture similarity assessments can be routinely applied in the benchmarking of orthology assignment software, in the identification of functionally diverged orthologs, and in the identification of entries in protein collections that most likely stem from a faulty gene prediction.
Regulatory required, classical toxicity studies for environmental hazard assessment are costly, time consuming, and often lack mechanistic insights about the toxic mode of action induced through a compound. In addition, classical toxicological non-human animal tests raise serious ethical concerns and are not well suited for high throughput screening approaches. Molecular biomarker-based screenings could be a suitable alternative for identifying particular hazardous effects (e.g. endocrine disruption, developmental neurotoxicity) in non-target organisms at the molecular level. This, however, requires a better mechanistic understanding of different toxic modes of action (MoA) to describe characteristic molecular key events and respective markers.
Ecotoxicgenomics, which uses modern day omic technologies and systems biology approaches to study toxicological responses at the molecular level, are a promising new way for elucidating
the processes through which chemicals cause adverse effects in environmental organisms. In this context, this PhD study was designated to investigate and describe MoA-characteristic
ecotoxicogenomic signatures in three ecotoxicologically important aquatic model organisms of different trophic levels (Danio rerio, Daphnia magna and Lemna minor).
Applying non-target transcriptomic and proteomic methodologies post chemical exposure, the aim was to identify robust functional profiles and reliable biomarker candidates with potential
predictive properties to allow for a differentiation among different MoA in these organisms. For the sublethal exposure studies in the zebrafish embryo model (96 hpf), the acute fish embryo toxicity test guideline (OECD 236) was used as conceptual framework. As different test compounds with known MoA, the thyroid hormone 3,3′,5-triiodothyronine (T3) and the thyrostatic 6-propyl-2-thiouracil (6-PTU), as well as six nerve- and muscle-targeting insecticides (abamectin, carbaryl, chlorpyrifos, fipronil, imidacloprid and methoxychlor) were evaluated. Furthermore, a novel sublethal immune challenge assay in early zebrafish embryos (48 hpf) was evaluated for its potential to assess immuno-suppressive effects at the gene expression level. Therefore, toxicogenomic profiles after an immune response inducing stimulus with and without prior clobetasol propionate (CP) treatment were compared. For the aquatic invertebrate D. magna, the study was performed with previously determined low effect concentrations (EC5 & EC20) of fipronil and imidacloprid according to the acute immobilization test in water flea (OECD 202). The aim was to compare toxicogenomic signatures of the GABA-gated chloride channel blocker (fipronil) and the nAChR agonist (imidacloprid). With similar low effect concentrations, a shortened 3 day version of the growth inhibition test with L. minor (OECD 221) was conducted to find molecular profiles differentiating between photosynthesis and HMG-CoA reductase inhibitory effects. Here, the biological interpretation of the molecular stress response profiles in L. minor due to the lack of functional annotation of the reference genome was particularly challenging. Therefore, an annotation workflow was developed based on protein sequence homology predicted from the genomic reference sequences.
With this PhD work, it was shown how transcriptomic, proteomic and computational systems biology approaches can be coupled with aquatic toxicological tests, to gain important mechanistic insights into adverse effects at the molecular level. In general, for the different investigated adverse effects for the different organisms, biomarker candidates were identified, which describe a potential functional link between impaired gene expressions and previously reported apical effects. For the assessed chemicals in the zebrafish embryo model, biomarker candidates for thyroid disruption as well as developmental toxicity targeting the heart and central nervous system were described. The biomarkers derived from nerve- and muscletargeting insecticides were associated with three major affected processes: (1) cardiac muscle cell development and functioning, (2) oxygen transport and hypoxic stress and (3) neuronal development and plasticity. To our knowledge, this is the first study linking neurotoxic insecticide exposure and affected expression of important regulatory genes for heart muscle (tcap, actc2) and forebrain (npas4a) development in a vertebrate model. The proposed immunosuppression assay found CP to affect innate immune induction by attenuating the response of genes involved in antigen processing, TLR signalling, NF-КB signalling, and complement activation ...
Forest wildflowers bloom earlier as Europe warms: lessons from herbaria and spatial modelling
(2022)
Today plants often flower earlier due to climate warming. Herbarium specimens are excellent witnesses of such long-term changes. However, the magnitude of phenological shifts may vary geographically, and the data are often clustered. Therefore, large-scale analyses of herbarium data are prone to pseudoreplication and geographical biases.
We studied over 6000 herbarium specimens of 20 spring-flowering forest understory herbs from Europe to understand how their phenology had changed during the last century. We estimated phenology trends with or without taking spatial autocorrelation into account.
On average plants now flowered over 6 d earlier than at the beginning of the last century. These changes were strongly associated with warmer spring temperatures. Flowering time advanced 3.6 d per 1°C warming. Spatial modelling showed that, in some parts of Europe, plants flowered earlier or later than expected. Without accounting for this, the estimates of phenological shifts were biased and model fits were poor.
Our study indicates that forest wildflowers in Europe strongly advanced their phenology in response to climate change. However, these phenological shifts differ geographically. This shows that it is crucial to combine the analysis of herbarium data with spatial modelling when testing for long-term phenology trends across large spatial scales.
Background: In times of global warming there is an urgent need to replace fossil fuel-based energy vectors by less carbon dioxide (CO2)-emitting alternatives. One attractive option is the use of molecular hydrogen (H2) since its combustion emits water (H2O) and not CO2. Therefore, H2 is regarded as a non-polluting fuel. The ways to produce H2 can be diverse, but steam reformation of conventional fossil fuel sources is still the main producer of H2 gas up to date. Biohydrogen production via microbes could be an alternative, environmentally friendly and renewable way of future H2 production, especially when the flexible and inexpensive C1 compound formate is used as substrate.
