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Acinetobacter baumannii is outstanding for its ability to cope with low water activities which significantly contributes to its persistence in hospital environments. The vast majority of bacteria are able to prevent loss of cellular water by amassing osmoactive compatible solutes or their precursors into the cytoplasm. One such precursor of an osmoprotectant is choline that is taken up from the environment and oxidized to the compatible solute glycine betaine. Here, we report the identification of the osmotic stress operon betIBA in A. baumannii. This operon encodes the choline oxidation pathway important for the production of the solute glycine betaine. The salt-sensitive phenotype of a betA deletion strain could not be rescued by addition of choline, which is consistent with the role of BetA in choline oxidation. We found that BetA is a choline dehydrogenase but also mediates in vitro the oxidation of glycine betaine aldehyde to glycine betaine. BetA was found to be associated with the membrane and to contain a flavin, indicative for BetA donating electrons into the respiratory chain. The choline dehydrogenase activity was not salt dependent but was stimulated by the compatible solute glutamate.
The original version of this Article contained errors where Table S5 and Table S6 were incorrectly cited. As the result, in the Methods section, under the subheading ‘Germline transformation, crossing setups and insertion junction sequencing’, “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S4).” now reads: “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S5).” And, under the subheading ‘Light sheet-based fluorescence microscopy’, “Metadata for the three datasets are provided in Table S5.” now reads: “Metadata for the three datasets are provided in Table S6.” In Data availability section, “Microscopy data can be accessed as described in Table S5.” now reads: “Microscopy data can be accessed as described in Table S6.” Additionally, in the Supplementary Information 8 file, the “Data Access” row was omitted in Table S6. The “Data Access” row now reads: Dataset (DS) DS0001 DS0002 DS0003 Dataset Access DOI: 10.5281/zenodo.4892363 DOI: 10.5281/zenodo.4892373 DOI: 10.5281/zenodo.4892381 The original Supplementary Information 8 file is provided below. Finally, the Supplementary Information 1 and 5 files published with this Article contained tracked changes, these have now been removed. The original Article and accompanying Supplementary Information files have been corrected.
Droughts impair plant growth, limit global net primary production and are predicted to increase in the course of climate change. Knowledge of the plant drought response on a molecular level can facilitate the selection of drought resistant genotypes and genetic engineering and thereby can help to implement strategies, such as assisted migration projects or crop improvement, in order to preserve natural and agricultural vegetation against droughts.
Studies on gene expression under drought stress were conducted in three species each of the genera Quercus and Panicum, to shed light on the molecular drought response in these species and identify drought responsive genes as a basis for technical applications.
In the genus Quercus, gene expression studies were conducted in the three major European forest trees Q. ilex, Q. pubescens and Q. robur, for which a distributional shift caused by climate change is predicted for the 21st century. RNA-Seq experiments were conducted in the three Quercus species for the first time, ortholog groups were assigned and unregulated genes, as well as drought responsive genes, were identified (Madritsch et al. 2019). For a set of the unregulated genes, a stable expression over the course of long-term drought periods was evaluated in order to enable an application as reference genes for normalizing qRT-PCR experiments (Kotrade 2019a). The reference genes were used in subsequent experiments to generate gene expression profiles over the course of a two-year drought experiment with consecutive drought periods for a set of twelve drought responsive genes and revealed a highly variable gene regulation under long-term drought stress in the Quercus species (Kotrade et al. 2019b).
In the genus Panicum, the gene expression in response to drought was examined in the two wild crop species, P. laetum and P. turgidum, and in the less drought tolerant species P. bisulcatum via RNA-Seq experiments (Kotrade et al. 2020 (in revision). The transcriptomes of the species were sequenced for the first time, ortholog groups were assigned and the gene regulation was compared across the species. The common grounds of the drought response in Panicum were determined by identifying similarities across the species, while the identification of differences between the species led to genes that might contribute to the higher drought tolerance of P. laetum and P. turgidum
A comparison across the two genera showed large differences in the gene regulation upon drought. This might be largely explained by different experimental setups that resulted in different drought conditions in the genera, such as drought intensity, drought duration and velocity of drought development.
The sequence information and the drought responsive genes identified in the Quercus and Panicum species can be used to develop marker assays for marker-assisted selection. The genes that putatively contribute to the higher drought tolerance of the two wild crop Panicum species should be considered as candidate targets in genetic engineering studies. Marker-assisted selection and genetic engineering can be applied, for example, in assisted migration projects to support natural vegetation in the course of climate change or to breed more drought tolerant crop strains to mitigate crop failure rates caused by droughts.
Mosquito breeding sites are complex aquatic environments with wide microbial diversity and physicochemical parameters that can change over time during the development of immature insect stages. Changes in biotic and abiotic conditions in water can alter life-history traits of adult mosquitos but this area remains understudied. Here, using microbial genomic and metabolomics analyses, we explored the metabolites associated with Aedes aegypti breeding sites as well as the potential contribution of Klebsiella sp., symbiotic bacteria highly associated with mosquitoes. We sought to address whether breeding sites have a signature metabolic profile and understand the metabolite contribution of the bacteria in the aquatic niches where Ae. aegypti larvae develop. An analysis of 32 mosquito-associated bacterial genomes, including Klebsiella, allowed us to identify gene clusters involved in primary metabolic pathways. From them, we inferred metabolites that could impact larval development (e.g., spermidine), as well as influence the quality assessment of a breeding site by a gravid female (e.g., putrescine), if produced by bacteria in the water. We also detected significant variance in metabolite presence profiles between water samples representing a decoupled oviposition event (oviposition by single females and manually deposited eggs) versus a control where no mosquito interactions occurred (PERMANOVA: p < 0.05; R2 = 24.64% and R2 = 30.07%). Five Klebsiella metabolites were exclusively linked to water samples where oviposition and development occurred. These data suggest metabolomics can be applied to identify compounds potentially used by female Ae. aegypti to evaluate the quality of a breeding site. Elucidating the physiological mechanisms by which the females could integrate these sensory cues while ovipositing constitutes a growing field of interest, which could benefit from a more depurated list of candidate molecules.
Background: Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results: A fed-batch system with plant biomass-derived sugars D-xylose, L-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and L-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion: This work has identified two additional CAZyme inducers for T. aurantiacus, L-arabinose and cellobiose, along with D-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.
RATIONALE: RBPs (RNA-binding proteins) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP SRSF4 (serine/arginine-rich splicing factor 4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. OBJECTIVE: To investigate the role of SRSF4 in the heart. METHODS AND RESULTS: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long noncoding RNAs, including GAS5 (growth arrest-specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide- resolution cross-linking and immuno-precipitation. GAS5 is a repressor of the GR (glucocorticoid receptor) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. CONCLUSIONS: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in patients with Cushing syndrome.
Aim: The identification of the mechanisms determining spatial variation in biological diversity along elevational gradients is a central objective in ecology and biogeography. Here, we disentangle the direct and indirect effects of abiotic drivers (climatic conditions, and land use) and biotic drivers (vegetation structure and food resources) on functional diversity and composition of bird and bat assemblages along a tropical elevational gradient. Location: Southern slopes of Mt. Kilimanjaro, Tanzania, East Africa. Methods: We counted birds and recorded bat sonotypes on 58 plots distributed in near-natural and anthropogenically modified habitats from 700 to 4,600 m above sea level. For the recorded taxa, we compiled functional traits related to movement, foraging and body size from museum specimens and databases. Further, we recorded mean annual temperature, precipitation, vegetation complexity as well as the number of fruits, flowers, and insect biomass as measures of resource availability on each study site. Results: Using path analyses, we found similar responses of bird and bat functional diversity to the variation in abiotic and biotic drivers along the elevational gradient. In contrast, the functional composition of both taxa showed distinct responses to abiotic and biotic drivers. For both groups, direct temperature effects were most important, followed by resource availability, precipitation and vegetation complexity. Main Conclusions: Our findings indicate that physiological and metabolic constraints imposed by temperature and resource availability determine the functional diversity of bird and bat assemblages, whereas the composition of individual functional traits is driven by taxon-specific processes. Our study illustrates that distinct filtering mechanisms can result in similar patterns of functional diversity along broad environmental gradients. Such differences need to be taken into account when it comes to conserving the functional diversity of flying vertebrates on tropical mountains.