Results: In this study, the versatile compound formate was used as substrate to drive H2 production by whole cells of the thermophilic acetogenic bacterium Thermoanaerobacter kivui which harbors a highly active hydrogen-dependent CO2 reductase (HDCR) to oxidize formate to H2 and CO2 and vice versa. Under optimized reaction conditions, T. kivui cells demonstrated the highest H2 production rates (qH2 = 685 mmol g−1 h−1) which were so far reported in the literature for wild-type organisms. Additionally, high yields (Y(H2/formate)) of 0.86 mol mol−1 and a hydrogen evolution rate (HER) of 999 mmol L−1 h−1 were observed. Finally, stirred-tank bioreactor experiments demonstrated the upscaling feasibility of the applied whole cell system and indicated the importance of pH control for the reaction of formate-driven H2 production.
Conclusions: The thermophilic acetogenic bacterium T. kivui is an efficient biocatalyst for the oxidation of formate to H2 (and CO2). The existing genetic tool box of acetogenic bacteria bears further potential to optimize biohydrogen production in future and to contribute to a future sustainable formate/H2 bio-economy.
Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
Entoloma (Agaricales, Basidiomycota) is a species-rich genus with approximately 2000 species known worldwide. In Central America, however, information about the species of this genus is sparse, despite the generally high biodiversity in this region. Recently, 124 specimens of Entoloma were collected in Panama, Chiriquí Province. In the present publication, the morphology of 20 species represented by more than one specimen is described and depicted with photographs, line drawings, and scanning electron micrographs. Molecular phylograms based on ITS or concatenated ITS and partial nc LSU rDNA sequences are provided. The taxonomic status of these species is evaluated and 17 species of Entoloma are described as new to science. Only one species could be assigned to an already known species, viz. Entoloma belouvense. Nolanea albertinae, described from Brazil, appeared similar and is combined in E. belouvense on varietal level. The identifications of two further species are uncertain. At least 30 other species, including potentially new species, cannot formally be described due to insufficient material. A preliminary key to the species of the genus Entoloma in Panama is provided. The spatial shape of the polyhedroid basidiospores of Entoloma spp. is discussed based on literature and the micrographs generated for the present study. Our re-evaluations indicate that the type of polyhedroid basidiospore and the structure of its base are not reliable as diagnostic characters for the delimitation of subgenera in Entoloma.
Forest species are affected by macroclimate, however, the microclimatic variability can be more extreme and change through climate change. Fungal fruiting community composition was affected by microclimatic differences. Here we ask whether differences in the fruiting community can be explained by morphological traits of the fruit body, which may help endure harsh conditions. We used a dead wood experiment and macrofungal fruit body size, color, and toughness. We exposed logs of two host tree species under closed and experimentally opened forest canopies in a random-block design for four years and identified all visible fruit bodies of two fungal lineages (Basidio- and Ascomycota). We found a consistently higher proportion of tough-fleshed species in harsher microclimates under open canopies. Although significant, responses of community fruit body size and color lightness were inconsistent across lineages. We suggest the toughness-protection hypothesis, stating that tough-fleshed fruit bodies protect from microclimatic extremes by reducing dehydration. Our study suggests that the predicted increase of microclimatic harshness with climate change will likely decrease the presence of soft-fleshed fruit bodies. Whether harsh microclimates also affect the mycelium of macrofungi with different fruit body morphology would complement our findings and increase predictability under climate change.
Vampire bats are the only mammals that feed exclusively on blood. To uncover genomic changes associated with this dietary adaptation, we generated a haplotype-resolved genome of the common vampire bat and screened 27 bat species for genes that were specifically lost in the vampire bat lineage. We found previously unknown gene losses that relate to reduced insulin secretion (FFAR1 and SLC30A8), limited glycogen stores (PPP1R3E), and a unique gastric physiology (CTSE). Other gene losses likely reflect the biased nutrient composition (ERN2 and CTRL) and distinct pathogen diversity of blood (RNASE7) and predict the complete lack of cone-based vision in these strictly nocturnal bats (PDE6H and PDE6C). Notably, REP15 loss likely helped vampire bats adapt to high dietary iron levels by enhancing iron excretion, and the loss of CYP39A1 could have contributed to their exceptional cognitive abilities. These findings enhance our understanding of vampire bat biology and the genomic underpinnings of adaptations to blood feeding.
Abstract
Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being lower in colder regions and in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylated regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.
Author summary
Variation within species is an important level of biodiversity, and it is key for future adaptation. Besides variation in DNA sequence, plants also harbour heritable variation in DNA methylation, and we want to understand the evolutionary significance of this epigenetic variation, in particular how much of it is under genetic control, and how much is associated with the environment. We addressed these questions in a high-resolution molecular analysis of 207 lines of the common plant field pennycress (Thlaspi arvense), which we collected across Europe, propagated under standardized conditions, and sequenced for their genetic and epigenetic variation. We found large geographic variation in DNA methylation, associated with both DNA sequence and climate of origin. Genetic variation was generally the stronger predictor of DNA methylation variation, but the strength of environmental association varied between different sequence contexts. Climate-of-origin was the strongest predictor in about one third of the differentially methylated regions in the CHH context, which suggests that epigenetic variation may play a role in the short-term climate adaptation of pennycress. As pennycress is currently being domesticated as a new biofuel and winter cover crop, our results may be relevant also for agriculture, particularly in changing environments.
Abstract
Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being higher in warmer regions and lower in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylation regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.
Author Summary: Variation within species is an important level of biodiversity, and it is key for future adaptation. Besides variation in DNA sequence, plants also harbour heritable variation in DNA methylation, and we want to understand the evolutionary significance of this epigenetic variation, in particular how much of it is under genetic control, and how much is associated with the environment. We addressed these questions in a high-resolution molecular analysis of 207 lines of the common plant field pennycress (Thlaspi arvense), which we collected across Europe, propagated under standardized conditions, and sequenced for their genetic and epigenetic variation. We found large geographic variation in DNA methylation, associated with both DNA sequence and climate of origin. Genetic variation was generally the stronger predictor of DNA methylation variation, but the strength of environmental association varied between different sequence contexts. Climate-of-origin was the strongest predictor in about one third of the differentially methylated regions in the CHH context, which suggests that epigenetic variation may play a role in the short-term climate adaptation of pennycress. As pennycress is currently being domesticated as a new biofuel and winter cover crop, our results may be relevant also for agriculture, particularly in changing environments.
In (eco-)toxicological studies the light/dark transition (LDT) test is one of the most frequently used behaviour assays with zebrafish eleutheroembryos. However, study results vary regarding data presentation and analysis and mostly focus on a limited amount of the recorded data. In this study, we investigated whether monitoring two behavioural outcomes (time and distance moved) together with analysing multiple parameters can improve test sensitivity and data interpretation. As a proof of principle 5-day old zebrafish (Danio rerio) eleutheroembryos exposed to either endocrine disruptors (EDs) or acetylcholine esterase (AChE) inhibitors were investigated. We analysed conventional parameters such as mean and sum and implemented additional endpoints such as minimum or maximum distance moved and new parameters assessing the bursting response of eleutheroembryos. Furthermore, changes in eleutheroembryonic behaviour during the moment of the light to dark transition were added. To improve data presentation control-normalised results were displayed in radar charts, enabling the simultaneous presentation of different parameters in relation to each other. This enabled us to identify parameters most relevant to a certain behavioural response. A cut off threshold using control data was applied to identify parameters that were altered in a biological relevant manner. Our approach was able to detect effects on different parameters that remained undetected when analysis was done using conventional bar graphs on - in most cases analysed - averaged, mean distance moved values. By combining the radar charts with additional parameters and by using control-based thresholds, we were able to increase the test sensitivity and promote a deeper understanding of the behaviour response of zebrafish eleutheroembryos in the LDT test and thereby increased its usability for behavioural toxicity studies.
The establishment and maintenance of protected areas(PAs) is viewed as a key action in delivering post-2020 biodiversity targets. PAs often need to meet a multitude of objectives, ranging from biodiversity protection to ecosystem service provision and climate change mitigation. As available land and conservation funding are limited, optimizing resources by selecting the most beneficial PAs is vital. Here we present a decision support tool that enables a flexible approach to PA selection on a global scale, allowing different conservation objectives to be weighted and prioritized according to user-specified preferences. We apply the tool across 1347 terrestrial PAs and highlight frequent trade-offs among different objectives, e.g., between biodiversity protection and ecosystem integrity. These results indicate that decision makers must usually decide among conflicting objectives. To assist this our decision support tool provides an explicitly value-based approach that can help resolve such conflicts by considering divergent societal and political demands and values.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2022)
Hyperparasitism on plant-parasitic fungi is a widespread but rarely studied phenomenon. Here, for the first time, we compile in a checklist information provided by peer-reviewed literature for fungi growing on colonies of black mildews (Meliolales, Ascomycota), a species-rich group of tropical and subtropical plant-parasitic microfungi. The checklist contains information on 189 species of contact-biotrophic microfungi in 82 genera. They belong to seven morphological groups: dematiaceous hyphomycetes, moniliaceous hyphomycetes, pycnidioid, perithecioid, catathecioid, and apothecioid fungi. By the fact that species accumulation curves do not reach saturation for any tropical country, it is evident that the knowledge of the diversity of hyperparasitic fungi on Meliolales is incomplete. A network analysis of records of hyperparasitic fungi, their host fungi and host plants shows that genera of hyperparasitic fungi are generalists concerning genera of Meliolales. However, most species of hyperparasitic fungi are restricted to meliolalean hosts. In addition to hyperparasitic fungi, diverse further microorganisms use meliolalean colonies as ecological niche. Systematic positions of most species are unknown because DNA sequence data are lacking for species of fungi hyperparasitic on Meliolales. We discuss the specific challenges of obtaining DNA sequence data from hyperparasitic fungi. In order to better understand the diversity, evolution and biology of hyperparasitic fungi, it is necessary to increase sampling efforts and to undertake further morphological, molecular, and ecological studies.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
NAD is a coenzyme central to metabolism that was also found to serve as a 5’-terminal cap of bacterial and eukaryotic RNA species. The presence and functionality of NAD-capped RNAs (NAD-RNAs) in the archaeal domain remain to be characterized in detail. Here, by combining LC-MS and NAD captureSeq methodology, we quantified the total levels of NAD-RNAs and determined the identity of NAD-RNAs in the two model archaea, Sulfolobus acidocaldarius and Haloferax volcanii. A complementary differential RNA-Seq (dRNA-Seq) analysis revealed that NAD transcription start sites (NAD-TSS) correlate with well-defined promoter regions and often overlap with primary transcription start sites (pTSS). The population of NAD-RNAs in the two archaeal organisms shows clear differences, with S. acidocaldarius possessing more capped small non-coding RNAs (sncRNAs) and leader sequences. The NAD-cap did not prevent 5’→3’ exonucleolytic activity by the RNase Saci-aCPSF2. To investigate enzymes that facilitate the removal of the NAD-cap, four Nudix proteins of S. acidocaldarius were screened. None of the recombinant proteins showed NAD decapping activity. Instead, the Nudix protein Saci_NudT5 showed activity after incubating NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into RNAs and the regulation of ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells.