Toxicogenomic differentiation of functional responses to fipronil and imidacloprid in Daphnia magna
(2021)
Active substances of pesticides, biocides or pharmaceuticals can induce adverse side effects in the aquatic ecosystem, necessitating environmental hazard and risk assessment prior to substance registration. The freshwater crustacean Daphnia magna is a model organism for acute and chronic toxicity assessment representing aquatic invertebrates. However, standardized tests involving daphnia are restricted to the endpoints immobility and reproduction and thus provide only limited insights into the underlying modes-of-action. Here, we applied transcriptome profiling to a modified D. magna Acute Immobilization test to analyze and compare gene expression profiles induced by the GABA-gated chloride channel blocker fipronil and the nicotinic acetylcholine receptor (nAChR) agonist imidacloprid. Daphnids were expose to two low effect concentrations of each substance followed by RNA sequencing and functional classification of affected gene ontologies and pathways. For both insecticides, we observed a concentration-dependent increase in the number of differentially expressed genes, whose expression changes were highly significantly positively correlated when comparing both test concentrations. These gene expression fingerprints showed virtually no overlap between the test substances and they related well to previous data of diazepam and carbaryl, two substances targeting similar molecular key events. While, based on our results, fipronil predominantly interfered with molecular functions involved in ATPase-coupled transmembrane transport and transcription regulation, imidacloprid primarily affected oxidase and oxidoreductase activity. These findings provide evidence that systems biology approaches can be utilized to identify and differentiate modes-of-action of chemical stressors in D. magna as an invertebrate aquatic non-target organism. The mechanistic knowledge extracted from such data will in future contribute to the development of Adverse Outcome Pathways (AOPs) for read-across and prediction of population effects.
Sleep is one of the fundamental requirements of all animals from nematodes to humans. It appears in different formats with shared features such as reduced muscle activities and reduced responsiveness to the environment. Despite the long history of sleep research, why a brain must be taken offline for a large portion of each day remains unknown. Moreover, sleep research focused on mammals and birds reveals two stages, rapid-eye-movement (REM) and slow-wave (SW) sleep, alternating during sleep. Whether these two stages of sleep exist in other vertebrates, particularly reptiles, is debated, as is the evolution of sleep in general.
Recordings from the brain of a lizard, the Australian bearded dragon Pogona vitticeps, indicate the presence of two electrophysiological states and provides a better picture of their sleep. Local field potential (LFP) signals, head velocity, eye movements, and heart rate during sleep match the pattern of REM and SW sleep in mammals. The SW and REM sleep patterns that we observed in lizards oscillated continuously for 6 to 10 hours with a period of 80-100 seconds when the ambient temperature was ~27°C. Lizard SW dynamics closely resemble those observed in rodent hippocampal CA1, yet originated from a brain area, the dorsal ventricular ridge (DVR), that does not correspond anatomically or transcriptomically to the mammalian hippocampus. This finding pushes back the probable evolution of these dynamics to the emergence of amniotes, at least 300 million years ago.
Unlike mammals and birds, REM and SW sleep in lizards occupy an almost equal amount of time during sleep. The clock-like alternation between these two sleep states was found initially by measuring the power modulation of two frequency bands, delta and beta. I recorded the full-band LFP and found an infra-slow oscillation (ISO) in the frequency range between 5 and 20 milli-Hz during sleep. The magnitude of ISO increased during sleep and decreased during both wakefulness and arousal during sleep. The up- and down-states of ISO were synchronized with the sleep state alternating rhythm but with a significant time lag dependent on the locations of the recording electrodes. Multi-site LFP recordings indicated that this ISO is a putative propagation wave sweeping extremely slowly, 30-67 µm/sec, from the posterior-dorsal pole to the anterior-ventral pole of the DVR.
Previous studies in other animals showed that brainstem areas such as the locus coeruleus, laterodorsal tegmentum, and periaqueductal gray are involved in sleep states regulation. It is sadly impossible to carry out in vivo recordings in the lizard brainstem without severely affecting them and their quality of life. I thus carried out ex vivo recordings in both DVR and brainstem. Pharmacological stimulation of the brainstem could reversibly silence one distinct EEG pattern characteristic of SW sleep, the sharp-wave and ripple complex, in DVR. An ISO could be recorded simultaneously in both DVR and brainstem. From data collected in both intact and split ex vivo brains, I concluded that there are independent ISO generators in at least two areas, the brainstem and the telencephalon. Their signals may normally be synchronized by long-range connections. The DVR ISO leads the brainstem ISO by ~29 sec. Optogenetic stimulation of brainstem neurons was able to disrupt the ISO in DVR reversibly.
In conclusion, the lizard brain offers a relatively simple model system to study sleep. Despite a diversity of results in different lizard species, my results revealed a number of new findings. Relevant for sleep research in general: 1) REM and SW sleep exist in a reptile. Since they also exist in birds and mammals, they probably existed in their common amniote ancestor, if not earlier. 2) REM and SW occupy equal amounts of time during sleep (50% duty cycle), a unique feature among all described sleep electrophysiological patterns, suggesting the possible existence of a simple central pattern generator of sleep, possibly ancestral. 3) I discovered the existence, in the local field potential, of an infra slow oscillation with extremely slow propagation, locked to the SW-REM alternating rhythm. The causes and mechanisms of this ISO remain to be understood. To my knowledge, the correlation between sleep states and a slow rhythm has only been reported in human scalp EEG recordings so far.
Recently, the potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and its potential transformation products 4-methyl-7-ethylaminocoumarin (C47T1) and 4-methyl-7-aminocoumarin (C47T2) were identified as novel environmental contaminants. We assessed for the first time the sources, distribution, and fate of these compounds in aquatic systems using the Holtemme River (Saxony-Anhalt, Germany), which is a hotspot for these contaminants. To this end, wastewater-treatment plant (WWTP) influent and effluent samples, surface water samples over 3 years, and the longitudinal profiles in water, sediment, and gammarids were analyzed. From the longitudinal profile of the river stretch, the WWTP of Silstedt was identified as the sole point source for these compounds in the River Holtemme, and exposure concentrations in the low micrograms per liter range could be recorded continuously over 3 years. Analysis of WWTP influent and effluent showed a transformation of approximately half of the C47 into C47T1 and C47T2 but no complete removal. A further attenuation of the three coumarins after discharge into the river could be largely attributed to dilution, while transformation was only approximately 20%, thus suggesting a significant persistence in aquatic systems. Experimentally derived partitioning coefficients between water and sediment organic carbon exceeded those predicted using the OPERA quantitative structure–activity relationship tools and polyparameter linear free-energy relationships by up to 93-fold, suggesting cation binding as a significant factor for their sorption behavior. Near-equilibrium conditions between water and sediment were not observed close to the emitting WWTP but farther downstream in the river. Experimental and predicted bioaccumulation factors for gammarids were closely matching, and the concentrations in field-sampled gammarids were close to steady state with exposure concentrations in the water phase of the river. Environ Toxicol Chem 2021;40:3078–3091. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
Background: The industrial production of various alcohols from organic carbon compounds may be performed at high rates and with a low risk of contamination using thermophilic microorganisms as whole-cell catalysts. Thermoanaerobacter species that thrive around 50–75 °C not only perform fermentation of sugars to alcohols, but some also utilize different organic acids as electron acceptors, reducing them to their corresponding alcohols. Results: We purified AdhE as the major NADH- and AdhB as the major NADPH-dependent alcohol dehydrogenase (ADH) from the cell extract of the organic acid-reducing Thermoanaerobacter sp. strain X514. Both enzymes were present in high amounts during growth on glucose with and without isobutyrate, had broad substrate spectra including different aldehydes, with high affinities (< 1 mM) for acetaldehyde and for NADH (AdhE) or NADPH (AdhB). Both enzymes were highly thermostable at the physiological temperature of alcohol production. In addition to AdhE and AdhB, we identified two abundant AdhA-type ADHs based on their genes, which were recombinantly produced and biochemically characterized. The other five ADHs encoded in the genome were only expressed at low levels. Conclusions: According to their biochemical and kinetic properties, AdhE and AdhB are most important for ethanol formation from sugar and reduction of organic acids to alcohols, while the role of the two AdhA-type enzymes is less clear. AdhE is the only abundant aldehyde dehydrogenase for the acetyl-CoA reduction to aldehydes, however, acid reduction may also proceed directly by aldehyde:ferredoxin oxidoreductase. The role of the latter in bio-alcohol formation from sugar and in organic acid reduction needs to be elucidated in future studies.
Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains (N/CDDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of “designer” NRPS from proteins that normally do not interact. The multiprotein “peptide-antimicrobial-Xenorhabdus” (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small CDD of PaxA cooperates with its preceding thiolation (T1) domain to bind the NDD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.
The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.
Acetogenic bacteria are already established as biocatalysts for production of high-value compounds from C1 substrates such as H2 + CO2 or CO. However, little is known about the physiology, biochemistry and bioenergetics of acetogenesis from formate, an interesting feedstock for biorefineries. Here, we analysed formate metabolism in the model acetogen Acetobacterium woodii. Cells grew optimally on 200 mM formate to an optical density of 0.6. Formate was exclusively converted to acetate (and CO2) with a ratio of 4.4:1. Transcriptome analyses revealed genes/enzymes involved in formate metabolism. Strikingly, A. woodii has two genes potentially encoding a formyl-THF synthetase, fhs1 and fhs2. fhs2 forms an operon with a gene encoding a potential formate transporter, fdhC. Deletion of fhs2/fdhC led to a reduced growth rate, formate consumption and optical densities. Acetogenesis from H2 + CO2 was accompanied by transient formate production; strikingly, formate reutilization was completely abolished in the Δfhs2/fdhC mutant. Take together, our studies gave the first detailed insights into the formatotrophic lifestyle of A. woodii.
Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Efficient engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) is an ongoing challenge. Here we describe a cloning and co-expression strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 220 mg L−1. Extending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into independently expressed and translated polypeptide chains. These synthetic type of NRPS (type S) enables easier access to engineering, overcomes cloning limitations, and provides a simple and rapid approach to building peptide libraries via the combination of different NRPS subunits.
Methanol is the simplest of all alcohols, is universally distributed in anoxic sediments as a result of plant material decomposition and is constantly attracting attention as an interesting substrate for anaerobes like acetogens that can convert bio-renewable methanol into value-added chemicals. A major drawback in the development of environmentally friendly but economically attractive biotechnological processes is the present lack of information on biochemistry and bioenergetics during methanol conversion in these bacteria. The mesophilic acetogen Eubacterium callanderi KIST612 is naturally able to consume methanol and produce acetate as well as butyrate. To grasp the full potential of methanol-based production of chemicals, we analysed the genes and enzymes involved in methanol conversion to acetate and identified the redox carriers involved. We will display a complete model for methanol-derived acetogenesis and butyrogenesis in Eubacterium callanderi KIST612, tracing the electron transfer routes and shed light on the bioenergetics during the process.
The constitution and regulation of effector repertoires shape host–microbe interactions. Ustilago maydis and Sporisorium reilianum are two closely related smut fungi, which both infect maize but cause distinct disease symptoms. Understanding how effector orthologs are regulated in these two pathogens can therefore provide insights into the evolution of different infection strategies. We tracked the infection progress of U. maydis and S. reilianum in maize leaves and used two distinct infection stages for cross-species RNA-sequencing analyses. We identified 207 of 335 one-to-one effector orthologs as differentially regulated during host colonization, which might reflect the distinct disease development strategies. Using CRISPR-Cas9-mediated gene conversion, we identified two differentially expressed effector orthologs with conserved function between two pathogens. Thus, differential expression of functionally conserved genes might contribute to species-specific adaptation and symptom development. Interestingly, another differentially expressed orthogroup (UMAG_05318/Sr10075) showed divergent protein function, providing a possible case for neofunctionalization. Collectively, we demonstrated that the diversification of effector genes in related pathogens can be caused both by alteration on the transcriptional level and through functional diversification of the encoded effector proteins.
Global landscapes are changing due to human activities with consequences for both biodiversity and ecosystems. For single species, terrestrial mammal population densities have shown mixed responses to human pressure, with both increasing and decreasing densities reported in the literature. How the impacts of human activities on mammal populations translates into altered global density patterns remains unclear. Here we aim to disentangle the effect of human impacts on large-scale patterns of mammal population densities using a global dataset of 6729 population density estimates for 468 mammal species (representing 59% and 44% of mammalian orders and families). We fitted a mixed effect model to explain the variation in density based on a 1-degree resolution as a function of the human footprint index (HFI), a global proxy of direct and indirect human disturbances, while accounting for body mass, trophic level and primary productivity (normalized vegetation index; NDVI). We found a significant positive relationship between population density and HFI, where population densities were higher in areas with a higher HFI (e.g. agricultural or suburban areas – no populations were located in very high HFI urban areas) compared to areas with a low HFI (e.g. wilderness areas). We also tested the effect of the individual components of the HFI and still found a consistent positive effect. The relationships remained positive even across populations of the same species, although variability among species was high. Our results indicate shifts in mammal population densities in human modified landscapes, which is due to the combined effect of species filtering, increased resources and a possible reduction in competition and predation. Our study provides further evidence that macroecological patterns are being altered by human activities, where some species will benefit from these activities, while others will be negatively impacted or even extirpated.
Thermoanaerobacter kivui ist ein thermophiles acetogenes Bakterium, das chemolithoautotroph auf CO2 unter Verwendung von molekularem H2 als Elektronendonor wächst und Acetat als Produkt über den Wood-Ljungdahl-Weg (WLP) bildet. Im WLP werden 2 Mol CO2 reduziert, um ein Mol Acetyl-CoA zu bilden. Erste Studien wurden durchgeführt, um die Physiologie von T. kivui zu verstehen. T. kivui wächst autotroph auf H2 + CO2 und nach Adaptation auch auf CO oder Syngas. T. kivui wächst ebenfalls auch in Minimalmedium ohne weitere Zugabe von Vitaminen, was es zu einem Biokatalysator mit hohem Potenzial für die Produktion von Chemikalien mit hohem Mehrwert macht. Heterotroph wächst T. kivui auf Glucose, Fructose, Mannose, Pyruvat oder Formiat. Kürzlich wurde beschrieben, dass T. kivui in der Lage ist, auf dem Zuckeralkohol Mannitol in Gegenwart und Abwesenheit von HCO3- (oder externem CO2) zu wachsen. Allerdings war das Wachstum in Abwesenheit von externem CO2 deutlich verlangsamt. Daher wurde in dieser Studie getestet, ob eine Zugabe von externem Formiat das "fehlende" CO2 kompensieren kann. In Kombination mit Formiat wurde das Wachstum auf Mannitol in CO2 und HCO3- freien definierten Medien bis zu einer maximalen OD600 von 2,34 und mit einer Verdopplungszeit von 2,0 ± 0,0 stimuliert, was dem Wachstumsverhalten auf Mannitol in Anwesenheit von CO2/HCO3- entsprach. In Abwesenheit von Formiat (oder CO2) erreichte T. kivui nur eine endgültige optische Dichte von bis zu 0,7 mit einer verlängerten Verdoppelungszeit von 5,2 ± 0,2 Stunden. Dieses Experiment zeigte die höhe metabolische Flexibilität von T. kivui durch die Nutzung von Formiat als Elektronenakzeptor, wenn kein oder nur wenig CO2 vorhanden ist.