Importance: This study reports the first characterization of 5’-terminally modified RNA molecules in Archaea and establishes that NAD-RNA modifications, previously only identified in the other two domains of life, are also prevalent in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii. We screened for NUDIX hydrolases that could remove the NAD-RNA cap and showed that none of these enzymes removed NAD modifications, but we discovered an enzyme that hydrolyzes ADPR-RNA. We propose that these activities influence the stabilization of NAD and its thermal degradation to potentially toxic ADPR products at elevated growth temperatures.
The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids.
The Mediterranean fruit fly (medfly), Ceratitis capitata, is an important model organism in biology and agricultural research with high economic relevance. However, information about its embryonic development is still sparse. We share nine long-term live imaging datasets acquired with light sheet fluorescence microscopy (484.5 h total recording time, 373 995 images, 256 Gb) with the scientific community. Six datasets show the embryonic development in toto for about 60 hours at 30 minutes intervals along four directions in three spatial dimensions, covering approximately 97% of the entire embryonic development period. Three datasets focus on germ cell formation and head involution. All imaged embryos hatched morphologically intact. Based on these data, we suggest a two-level staging system that functions as a morphogenetic framework for upcoming studies on medfly. Our data supports research on wild-type or aberrant morphogenesis, quantitative analyses, comparative approaches to insect development as well as studies related to pest control. Further, they can be used to test advanced image processing approaches or to train machine learning algorithms and/or neuronal networks.
The success of the increasing use of technology in education is highly dependent on learner acceptance. Although the Technology Acceptance Model (TAM) is dominant in research for surveying acceptance of technology, it does not allow the prediction of a successful first time use of technology. The successful first time use can be determined with the survey of technology affinity, as it corresponds to the expression of certain personality traits of users and is thus detached from the specific technology. Since there are no measurement instruments for the educational sector so far and existing instruments for measuring technology affinity do not meet the specific requirements for use in the educational context (e.g., limited time for questioning), we present the single item Inclusion of Technology Affinity in Self-Scale (ITAS). In study 1 we provide evidence of convergent and discriminant validity within the general population so that a generalization of its applicability is possible. In study 2 we subsequently tested ITAS in the actual target group, the educational sector. The high correlations of the ITAS with the ATI and the control instrument TA-EG (ranging from rs = 0.679 to rs = 0.440) show that ITAS is suitable for use in research. Furthermore, the newly developed instrument convinces with its low complexity, the graphical component, which requires little text understanding and the high time saving. This research thus can contribute to the investigation of technology affinity in the educational sector helping educators to conduct technical activities with their learning group, to predict possible difficulties and adjust their planning accordingly.
Aim
How species respond to climate change is influenced by their sensitivity to climatic conditions (i.e. their climatic niche) and aspects of their adaptive capacity (e.g. their dispersal ability and ecological niche). To date, it is largely unknown whether and how species’ sensitivity to climate change and their adaptive capacity covary. However, understanding this relationship is important to predict the potential consequences of a changing climate for species assemblages. Here, we test how species’ sensitivity to climate change and trait-based measures of their ecological adaptive capacity (i) vary along a broad elevational gradient and (ii) covary across a large number of bird species.
Location
A Neotropical elevational gradient (300–3600 m.a.s.l.) in the Manú Biosphere Reserve, south-east Peru.
Methods
We focus on 215 frugivorous bird species along a Neotropical elevational gradient. We approximate species’ sensitivity to climate change by their climatic niche breadth, based on species occurrences across South America and bioclimatic variables. In addition, we use a trait-based approach to estimate the dispersal ability of species (approximated by their wing pointedness), their dietary niche breadth (approximated by bill width) and their habitat niche breadth (the number of used habitat classes).
Results
We found that (i) species’ climatic niche breadth increased with elevation, while their trait-based dispersal ability and dietary niche breadth decreased with elevation, and (ii) sensitivity to climate change and trait-based adaptive capacity were not related across species.
Main conclusions
These results suggest different mechanisms of how species in lowland and highland assemblages might respond to climate change. The independent variation of species’ sensitivity to climate change and their trait-based adaptive capacity suggests that accounting for both dimensions will improve assessments of species’ susceptibility to climate change and potential impacts of climate change on diverse species assemblages.
Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish
(2022)
The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring.
To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish.
Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish.
To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively.
On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling.
Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.
Lifespan Extension of Podospora anserina Mic60-Subcomplex Mutants Depends on Cardiolipin Remodeling
(2022)
Function of mitochondria largely depends on a characteristic ultrastructure with typical invaginations, namely the cristae of the inner mitochondrial membrane. The mitochondrial signature phospholipid cardiolipin (CL), the F1Fo-ATP-synthase, and the ‘mitochondrial contact site and cristae organizing system’ (MICOS) complex are involved in this process. Previous studies with Podospora anserina demonstrated that manipulation of MICOS leads to altered cristae structure and prolongs lifespan. While longevity of Mic10-subcomplex mutants is induced by mitohormesis, the underlying mechanism in the Mic60-subcomplex deletion mutants was unclear. Since several studies indicated a connection between MICOS and phospholipid composition, we now analyzed the impact of MICOS on mitochondrial phospholipid metabolism. Data from lipidomic analysis identified alterations in phospholipid profile and acyl composition of CL in Mic60-subcomplex mutants. These changes appear to have beneficial effects on membrane properties and promote longevity. Impairments of CL remodeling in a PaMIC60 ablated mutant lead to a complete abrogation of longevity. This effect is reversed by supplementation of the growth medium with linoleic acid, a fatty acid which allows the formation of tetra-octadecanoyl CL. In the PaMic60 deletion mutant, this CL species appears to lead to longevity. Overall, our data demonstrate a tight connection between MICOS, the regulation of mitochondrial phospholipid homeostasis, and aging of P. anserina.
The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.
The tremendous body of knowledge about genetics, cell biology, and metabolism of Saccharomyces cerevisiae, as well as its long history and robustness in industrial fermentations, have made this yeast one of the most popular microbial cell factories. Novel genetic tools have enabled the rapid construction of strains producing various platform chemicals, fuels, or pharmaceuticals. The relevance of synthetic biology approaches, such as the construction of fully synthetic genomes and artificial cellular compartments are not only relevant for biotechnological applications but can also lead to new insight into basic principles of life.
Filamentous enzymes have been found in all domains of life, but the advantage of filamentation is often elusive1. Some anaerobic, autotrophic bacteria have an unusual filamentous enzyme for CO2 fixation—hydrogen-dependent CO2 reductase (HDCR)2,3—which directly converts H2 and CO2 into formic acid. HDCR reduces CO2 with a higher activity than any other known biological or chemical catalyst4,5, and it has therefore gained considerable interest in two areas of global relevance: hydrogen storage and combating climate change by capturing atmospheric CO2. However, the mechanistic basis of the high catalytic turnover rate of HDCR has remained unknown. Here we use cryo-electron microscopy to reveal the structure of a short HDCR filament from the acetogenic bacterium Thermoanaerobacter kivui. The minimum repeating unit is a hexamer that consists of a formate dehydrogenase (FdhF) and two hydrogenases (HydA2) bound around a central core of hydrogenase Fe-S subunits, one HycB3 and two HycB4. These small bacterial polyferredoxin-like proteins oligomerize through their C-terminal helices to form the backbone of the filament. By combining structure-directed mutagenesis with enzymatic analysis, we show that filamentation and rapid electron transfer through the filament enhance the activity of HDCR. To investigate the structure of HDCR in situ, we imaged T. kivui cells with cryo-electron tomography and found that HDCR filaments bundle into large ring-shaped superstructures attached to the plasma membrane. This supramolecular organization may further enhance the stability and connectivity of HDCR to form a specialized metabolic subcompartment within the cell.
In Europe, the sugar refinery is largely based on sugar beets. This route for obtaining household sugar results in a large amount of biomass waste, consisting mainly of the insoluble beet resi-dues, e.g., cell wall fragments. To a vast moiety this debris consists of the polymer pectin (up to 20% in the dry total solids). The structure of pectin is based on a backbone of D-galacturonic acid units (GalA), but also contains various other sugar monomers, predominantly L-arabinose, D-galactose, L-rhamnose and D-xylose. The amount of GalA adds up to a moiety of up to 70% with-in this sugar cocktail. So far, this debris is only fed to cattle or simply burnt. In nature, pectin is a common substrate for various organisms. The degradation of pectin-rich biomass is often per-formed by filamentous fungi like Hypocrea jecorina (also known as Trichoderma reesei) and As-pergillus niger, which evolved pectinases to degrade the pectin backbone and pathways to con-sume the monomer GalA as a sole carbon source. The fungal catabolism of pectin residues starts with the reduction of GalA to L-galactonate (GalOA) by a GalA-reductase. Even though filamen-tous fungi are native hosts of the GalA-catabolism and certain engineering approaches have al-ready been demonstrated, this class of organisms remains challenging with regard to bioreactor cultivation and tedious genetic accessibility. In contrast, the yeast S. cerevisiae is well known in fermentation processes and easily modified by a versatile set of genetic tools. So far, first ap-proaches have already been conducted to transfer the GalA utilization pathways into S. cerevisiae, but these approaches indicated limitations regarding GalA-uptake and redox cofac-tor replenishment due to the relatively high oxidative state of GalA compared to other sugars like glucose and galactose. Furthermore, the generally strongly increased demand for redox co-factors must be met by GalA reduction by finding new cofactor sources or redirecting reactions of the core metabolism.
This work aimed at the production of GalOA, which is the first intermediate of the fungal GalA catabolism. This compound shows an interesting range of potential applications, for instance as a food and cosmetic additive. To overcome the oxidized character of GalA, the presence of a more reduced co-substrate as a redox donor and as a carbon and energy source was required. To further enhance the reduction of GalA, modulation of the redox-cofactor supply and enzyme engineering were performed.