Genomanalysen ergaben, dass T. kivui ein Trehalose- und Maltose-Transportsystem-Permeaseprotein (MalF) besitzt. Darüber hinaus verfügt T. kivui über Trehalose- und Maltosehydrolase-Gene, die als Kojibiose-Phosphorylase annotiert sind. Obwohl in der Originalveröffentlichung beschrieben wurde, dass der Organismus nicht auf Maltose oder Trehalose wachsen kann, konnte T. kivui im Laufe dieser Arbeit an das Wachstum auf Maltose und Trehalose adaptiert werden. Nach dem Transfer von einer Glukose-Vorkultur auf ein Medium mit 25 mM Maltose oder 25 mM Trehalose als alleinige C-Quelle wurde kein Wachstum erzielt. Bei Verwendung der gleichen Vorkultur in einem Medium mit höherer Konzentration (50 mM) Maltose oder Trehalose, begannen die Zellen zu wachsen. Bei Verwendung dieser adaptierten kulturen als Vorkultur wuchsen die Zellen in Gegenwart von in 25 mM Maltose oder Trehalose bis zu einer maximalen OD600 von 1,12 bzw. 0,73. Die Adaptation hing mit der Tatsache zusammen, dass der Organismus eine höhere Konzentration benötigt, um sich an diese Kohlenstoffquellen zu gewöhnen. Durch diese Daten wird das heterotrophe Potenzial von T. kivui erhöht.
Um die Bedeutung der wasserstoffabhängigen Kohlendioxidreduktase (HDCR) während des Wachstums auf Formiat oder auf H2 + CO2 im Stoffwechsel von T. kivui zu verstehen, wurden Studien auf molekularer Ebene durchgeführt. Die HDCR nutzt H2 direkt für die Reduktion von CO2 zu Formiat im ersten Schritt des Wood-Ljungdahl-Wegs (WLP). Um die Rolle der HDCR in dieser Reaktion zu untersuchen, wurde das hdcr-Gencluster mit Hilfe des kürzlich entwickelten Mutagenesytems für T. kivui deletiert. In Wachstumstudien konnte anschliessend gezeigt werden, dass die ߡhdcr-Deletionsmutante nicht mehr auf Formiat oder H2 + CO2 als alleiniger Kohlenstoffquelle wachsen konnte. Nach Komplementation der Mutante mit dem hdcr-Gene in cis wuchsen die Kulture wieder auf Formiat oder H2 + CO2. Diese Experimente zeigten, dass die HDCR für das Wachstum auf H2 + CO2 oder Formiat essentiell ist. Interessanterweise konnte in der ߡhdcr-Mutante ebenfalls ein verändertes Wachstum auf Glukose als alleiniger C-Quelle festgestellt werden. Die T. kivui ߡhdcr-Mutante wuchs nur bis zu einer OD600 von 0,2, während der Wildtyp und der hdcr-komplementierte Stamm bis zu einer OD600 von 2,64 bzw. 2,4 wuchsen. Damit wurde bewiesen, dass die HDCR auch für die vollständige Glukoseoxidation in T. kivui erforderlich ist. Durch die Zugabe von Formiat wurde das Wachstum vollständig wiederhergestellt, ähnlich wie beim Wildtyp. Dies belegt wieder die Nutzung Formiat als terminalen Elektronenakzeptor. Auch auf Mannitol oder Pyruvat konnte die Mutanten nur in Gegenwart von Formiat wachsen. Der Substratverbrauch und die Produktbildung der T. kivui ߡhdcr-Mutante wurden in einem Zellsuspensionsexperiment untersucht. Die Zellen verbrauchten Formiat nur in Gegenwart von Glukose und produzierten Acetat mit einem Acetat/Substrat-Verhältnis von etwas mehr als 3,0, während die Acetatproduktion nur 12 mM betrug, wenn Glukose als alleiniges Substrat verwendet wurde. Diese Ergebnisse zeigen eine enge Kopplung der Oxidation von Multikohlenstoffsubstraten an den WLP.
T. kivui ist eines der wenigen Acetogenen, die CO als einzige Kohlenstoff- und Energiequelle nutzen können. ...
More than 2 million tons of glycerol are produced during industrial processes each year and, therefore, glycerol is an inexpensive feedstock to produce biocommodities by bacterial fermentation. Acetogenic bacteria are interesting production platforms and there have been few reports in the literature on glycerol utilization by this ecophysiologically important group of strictly anaerobic bacteria. Here, we show that the model acetogen Acetobacterium woodii DSM1030 is able to grow on glycerol, but contrary to expectations, only for 2–3 transfers. Transcriptome analysis revealed the expression of the pdu operon encoding a propanediol dehydratase along with genes encoding bacterial microcompartments. Deletion of pduAB led to a stable growth of A. woodii on glycerol, consistent with the hypothesis that the propanediol dehydratase also acts on glycerol leading to a toxic end-product. Glycerol is oxidized to acetate and the reducing equivalents are reoxidized by reducing CO2 in the Wood–Ljungdahl pathway, leading to an additional acetate. The possible oxidation product of glycerol, dihydroxyacetone (DHA), also served as carbon and energy source for A. woodii and growth was stably maintained on that compound. DHA oxidation was also coupled to CO2 reduction. Based on transcriptome data and enzymatic analysis we present the first metabolic and bioenergetic schemes for glycerol and DHA utilization in A. woodii.
Objectives: The four-dimensional ultrasound (4D-US) enables imaging of the aortic segment and simultaneous determination of the wall expansion. The method shows a high spatial and temporal resolution, but its in vivo reliability is so far unknown for low-measure values. The present study determines the intraobserver repeatability and interobserver reproducibility of 4D-US in the atherosclerotic and non-atherosclerotic infrarenal aorta. Methods: In all, 22 patients with non-aneurysmal aorta were examined by an experienced examiner and a medical student. After registration of 4D images, both the examiners marked the aortic wall manually before the commercially implemented speckle tracking algorithm was applied. The cyclic changes of the aortic diameter and circumferential strain were determined with the help of custom-made software. The reliability of 4D-US was tested by the intraclass correlation coefficient (ICC). Results: The 4D-US measurements showed very good reliability for the maximum aortic diameter and the circumferential strain for all patients and for the non-atherosclerotic aortae (ICC >0.7), but low reliability for circumferential strain in calcified aortae (ICC = 0.29). The observer- and masking-related variances for both maximum diameter and circumferential strain were close to zero. Conclusions: Despite the low-measured values, the high spatial and temporal resolution of the 4D-US enables a reliable evaluation of cyclic diameter changes and circumferential strain in non-aneurysmal aortae independent from the observer experience but with some limitations for calcified aortae. The 4D-US opens up a new perspective with regard to noninvasive, in vivo assessment of kinematic properties of the vessel wall in the abdominal aorta.