Im Rahmen dieser Arbeit wurden verschiedene metabolische Anpassungsmechanismen des humanpathogenen Bakteriums Acinetobacter baumannii an seinen Wirt untersucht. Im ersten Teil wurde die Rolle von verschiedenen Trimethylammoniumverbindungen (Cholin, Glycinbetain und Carnitin) und den zugehörigen Aufnahmesystemen, sowie ihren Stoffwechselwegen während dieses Prozesses analysiert. Für die Analyse der Transportsysteme wurde eine markerlose Vierfachmutante (Δbcct) von A. baumannii generiert, sodass alle bekannten Transportsysteme für die genannten Verbindungen deletiert vorlagen. Wachstumsversuche mit dieser Mutante zeigten, dass es in A. baumannii keine weiteren Transporter für die Aufnahme von Cholin gibt, jedoch weitere primär aktive oder sekundär aktive Transporter für die Aufnahme von Glycinbetain. Weiterhin konnten innerhalb dieser Arbeit die KM-Werte der Transporter bestimmt werden. Verschiedene Virulenz- und Infektionsanalysen führten zu dem Schluss, dass die Transporter keine Rolle bei der Virulenz von A. baumannii spielen. In Genomanalysen konnten die Gene, die für die Enzyme des Oxidationsweges von Cholin zu Glycinbetain kodieren identifiziert werden (Cholin-Dehydrogenase (betA), GlycinbetainAldehyd-Dehydrogenase (betB) und ein potenzieller Regulator (betI)). Es wurden Deletionsmutanten innerhalb dieses Genclusters generiert, mit dessen Hilfe gezeigt werden konnte, dass Cholin unter Salzstress ausschließlich als Vorläufer für das kompatible Solut Glycinbetain fungiert und nicht als kompatibles Solut von A. baumannii genutzt werden kann. Virulenz- und Infektionsstudien mit den Deletionsmutanten zeigten, dass der Cholin-Oxidationsweg keine Rolle bei der Virulenz von A. baumannii spielt.
Die Cholin-Dehydrogenase BetA wurde zusätzlich in E. coli produziert und anschließend mittels NiNTA-Affinitätschromatographie aufgereinigt. Die biochemische Charakterisierung des Enzyms zeigte, dass BetA membranständig ist und die höchste Aktivität bei einem pH-Wert von 9,0 hat. Salze wie NaCl oder KCl hatten keinen Effekt auf die Aktivität des Enzyms, während Glutamat die Aktivität stimulierte.
Weiterhin konnte FAD als Cofaktor identifiziert werden und der KM-Wert ermittelt werden. Zudem konnte gezeigt werden, dass die Oxidation von Cholin zu Glycinbetain unter isoosmotischen Bedingungen zu einem Anstieg der ATP-Konzentration in A. baumannii-Zellsuspensionen führt und damit, dass Cholin als alternative Energiequelle genutzt wird. Das Phospholipid Phosphatidylcholin konnte als natürliche Cholinquelle identifiziert werden. Eine Rolle der Phospholipasen D bei der Abspaltung der Cholin-Kopfgruppe des Phosphatidylcholins konnte ausgeschlossen werden. Die Gene für die Oxidation von Cholin zu Glycinbetain werden ausschließlich in Anwesenheit von Cholin exprimiert, jedoch unabhängig von der extrazellulären Salzkonzentration. Diese Studien zeigten, dass der Cholin-Oxidationsweg eine Rolle in der metabolischen Adaptation von A. baumannii an den Wirt spielt. Phosphatidylcholin kann hier als natürliche Cholinquelle im Wirt genutzt werden, da die Wirtsmembranen aus bis zu 70 % Phosphatidylcholin bestehen. Transportstudien mit Carnitin führten zu dem Schluss, dass der Transporter Aci01347 aus A. baumannii neben Cholin ebenfalls Carnitin transportiert. Wachstumsversuche mit einer aci01347-Mutante bestätigen, dass Aci01347 essenziell für die Aufnahme und anschließende Verwertung von Carnitin als Kohlenstoffquelle ist. Es konnte weiterhin gezeigt werden, dass das Transportergen mit essenziellen Genen für den Carnitin-Abbau in einem Operon liegt. Für die Analyse des Abbauweges von Carnitin wurden markerlose Deletionsmutanten innerhalb des Operons generiert. In Wachstumsstudien mit diesen Mutanten konnte der Abbauweg aufgeklärt werden und der Regulator des Operons identifiziert werden. Carnitin wird hier über Trimethylamin und Malat-Semialdehyd zu D-Malat umgewandelt und anschließend über Pyruvat in den TCA-Zyklus eingespeist. Der Regulator wurde zusätzlich in E. coli produziert und mittels Ni-NTA-Affinitätschromatographie aufgereinigt. Mithilfe von EMSA-Studien konnte die Bindestelle des Regulators auf eine 634 Bp lange DNA-Sequenz stromaufwärts des CarnitinOperons eingegrenzt werden. Durch Transkriptomanalysen konnte gezeigt werden, dass bei Wachstum mit Acetylcarnitin, Carnitin und D-Malat die Expression des Carnitin-Operons induziert wurde. Darüber hinaus wurden die Gene konservierter Aromatenabbauwege wie z. B. des Homogentisatweges, des Phenylacetatweges und des Protocatechuat-Abbaus, verstärkt exprimiert. In G. mellonellaVirulenzstudien konnte eine Rolle des Abbaus von Carnitin bei der Virulenz von A. baumannii nachgewiesen werden. Zusätzlich konnte dieser Effekt dem entstehenden Trimethylamin zugesprochen werden...