Genetic engineering of Saccharomyces cerevisiae for improved cytosolic isobutanol biosynthesis
(2021)
The finite nature of fossil resources and the environmental problems caused by their excessive usage requires alternative approaches. The transformation from a fossil based economy to one based on renewable biomass is called a “bioeconomy”. To substitute fossil resources, various microorganisms have already been modified for the biosynthesis of valuable chemicals from biomass. However, the development of such efficient microorganisms at an industrial scale, remains a major challenge. The most prominent and robust microorganism for industrial production is the yeast Saccharomyces cerevisiae, which is known to produce ethanol that is used as renewable biofuel. However, S. cerevisiae is also naturally able to produce isobutanol in small amounts. Isobutanol is favoured as a biofuel compared to ethanol due to its higher octane number and lower hygroscopicity, which makes it more suitable for application in conventional combustion engines. In S. cerevisiae, the biosynthesis of isobutanol is permitted by the combination of mitochondrial valine synthesis (catalysed by Ilv2, Ilv5 and Ilv3) and its cytosolic degradation (catalysed by Aro10 and Adh2). The different compartmentalisation of the two pathways limit isobutanol biosynthesis. Thus, Brat et al. (2012) were able to increase the isobutanol yield up to 15 mg/gGlc by cytosolic re localisation of the enzymes Ilv2Δ54, Ilv5Δ48 and Ilv3Δ19 (cyt-ILV), with simultaneous deletion of ilv2. This corresponds to approximately 3.7% of the theoretical yield of 410 mg/gGlc, implying existing limitations in isobutanol biosynthesis, which have been investigated in this work.
For yet unknown reasons, isobutanol was only produced by S. cerevisiae in a valine free medium, according to Brat et al. (2012). This work shows that this can be attributed to the catalytic activity of Ilv2Δ54, which acted as growth inhibitor to S. cerevisiae. By this logic, a negative selection on the ILV2∆54 gene was exerted, which made the ilv2 deletion and simultaneous valine exclusion necessary to maintain the functional expression of toxic ILV2∆54. Furthermore, it was shown that valine exclusion is not mandatory due to the feedback regulation of Ilv2, permitted by Ilv6. Rather, increased isobutanol yield was observed when cytosolic Ilv6∆61 was expressed in the valine free medium, which is explained by the enhanced regulation of Ilv2Δ54 by Ilv6∆61 when BCAA are absent. Isobutanol biosynthesis is neither redox nor NAD(P)H co factor balanced. It was seen that co factor imbalance could be mitigated by the expression of an NADH oxidase (NOX), but not by expression of the NADH dependent ilvC6E6, since the latter showed low in vivo activity. Furthermore, it was seen that NAD(H) imbalance did already limit isobutanol biosynthesis, but the NADP(H) imbalance did not. Another limitation of cytosolic isobutanol biosynthesis is the secretion of the intermediate 2‑dihydroxyisovalerate, which then no longer is taken up by S. cerevisiae, causing a reduced isobutanol yield. This is attributed to insufficient Ilv3∆19 activity, due to poor iron sulphur cluster apo protein maturation. Therefore, it was aimed to replace Ilv3∆19 by heterologous dihydroxyacid dehydratases. Even though some of the enzymes were functionally expressed, none showed better in vivo activity than Ilv3∆19. Therefore, the Ilv3∆19 apo protein maturation was improved. This was achieved by the genomic deletion of fra2 or pim1 as well as by the cytosolic expression of Grx5∆29.
In addition to the isobutanol pathway, S. cerevisiae was optimised for isobutanol biosynthesis by rational and evolutionary engineering. For this purpose, the genes which are necessary for isobutanol production were integrated into the ilv2 locus, and the resulting strain was evolved in a medium containing the toxic amino acid analogue norvaline. Evolved single colonies were isolated, which presented improved growth and increased isobutanol yields (0.59 mg/gGlc) in a valine free medium, as compared to the initial strain. This is explained by a gene dosage effect which occurred during the evolutionary engineering experiment. In collaboration with Dr. Wess, the genes ilv2, bdh1/2, leu4/9, ecm31, ilv1, adh1, gpd1/2 and ald6 were cumulatively deleted in CEN.PK113 7D to block competing metabolic pathways. The resulting strain JWY23 achieved isobutanol yields up to 67.3 mg/gGlc, when expressing the cyt ILV enzymes from a multi copy vector. The most promising approaches of this work, namely the deletion of fra2 and the expression of Grx5∆29, Ilv6∆61, and NOX, were confirmed in this JWY23 strain. The highest isobutanol yield from this work was observed at 72 mg/gGlc for Ilv6∆61 and cyt ILV enzymes expressing JWY23, which corresponds to 17.6% of the theoretical isobutanol yield.
Isobutyric acid (IBA) is a by product of isobutanol biosynthesis, but it is also considered a valuable platform chemical. Therefore, the approaches that improved isobutanol biosynthesis were applied to the biosynthesis of IBA in S. cerevisiae. The highest IBA yield of 9.8 mg/gGlc was observed in a valine free medium by expression of cyt ILV enzymes, NOX and Ald6 in JWY04 (CEN.PK113 7D Δilv2; Δbdh1; Δbdh2; Δleu4; Δleu9; Δecm31; Δilv1). This corresponded to an 8.9 fold increase compared with the control and is, to our best knowledge, the highest IBA yield reported to date for S. cerevisiae.
Die Studien im Rahmen dieser Arbeit wurden am Modellorganismus Anabaena sp. PCC 7120 (Anabaena) durchgeführt, einem filamentösen Süßwasser-Cyanobakterium. Cyanobakterien sind photosynthetische, Gram-negative Organismen. Sie besitzen eine das Zytosol begrenzende Plasmamembran und eine Äußere Membran. TonB-abhängige Transporter (TBDTs) und Porine der Äußeren Membran bewerkstelligen und regulieren die Aufnahme von Nährstoffen. Typischerweise wenig abundante Substrate für den TBDT-vermittelten, aktiven Transport sind beispielsweise eisenhaltige Siderophore oder VitaminB12. Kleinere gelöste und abundante Stoffe wie Salze oder andere Ionen gelangen hingegen passiv durch Porine in das Periplasma.
In Anabaena wurden neun putative Porine identifiziert. Sieben hiervon wiesen eine porinspezifische Domänenstruktur auf (Alr0834, Alr2231, All4499, Alr4550, Alr4741, All5191 und All7614), und wurden im Rahmen dieser Arbeit näher betrachtet. Die Expression dieser sieben Gene wurde vergleichend untersucht, nachdem der Wildtyp in Standardmedium oder in Medium indem jeweils Mangan, Eisen, Kupfer oder Zink fehlte angezogen wurde. Außerdem wurde das Wachstum der einzelnen Porinmutanten im Vergleich zum Wildtyp auf Festmedium mit hohen Konzentrationen von Salzen, Antibiotika oder anderen Stoffen analysiert. Hierbei konnten den einzelnen Mutanten teilweise spezifische phänotypische Eigenschaften zugeschrieben werden. Zusammengefasst kann anhand der Analysenergebnisse vermutet werden, dass Alr4550 eine besondere Rolle in der Wahrung der Zellhüllenstabilität oder -integrität spielt, wohingegen das Fehlen von Alr5191 auf unbekannte Weise die Fixierung von Stickstoff zu erschweren scheint. Die alr2231-Mutante zeigte eine Resistenz gegenüber hohen Zinkkonzentrationen, was die Vermutung zulässt, dass Zink ein Substrat von Alr2231 darstellt. Für weitere Porine kann ebenfalls ein Zusammenhang zum Transport von Kupfer oder Mangan vermutet werden.
Neben Porinen wurden ebenfalls TonB-ähnliche Proteine in Anabaena untersucht. TonB ist ein plasmamembranständiges Protein, das in Komplex mit ExbB und ExbD die Energie für Transportprozesse über die Äußere Membran bereitstellt. Hierfür bindet TonB C-terminal an TBDTs und induziert dort Strukturänderungen, welche den Substratimport ins Periplasma ermöglichen. Als Energiequelle wird der Protonengradient genutzt, der über die Plasmamembran besteht. In Anabaena wurden vier putative TonB Proteine identifiziert, die sich jeweils in Länge und Domänenstruktur unterscheiden. Im Rahmen dieser Arbeit konnte durch Substrattransport-Experimente und Wachstumsanalysen gezeigt werden, dass TonB3 an der Aufnahme zweier Siderophore (Schizokinen und dem Xenosiderophor Ferrichrom) beteiligt ist, da die entsprechende Mutante sich als unfähig erwies diese zu als Eisenquelle nutzbar zu machen. Daneben wies TonB3 weitere Merkmale auf, die auch TonB-Proteinen anderer Organismen zugeschrieben wurden (Wachstumsdefizit der Mutante unter Eisenmangel, eisenabhängiges Expressionsprofil). Interessanterweise zeigte sich, dass das Siderophor Ferrichrom ebenfalls nicht als Eisenquelle für die tonB4-Mutante zur Verfügung stand, was zum Beispiel auf eine Beteiligung von TonB4 an dessen Transport hinweisen könnte.
TonB1, welches sich durch ein inkomplettes TBDT-Interaktionsmotiv auszeichnet, und TonB2 konnte keine Beteiligung am Siderophoretransport zugeschrieben werden, jedoch zeigten Mutanten der einzelnen Gene spezifische phänotypische Eigenschaften. Die tonB1-Mutante stach hervor durch ein vergleichsweise stark verzögertes Wachstum unter diazotrophen Bedingungen. Es konnte gezeigt werden, dass sowohl die Nitrogenaseaktivität als auch die expression vermindert war im tonB1-Mutantenstamm. Außerdem zeigten die Heterozysten dieser Mutante, die auf die Stickstoffixierung spezialisierten Zellen, eine abnormale Morphologie. Da die Expression von tonB1 jedoch nach dem Überführen von Wildypzellen in stickstoffreies Medium nicht erhöht war, kann eine direkte Beteiligung von TonB1 an der Heterozystendifferenzierung als unwahrscheinlich betrachtet werden. Die Zelleinschnürungen zwischen Heterozysten und vegetativen Zellen waren in I-tonB1 weniger ausgeprägt als im Wildtyp, was durch eine Anfärbung der Zellwand mit einem Fluoreszenzmarker gezeigt werden konnte. Ebenfalls konnte anhand des fluoreszierenden Markers Calcein gezeigt werden, dass die molekulare Diffusionsgeschwindigkeit zwischen Heterozysten und vegetativen Zellen, und auch zwischen zwei benachbarten vegetativen Zellen, in der tonB1-Mutante erhöht ist. Deswegen kann hier vermutlich vermehrt die Nitrogenase schädigender Sauerstoff in Heterozysten eindringen. Die aufgezählten Ergebnisse deuten auf eine Funktion von SjdR im Aufbau der Septumsstrukturen hin, beispielsweise durch Regulation der Peptidoglykansynthese oder -verteilung, weswegen TonB1 umbenannt wurde in SjdR (Septal junction disc regulator).
Die Untersuchung der tonB2-Mutante zeigte bei dieser eine veränderte Pigmentierung, eine vermehrte Lipopolysaccharidproduktion und Filamentaggregation sowie eine erhöhte Resistenz gegenüber bestimmten Antibiotika oder Detergenzien. Letzteres könnte auf die ebenfalls in der tonB2-Mutante beobachtete verringerte Porinexpression zurückgeführt werden. Es wurde außerdem eine vermehrte Anreicherung von Kupfer und Molybdän in der Mutante gemessen, was ein Grund für die Veränderte Pigmentierung sein könnte und ebenfalls die Porinexpression beeinflussen könnte. Insgesamt scheint sich das Fehlen von TonB2 auf die Integrität der Äußeren Membran auszuwirken. Daher kann für TonB2, eine Funktion in Anlehnung an das Tol-system vermutet werden.
Climate change causes increased tree mortality leading to canopy loss and thus sun-exposed forest floors. Sun exposure creates extreme temperatures and radiation, with potentially more drastic effects on forest organisms than the current increase in mean temperature. Such conditions might potentially negatively affect the maturation of mushrooms of forest fungi. A failure of reaching maturation would mean no sexual spore release and, thus, entail a loss of genetic diversity. However, we currently have a limited understanding of the quality and quantity of mushroom-specific molecular responses caused by sun exposure. Thus, to understand the short-term responses toward enhanced sun exposure, we exposed mushrooms of the wood-inhabiting forest species Lentinula edodes, while still attached to their mycelium and substrate, to artificial solar light (ca. 30°C and 100,000 lux) for 5, 30, and 60 min. We found significant differentially expressed genes at 30 and 60 min. Eukaryotic Orthologous Groups (KOG) class enrichment pointed to defense mechanisms. The 20 most significant differentially expressed genes showed the expression of heat-shock proteins, an important family of proteins under heat stress. Although preliminary, our results suggest mushroom-specific molecular responses to tolerate enhanced sun exposure as expected under climate change. Whether mushroom-specific molecular responses are able to maintain fungal fitness under opening forest canopies remains to be tested.
1. Plant-fungal interactions are important for plant community assembly, but quantifying these relationships remains challenging. High throughput sequencing of fungal communities allows us to identify plant-fungal associations at a high level of resolution, but often fails to provide information on taxonomic and functional assignment of fungi. 2. We transplanted seeds of Pinus cembra across an elevational gradient (1850–2250 m a.s.l.) and identified environmental factors and known fungal associates important for seedling establishment and survival. We then applied null model tests to identify taxonomically unassigned fungi associated with pine recruitment. 3. Early seedling establishment was determined by abiotic environmental factors, while seedling survival was predominantly affected by biotic environmental factors (i.e., the abundance of a fungal pathogen known from literature and the distance to adult trees). Null model tests identified known mycorrhizal partners and a large number of unknown operational taxonomic units (OTUs) associated with seedling survival, including saprotrophic and pathogenic species. These results highlight that unknown fungal OTUs, which are usually discarded from analyses, could play a crucial role for plant survival. 4. Synthesis. We conclude that high throughput metabarcoding paired with null model tests, is a valuable approach for identifying hidden plant-fungal associations within large and complex DNA metabarcoding datasets. Such an approach can be an important tool in illuminating the black box of plant-microbe interactions, and thus understanding ecosystem dynamics.
The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2 + CO2, formate, pyruvate, lactate or alanine. In the absence of Na+, the Wood–Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities which significantly contributes to its persistence in hospital environments. The vast majority of bacteria are able to prevent loss of cellular water by amassing osmoactive compatible solutes or their precursors into the cytoplasm. One such precursor of an osmoprotectant is choline that is taken up from the environment and oxidized to the compatible solute glycine betaine. Here, we report the identification of the osmotic stress operon betIBA in A. baumannii. This operon encodes the choline oxidation pathway important for the production of the solute glycine betaine. The salt-sensitive phenotype of a betA deletion strain could not be rescued by addition of choline, which is consistent with the role of BetA in choline oxidation. We found that BetA is a choline dehydrogenase but also mediates in vitro the oxidation of glycine betaine aldehyde to glycine betaine. BetA was found to be associated with the membrane and to contain a flavin, indicative for BetA donating electrons into the respiratory chain. The choline dehydrogenase activity was not salt dependent but was stimulated by the compatible solute glutamate.
An exploration of the relationship between recruitment communication and foraging in stingless bees
(2021)
Social information is widely used in the animal kingdom and can be highly adaptive. In social insects, foragers can use social information to find food, avoid danger, or choose a new nest site. Copying others allows individuals to obtain information without having to sample the environment. When foragers communicate information they will often only advertise high-quality food sources, thereby filtering out less adaptive information. Stingless bees, a large pantropical group of highly eusocial bees, face intense inter- and intra-specific competition for limited resources, yet display disparate foraging strategies. Within the same environment there are species that communicate the location of food resources to nest-mates and species that do not. Our current understanding of why some species communicate foraging sites while others do not is limited. Studying freely foraging colonies of several co-existing stingless bee species in Brazil, we investigated if recruitment to specific food locations is linked to 1) the sugar content of forage, 2) the duration of foraging trips, and 3) the variation in activity of a colony from 1 day to another and the variation in activity in a species over a day. We found that, contrary to our expectations, species with recruitment communication did not return with higher quality forage than species that do not recruit nestmates. Furthermore, foragers from recruiting species did not have shorter foraging trip durations than those from weakly recruiting species. Given the intense inter- and intraspecific competition for resources in these environments, it may be that recruiting species favor food resources that can be monopolized by the colony rather than food sources that offer high-quality rewards.