The change in allele frequencies within a population over time represents a fundamental process of evolution. By monitoring allele frequencies, we can analyze the effects of natural selection and genetic drift on populations. To efficiently track time-resolved genetic change, large experimental or wild populations can be sequenced as pools of individuals sampled over time using high-throughput genome sequencing (called the Evolve & Resequence approach, E&R). Here, we present a set of experiments using hundreds of natural genotypes of the model plant Arabidopsis thaliana to showcase the power of this approach to study rapid evolution at large scale. First, we validate that sequencing DNA directly extracted from pools of flowers from multiple plants -- organs that are relatively consistent in size and easy to sample -- produces comparable results to other, more expensive state-of-the-art approaches such as sampling and sequencing of individual leaves. Sequencing pools of flowers from 25-50 individuals at ∼40X coverage recovers genome-wide frequencies in diverse populations with accuracy r > 0.95. Secondly, to enable analyses of evolutionary adaptation using E&R approaches of plants in highly replicated environments, we provide open source tools that streamline sequencing data curation and calculate various population genetic statistics two orders of magnitude faster than current software. To directly demonstrate the usefulness of our method, we conducted a two-year outdoor evolution experiment with A. thaliana to show signals of rapid evolution in multiple genomic regions. We demonstrate how these laboratory and computational Pool-seq-based methods can be scaled to study hundreds of populations across many climates.
Out-of-school laboratories, also called student labs, are an advantageous opportunity to teach biological subjects. Particularly in the case of complex fields such as neurobiology, student labs offer the opportunity to learn about difficult topics in a practical way. Due to numerous advantages, digital student labs are becoming increasingly popular nowadays. In this study, we investigated the effect of an electrophysiological setup for a virtual experiment with and without hands-on elements on participant motivation and technology acceptance. For this purpose, 235 students were questioned during a student laboratory day. The surveyed students showed high motivation and technology acceptance for the virtual experiment. In the comparison, the electrophysiological setup with hands-on elements performs better in the intrinsic components than the setup without hands-on elements: Thus, the hands-on approach is rated as more interesting and the perceived enjoyment scores higher. Nevertheless, both experimental groups show high values, so that the results of the study support the positive influence of digital laboratory as well as a positive influence of hands-on elements.
Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
Mutational analysis of ribosomal DNA and maturation-scheme analysis of ribosomal RNA in A. thaliana
(2022)
Ribosome biogenesis is a fundamental cellular process beginning with long precursor rRNA transcription from multi-copies of repetitive 45S ribosomal DNAs. At the subunit level, the primary pre-rRNA transcript encapsuled in 90S protein-RNA complex undergoes decisive splitting in two chief ways for further maturation into large (LSU) and small (SSU) ribosomal subunit. The usage of specific rDNA copies from defined chromosomes and their selective role during growth and development have been a topic of interest owing to its contribution to specialized ribosome theory which proposes non-monolithic functions for ribosomes and thereby their mRNA translation potential. Dual-guide CRISPR/Cas9 mediated disruption of rDNA regions resulted in stable disruption of up to 2.5% and 5% of all rDNA copies in hetero- and homozygous (ploop KD) conditions, respectively. At the RNA level, the mutation excised a critical structural element, P-loop on the LSU 25S rRNA. Mutation caused a dosage dependent defect with homozygosity leading to severe developmental defects through vegetative and reproductive growth phases which is manifested in their proteome by means of disregulation through both increase and decrease of several gene ontological categories of proteins in mutants. Interestingly, the mutation on chromosome 4 triggered dosage compensation through rRNA expression from chromosome 2 further compounded by ectopic rRNA biogenesis defects. The mutated copies however are not incorporated in the translating ribosomes and as a direct or indirect consequence led to elevated basal autophagic levels in the mutants.
The primary 35S transcript is known to undergo two modes of initial cleavages at the pre-rRNA level that aid in their subsequent maturation. Root cell culture (RCC) studies shows that these cells contain a novel ITS2-first cleaved precursor even under control growth conditions, P-C2 adding a third maturation means for the 35S pre-rRNA. This maturation path is further known to be triggered under elevated growth temperature forming a novel adaptive response in Arabidopsis and two other crop plants, tomato, and rice. Taken together, the pulse-chase labeling analysis of control and stressed tissues uncovers the fine-tuned pre-rRNA schematics with crossovers between multiple maturation paths.
Na+ homeostasis in Acinetobacter baumannii is facilitated via the activity of the Mrp antiporter
(2022)
The human opportunistic pathogen Acinetobacter baumannii is a global threat to healthcare institutions worldwide, since it developed very efficient strategies to evade host defence and to adapt to the different environmental conditions of the host. This work focused on the importance of Na+ homeostasis in A. baumannii with regards to pathobiological aspects. In silico studies revealed a homologue of a multicomponent Na+/H+ antiporter system. Inactivation of the Mrp antiporter through deletion of the first gene (mrpA′) resulted in a mutant that was sensitive to increasing pH values. Furthermore, the strain was highly sensitive to increasing Na+ and Li+ concentrations. Increasing Na+ sensitivity is thought to be responsible for growth impairment in human fluids. Furthermore, deletion of mrpA′ is associated with energetic defects, inhibition of motility and survival under anoxic and dry conditions.
Nanoplastics affect the inflammatory cytokine release by primary human monocytes and dendritic cells
(2022)
So far, the human health impacts of nano- and microplastics are poorly understood. Thus, we investigated whether nanoplastics exposure induces inflammatory processes in primary human monocytes and monocyte-derived dendritic cells. We exposed these cells in vitro to nanoplastics of different shapes (irregular vs. spherical), sizes (50–310 nm and polydisperse mixtures) and polymer types (polystyrene; polymethyl methacrylate; polyvinyl chloride, PVC) using concentrations of 30–300 particles cell−1. Our results show that irregular PVC particles induce the strongest cytokine release of these nanoplastics. Irregular polystyrene triggered a significantly higher pro-inflammatory response compared to spherical nanoplastics. The contribution of chemicals leaching from the particles was minor. The effects were concentration-dependent but varied markedly between cell donors. We conclude that nanoplastics exposure can provoke human immune cells to secrete cytokines as key initiators of inflammation. This response is specific to certain polymers (PVC) and particle shapes (fragments). Accordingly, nanoplastics cannot be considered one homogenous entity when assessing their health implications and the use of spherical polystyrene nanoplastics may underestimate their inflammatory effects.