Obligate endoparasitic oomycetes are known to ubiquitously occur in marine and freshwater diatoms, but their diversity is still largely unexplored. Many of these parasitoids are members of the early-diverging oomycete lineages (Miracula, Diatomophthora), others are within the Leptomitales of the Saprolegniomycetes (Ectrogella, Lagenisma) and some have been described in the Peronosporomycetes (Aphanomycopsis, Lagenidium). Even though some species have been recently described and two new genera were introduced (Miracula and Diatomophthora), the phylogeny and taxonomy of most of these organisms remain unresolved. This is contrasted by the high number of sequences from unclassified species, as recently revealed from environmental sequencing, suggesting the presence of several undiscovered species. In this study, a new species of Miracula is reported from a marine centric diatom (Minidiscus sp.) isolated from Skagaströnd harbor in Northwest Iceland. The morphology and life cycle traits of this novel oomycete parasite are described herein, and its taxonomic placement within the genus Miracula is confirmed by molecular phylogeny. As it cannot be assigned to any previously described species, it is introduced as Miracula islandica in this study. The genus Miracula thus contains three described holocarpic species (M. helgolandica, M. islandica, M. moenusica) to which likely additional species will need to be added in the future, considering the presence of several lineages known only from environmental sequencing that clustered within the Miracula clade.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Acinetobacter baumannii is an opportunistic pathogen, which has become a rising threat in healthcare facilities worldwide due to increasing antibiotic resistances and optimal adaptation to clinical environments and the human host. We reported in a former publication on the identification of three phopholipases of the phospholipase D (PLD) superfamily in A. baumannii ATCC 19606T acting in concerted manner as virulence factors in Galleria mellonella infection and lung epithelial cell invasion. This study focussed on the function of the three PLDs. A Δpld1-3 mutant was defect in biosynthesis of the phospholipids cardiolipin (CL) and monolysocardiolipin (MLCL), whereas the deletion of pld2 and pld3 abolished the production of MLCL. Complementation of the Δpld1-3 mutant with pld1 restored CL biosynthesis demonstrating that the PLD1 is implicated in CL biosynthesis. Complementation of the Δpld1-3 mutant with either pld2 or pld3 restored MLCL and CL production leading to the conclusion that PLD2 and PLD3 are implicated in CL and MLCL production. Mutant studies revealed that two catalytic motifs are essential for the PLD3-mediated biosynthesis of CL and MLCL. The Δpld1-3 mutant exhibited a decreased colistin and polymyxin B resistance indicating a role of CL in cationic antimicrobial peptides (CAMPs) resistance.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K+, in osmostress response. Growth of A. baumannii was K+-dependent and the K+-dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K+ and K+ accumulation increased with the salinity of the medium. K+ uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K+ concentration: mannitol and glutamate concentrations increased with increasing K+ concentrations whereas trehalose was highest at low K+. After osmotic upshock, cells first accumulated K+ followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K+ and glutamate. These experiments demonstrate K+ uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K+ and different organic solutes.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities and therefore its adaptation mechanism to osmotic stress. Here we report on the identification and characterization of five different secondary active compatible solute transporters, belonging to the betaine-choline-carnitine transporter (BCCT) family. Our studies revealed two choline-specific and three glycine betaine-specific BCCTs. Activity of the BCCTs was differentially dependent to the osmolality: one choline and one betaine transporter were osmostress-independent. Addition of choline to resting cells of Acinetobacter grown in the presence of the co-substrate choline or with phosphatidylcholine as sole carbon source led to ATP synthesis in the wild type but not in the BCCT quadruple mutant. This indicates that the BCCTs are essential to transport the energy substrate choline. The role of the different BCCTs in osmostress resistance and in metabolic adaptation of A. baumannii to the human host is discussed.
A1AO ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg−1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na+)-binding site. Indeed, ATP hydrolysis was strictly Na+-dependent. N,N′-dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+. Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD-4 for a common binding site. After incorporation of the A1AO ATP synthase into liposomes, ATP-dependent primary transport of 22Na+ as well as ΔµNa+-driven ATP synthesis could be demonstrated. The Na+ A1AO ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases.
Understanding hominin expansions requires the comprehension of movement processes at different scales. In many models of hominin expansion these processes are viewed as being determined by large-scale effects, such as changes in climate and vegetation spanning continents and thousands or even millions of years. However, these large-scale patterns of expansions also need to be considered as possibly resulting from the accumulation of small-scale decisions of individual hominins. Moving on a continental scale may for instance involve crossing a water barrier. We present a generalized agent-based model for simulating the crossing of a water barrier where the agents represent the hominin individuals. The model can be configured to represent a variety of movement modes across water. Here, we compare four different behavioral scenarios in conjunction with a set of water barrier configurations, in which agents move in water by either paddling, drifting, swimming or rafting. We introduce the crossing-success-rate (CSR) to quantify the performance in water crossing. Our study suggests that more focus should be directed towards the exploration of behavioral models for hominins, as directionality may be a more powerful factor for crossing a barrier than environmental opportunities alone. A prerequisite for this is to perceive the opposite shore. Furthermore, to provide a comprehensive understanding of hominin expansions, the CSR allows for the integration of results obtained from small-scale simulations into large-scale models for hominin expansion.
Young trees of deciduous Quercus robur and Q. petraea and evergreen Q. ilex were grown together in a competition lysimeter experiment to assess i) – whether the observed growth differences between evergreen Q. ilex and the deciduous Q. robur and Q. petraea on sandy soil in the field and ii) – whether the different natural distribution of Q. robur and Q. petraea could be attributed to physiological differences between the species under experimental drought stress (DS). Half of the plants were subjected to long-term DS in two consecutive years and monitored for physiological and growth parameters. In the first year, water withholding for more than three months did not lead to significant drought stress, probably because of a sufficient residual water volume in the lysimeter for the relatively small plants. However, in the second year, 2018, which was warmer, the bigger plants now competed for the residual water and clear drought stress symptoms developed for more than two months in all trees in the DS lysimeter basin. Growth was only moderately (and mostly not significantly) affected by the DS in the second year, except for a smaller total leaf area in DS Q. ilex as compared to DS Q. robur and Q. petraea and smaller root collar diameter in DS Q. ilex compared to DS Q. robur. Under DS, the deciduous species revealed significant decreases in ΔVIP, indicating a negative effect on electron transport through PS I. Pn, PIabs and water relations parameters (ΨPD and LWC) all decreased to various extents under DS in all three species, leading to clear separation of the deciduous from the evergreen species by PCA. However, PCA did not separate the two deciduous species from each other. It is concluded that longer root growth in the two deciduous species as compared to Q. ilex ameliorates DS effects in Q. robur and Q. petraea and may be the key to understand the better performance of deciduous oaks on sandy soil in the field.