Microbial production of chemicals is a sustainable alternative to conventional industrial processes. However, the implementation of exogenous metabolic pathways is hampered by slow diffusion rates, competing pathways, or secretion of intermediates. Pre-existing organelles have been harnessed to overcome these problems, but these approaches suffer from interference with endogenous pathways. We have developed a new concept for the compartmentalization of enzymatic pathways in ER-derived vesicles.
Echolocation behavior, a navigation strategy based on acoustic signals, allows scientists to explore neural processing of behaviorally relevant stimuli. For the purpose of orientation, bats broadcast echolocation calls and extract spatial information from the echoes. Because bats control call emission and thus the availability of spatial information, the behavioral relevance of these signals is undiscussable. While most neurophysiological studies, conducted in the past, used synthesized acoustic stimuli that mimic portions of the echolocation signals, recent progress has been made to understand how naturalistic echolocation signals are encoded in the bat brain. Here, we review how does stimulus history affect neural processing, how spatial information from multiple objects and how echolocation signals embedded in a naturalistic, noisy environment are processed in the bat brain. We end our review by discussing the huge potential that state-of-the-art recording techniques provide to gain a more complete picture on the neuroethology of echolocation behavior.
tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.
The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. 13C-detected NMR experiments, acquired simultaneously to 1H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1–248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.
Non-ribosomal peptide synthetases (NRPSs) are modular biosynthetic megaenzymes producing many important natural products and refer to a specific set of peptides in bacteria’s and fungi’s secondary metabolism. With the actual purpose of providing advantages within their respective ecological niche, the bioactivity of the structurally highly diverse products ranges from, e.g., antibiotic (e.g., vancomycin) to immunosuppressive (e.g., cyclosporin A) to cytostatic (e.g., echinomycin or thiocoralin) activity.
An NRPS module consists of at least three core domains that are essential for the incorporation of specific substrates with the 'multiple carrier thiotemplate mechanism' into a growing peptide chain: an adenylation (A) domain selects and activates a cognate amino acid; a thiolation (T) domain shuffles the activated amino acid and the growing peptide chain, which are attached at its post-translationally 4ʹ-phosphopantetheine (4'-PPant) group, between the active sites; a condensation (C) domain links the upstream and downstream substrates. NRPS synthesis is finished with the transfer of the assembled peptide to the C-terminal chain-terminating domain. Accordingly, the intermediate is either released by hydrolysis as a linear peptide chain or by an intramolecular nucleophilic attack as a cyclic peptide.
The NRPS’s modular character seems to imply straightforward engineering to take advantage of their features but appears to be more challenging. Since the pioneering NRPS engineering approaches focused on the reprogramming and replacement of A domains, several working groups developed advanced methods to perform a complete replacement of subdomains or single or multiple catalytic domains.
The first part of this work focusses parts of the publication with the title 'De novo design and engineering of non-ribosomal peptide synthetases', which follows up assembly line engineering with the development of a new guideline. Thereby, the pseudodimeric V-shaped structure of the C domain is exploited to separate the N-terminal (CDSub) and C-terminal (CASub) subdomains alongside a four-AA-long linker. This results in the creation of self-contained, catalytically active CASub-A-T-CDSub (XUC) building blocks. As an advantage over the previous XU concept, the characteristics (substrate- and stereoselectivity) assigned to the C domain subunits are likewise exchanged, and thus, no longer represent a barrier. Furthermore, with the XUC concept, no important interdomain interfaces are disrupted during the catalytic cycle of NRPS, allow to expect much higher production titers. Moreover, the XUC concept shows a more flexible application within its genus origin of building blocks to create peptide libraries. Additionally, with this concept only 80 different XUC building blocks are needed to cover the entire proteinogenic amino acid spectrum.
The second part of this work addresses the influence of the C domain on activity and specificity of A domains. In a comprehensive analysis, a clear influence of different C domains on the in vitro activation rate and the in vivo substrate spectrum could be observed. Further in situ and in silico characterizations indicate that these influences are neither the result of the respective A domains promiscuity nor the C domain’s proofreading, but due to an 'extended gatekeeping' function of the C domain. This novel term of an 'extended gatekeeping' function describes the very nature of interfaces that C domains can form with an A domain of interest. Therefore, the C-A interface is assumed to have a more significant contribution to a selectivity filter function.
The third part of this work combines the NRPS engineering with phylogenetic/evolutionary perspectives. At first, the C-A interface could be precisely defined and further identified to encode equivalent information corresponding to the complete C-A didomain. Moreover, the comparison of NRPSs topology reveals hints for a co-evolutionary relatedness of the C-A didomain and could be shown to reassemble even after separation. In this regard, based on a designed CAopt.py algorithm, the reassembling-compatibility of hybrid interfaces could be determined by scoring of the co-expressed NRPS hybrids. This algorithm also enables the randomization of the interface sequences, thus, leading to the identification of more functional interface variant, which cause significantly higher peptide production and could even be applied to other native and hybrid interfaces.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components in S. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.