The Southern Ocean (SO) continental shelf and deep sea are environments characterised by different benthic communities. Their structure and composition are driven and shaped by different variables: whilst on the continental shelf physical environmental variables are the main drivers shaping faunal abundance, structure and composition, the deep-sea fauna is most problably driven by biological variables such as predation and competition. Among shelf and deep-sea benthic communities, peracarids (e.g. amphipods and isopods) are one of the most dominant groups, showing high levels of abundance and diversity in both environments. Knowledge on their assemblage structure and composition in the SO remains limited, as well as the knowledge of the environmental variables that influence them. Therefore, the aim of our study was to investigate peracarid assemblages from the SO continental shelf and deep sea and to assess the main drivers shaping their assemblage structure along a wide bathymetric gradient (from 160 m to about 6000 m depth) and at a large geographic scale. We analysed the spatial distribution of 183,606 peracarids sampled using an epibenthic sledge (EBS) during nine different expeditions in the SO, covering a latitudinal range of 77° to 41° South. Depth was identified as the main driver shaping peracarid abundance pattern, their assemblage structure from the continental shelf (<1499 m) was dissimilar to that from the deep sea (>1500 m). Also, depth was differently correlated with different peracarid orders: while isopod abundances increased with depth, amphipods and mysids were negatively correlated; no correlation was found with cumaceans and tanaidaceans. The dissimilar peracarid assemblage structure between the SO continental shelf and the SO deep sea can be due to the assumption that there are different driving forces shaping benthic assemblages from these two environments (physical variables on the continental shelf, biological interactions in the deep sea). As a result, we also suggest that environmental changes due to climate change (e.g. temperature, ice coverage, productivity) would have different consequences depending on the bathymetric range considered.
Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, partial and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and their severe discomfort.
FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms—that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3′ region of FAD1 mRNA was analyzed by 3′RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3′UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3′UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3′UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.
In recent years, several neuronal differentiation protocols were published that circumvent the requirement of embryoid body (EB) formation under serum-deprivation and simplified medium conditions. But a neuronal default model to establish an approach that works efficiently for all pluripotent cells and neuronal precursors is still lacking. Whether such a default neural mechanism exist and how this is implemented across a broad spectrum of cell source, is addressed in several studies and still controversially discussed. It was proposed that the default neuronal fate is initiated in the absence of extrinsic signals and is achieved by eliminating extracellular inhibitors of neuroectodermal fate and suppressing cell-cell signalling through limited cell density. Previous studies reported that ESC and ECC grown at low density and in absence of exogenous factors or feeder layers die within 24 h but acquire a neural identity as indicated by expression of the neural marker Nestin. Thus, this application is not suitable for generating neural cultures. Furthermore, it was reported that P19 cells survive and express neuroectodermal marker genes in serum-free DMEM/F12 medium containing transferrin, insulin, and selenite, although no neurites were identified.
Based on this background, in this study, a novel approach to induce neuronal differentiation in vitro was developed that implements a nutrient-poor environment, which, in contrast to previous studies, ensures the survival of neuronally differentiated cells over a long period of time and allows normal formation of neurites. Neither the formation of free-floating aggregates nor supplementation of growth factors or known inducers was required to establish a reliable neuronal differentiation protocol. A simple medium, consisting of DMEM/F12+N2 that was highly diluted in salt solution, was sufficient to drive a fast neuronal differentiation in monolayer cultures. Serum deprivation and strong dilution of DMEM/F12+N2 medium cause a nutrient-poor environment in which the influence of growth factors and inducers is minimized. This medium creates a metabolically defined environment that is presumably free of extrinsic signals that prevent the decision of neuronal fate. Analysis of the medium components discovered no actual inducer. Hence, it was suggested that the metabolic composition of the medium exclusively covers specific cell requirements of neurons, therefore ensures their survival, and drives the switch from pluripotent cells to neurons. The self-developed method was established by usage of the murine embryonal carcinoma cell line P19 and could be transferred to murine ESC. Consequently, the method could provide a feasible protocol for a generally valid neuronal default model.
The established protocol provides several advantages such as the possibility to generate stable pure neuronal cultures by a fast, simple, and highly reproducible one-step induction under defined medium conditions with a minimum of exogen effectors. The method is characterised by clear and steady medium conditions that makes the investigation of specific cell requirements during differentiation accessible. It is therefore expected to be a useful tool to investigate the molecular basis of neuronal differentiation as well as for high throughput screenings. The phenotype of mature postmitotic neurons was arising within one week and cultures were shown to stay stable at least for three weeks. The neuronal identity was confirmed by expression of neuronal markers through immunofluorescence staining and mass spectrometry analysis. Furthermore, increased levels of axon markers were detected in early neuronal differentiation and functionality of the synapses of the P19-derived neurons was ascertained by detection of calcium activity. Axonal laser ablation, immediately followed by fast regrowth of connections in the neuronal network, revealed a strong regeneration potential under the given conditions. Furthermore, the generated neurons showed a morphologically distinct phenotype and the formation of neural rosettes. Immunofluorescence staining demonstrated the generation of pure and homogeneous neuronal cultures, free of glial cells.
Retinoic acid (RA) plays an essential role in cell signalling during embryogenesis and efficiently induces neuronal differentiation in vitro in a concentration dependent manner. Neither retinol nor retinoic acid was included in any of the components of the self-prepared medium in this work. However, I observed, dependence on RARβ- and/or RARγ-regulated RA signalling in serum-free monolayer cultures. Nevertheless, neuronal differentiation in serum-free monolayer cultures was assumed to be RARα-independent because (i) RARα was slightly downregulated after neuronal induction, (ii) the truncated RARα of the RAC65 mutant had no effect on induction efficiency, and (iii) a pan-RAR inhibitor suppressed neuronal differentiation. In contrast to serum-free monolayer cultures, the truncated RARα prevented neuronal differentiation by application of the conventional protocol where cells are grown in free floating cell aggregates in serum-containing medium. Proteome analysis of P19 cells, treated by the self-developed differentiation protocol over five days showed increased levels of cellular RA binding proteins that mediate the cellular RA transport and are involved in canonical as well as non-canonical RA signalling.
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Acetogenic bacteria are a polyphyletic group of organisms that fix carbon dioxide under anaerobic, non-phototrophic conditions by reduction of two mol of CO2 to acetyl-CoA via the Wood–Ljungdahl pathway. This pathway also allows for lithotrophic growth with H2 as electron donor and this pathway is considered to be one of the oldest, if not the oldest metabolic pathway on Earth for CO2 reduction, since it is coupled to the synthesis of ATP. How ATP is synthesized has been an enigma for decades, but in the last decade two ferredoxin-dependent respiratory chains were discovered. Those respiratory chains comprise of a cytochrome-free, ferredoxin-dependent respiratory enzyme complex, which is either the Rnf or Ech complex. However, it was discovered already 50 years ago that some acetogens contain cytochromes and quinones, but their role had only a shadowy existence. Here, we review the literature on the characterization of cytochromes and quinones in acetogens and present a hypothesis that they may function in electron transport chains in addition to Rnf and Ech.
Im Alter von 77 Jahren verstarb am 5.7.2014 der Mikrobiologe Prof. Friedrich Willi Pons. Nach einem Studium der Biologie und Chemie spezialisierte er sich auf Genetik in der Pionierzeit der Molekularen Biologie in einem sehr guten Umfeld mit den Kollegen B. Rajewsky, Th. Wieland, G. Pfleiderer, R. W. Kaplan, A. Kleinschmidt, H. Zahn. Seine Promotion zur Untersuchung der DNS einiger Serratia-Stämme und deren Phagen bei Prof. R. W. Kaplan fand 1965 sehr viel wissenschaftliche Beachtung.
[Nachruf] Abbas Gholami
(2013)
Dr. Abbas Gholami ist am 28. August 2013 verstorben. Geboren 1945 und aufgewachsen in Quchan, Persien, führte ihn seine Sehnsucht als 18-Jährigen nach Deutschland, nach Frankfurt. Hier nahm er das Chemiestudium an der Goethe-Universität auf und beendete es als Diplomchemiker. Eine Dissertation auf dem Alkaloidgebiet bei Prof. Teuber folgte und 1979 wurde er promoviert.
Zottelige Landschaftspfleger
(2013)