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Thermoanaerobacter kivui is a thermophilic acetogen that can grow on carbon monoxide as sole carbon and energy source. To identify the gene(s) involved in CO oxidation, the genome sequence was analyzed. Two genes potentially encoding CO dehydrogenases were identified. One, cooS, potentially encodes a monofunctional CO dehydrogenase, whereas another, acsA, potentially encodes the CODH component of the CODH/ACS complex. Both genes were cloned, a His-tag encoding sequence was added, and the proteins were produced from a plasmid in T. kivui. His-AcsA copurified by affinity chromatography with AcsB, the acetyl-CoA synthase of the CO dehydrogenase/acetyl CoA synthase complex. His-CooS copurified with CooF1, a small iron-sulfur center containing protein likely involved in electron transport. Both protein complexes had CO:ferredoxin oxidoreductase as well as CO:methyl viologen oxidoreductase activity, but the activity of CooSF1 was 15-times and 231-times lower, respectively. To underline the importance of CooS, the gene was deleted in the CO-adapted strain. Interestingly, the ∆cooS deletion mutant did not grow on CO anymore. These experiments clearly demonstrated that CooS is essential for growth of T. kivui on CO. This is in line with the hypothesis that CooS is the CO-oxidizing enzyme in cells growing on CO.
Neuroligin-3 (Nlgn3), a neuronal adhesion protein implicated in autism spectrum disorder (ASD), is expressed at excitatory and inhibitory postsynapses and hence may regulate neuronal excitation/inhibition balance. To test this hypothesis, we recorded field excitatory postsynaptic potentials (fEPSPs) in the dentate gyrus of Nlgn3 knockout (KO) and wild-type mice. Synaptic transmission evoked by perforant path stimulation was reduced in KO mice, but coupling of the fEPSP to the population spike was increased, suggesting a compensatory change in granule cell excitability. These findings closely resemble those in neuroligin-1 (Nlgn1) KO mice and could be partially explained by the reduction in Nlgn1 levels we observed in hippocampal synaptosomes from Nlgn3 KO mice. However, unlike Nlgn1, Nlgn3 is not necessary for long-term potentiation. We conclude that while Nlgn1 and Nlgn3 have distinct functions, both are required for intact synaptic transmission in the mouse dentate gyrus. Our results indicate that interactions between neuroligins may play an important role in regulating synaptic transmission and that ASD-related neuroligin mutations may also affect the synaptic availability of other neuroligins.
Bei den meisten erwachsenen Säugetieren führt ein Herzinfarkt zu Fibrose und Verlust von funktionellem Herzgewebe. Einige Wirbeltiere, wie der Zebrabärbling, besitzen jedoch die bemerkenswerte Fähigkeit, nach einer Schädigung ihres Herzgewebes verlorenes Gewebe zu regenerieren und so schädliche Folgen zu verhindern. Die lokale Immunantwort auf eine Verletzung wird zunehmend als eine wichtige Determinante für das regenerative Potential eines Gewebes gesehen. Das Komplementsystem ist Teil des humoralen Immunsystems. Historisch ist es als eine Sammlung von Protein bekannt, den Komplementkomponenten, die in der Leber synthetisiert werden und im Blutkreislauf zirkulieren. Bei Exposition gegenüber einem Auslöser, wie z. B. einem Pathogen, wird eine Komplementkomponentproteinspaltungskaskade initiiert, die dazu führen kann, dass Immunzellen rekrutiert werden, und, dass die Phagozytose erleichtert, ggf. die Zielzelle lysiert wird. Studien legen nahe, dass das Komplementsystem an zellulären Prozessen beteiligt sei, die für Entwicklungs- und Krankheitsprozesse entscheidend sind, wie etwa Proliferation und Dedifferenzierung. Es gibt Hinweise, dass das Komplementsystem eine Rolle bei Krebserkrankungen und bei regenerativen Prozessen spielen könnte. In verschiedenen Arten wurde eine lokale verletzungsinduzierte Expression von komplementkomponentkodierenden Genen in regenerierendem Gewebe beobachtet.
Einzelne Studien legen nahe, dass Funktionsverlust einzelner Komplementkomponenten regenerative Prozesse beeinträchtigt.
Offene Fragen bleiben jedoch: Ist die lokale Expression von mehreren komplementkomponentkodierenden Genen ein Merkmal von regenerierendem Gewebe, das sie von Geweben unterscheidet, welchem die Fähigkeit zur Regeneration fehlt? Und welche Rolle könnte das Komplementsystem und seine Komponenten während des regenerativen Prozesses spielen? Um diesen Fragen nachzugehen, wurde eine Expressionsanalyse von Zebrabärblingsgewebe nach Verletzung mittels RT-qPCR und in situ Hybridisierung durchgeführt: kardiale Kryoverletzung, Larvenrumpfamputation und Schwanzflossenamputation. Ich beobachtete, dass mehrere komplementkomponentkodierende Gene in diesen Geweben nach Verletzung induziert wurden. Die Interpretation veröffentlichter single cell RNAseq Datensätze legt nahe, dass diese komplementkomponentenkodierenden Gene von verschiedenen Zelltypen exprimiert werden, darunter Immunzellen, Epikardzellen und Fibroblasten. Um transkriptionelle Unterschiede zwischen regenerierendem und nicht regenerierendem Gewebe zu identifizieren, verwendete ich ein nicht regeneratives Zebrabärblingmodell, die il11ra- Mutante. Dieser Mutante fehlt die Fähigkeit, verschiedene Organe zu regenerieren, das ist der Fall beim Herzen, dem larvalen Rumpf, und der Schwanzflosse. Ich stellte fest, dass die Mehrheit der verletzungsinduzierten komplementkomponentkodierenden Gene il11ra nachgeschaltet war. Darüber hinaus zeigten Experimente unter Verwendung chemischer Inhibitoren, dass speziell die Expression der komplementkomponentkodierenden Gene c3a.1,
c4b und c7a im Larvenrumpfamputationsmodell durch den Il11-Stat3-Signalweg moduliert wird.
Zur Klärung der Frage, ob das Komplementsystem und/ oder seine Komponenten eine Rolle während der Regeneration spielen, wurden verschiede Funktionsverlustmodelle generiert und im larvalen Rumpfamputationsmodell auf mögliche Aberrationen getestet. Zum einen generierte ich Überexpressionslinien von endogenen Inhibitoren der Komplementproteinspaltungskaskade. Überexpression eines etablierten Komplementsysteminhibitors rca2.1/ tecrem führte zu einer im Vergleich zu Wildtyp- Geschwistern verringerten Regeneration des larvalen Rumpfs. Zum anderen generierte ich Funktionsverlustmutanten von individuellen Komplementkomponenten durch CRISPR/Cas9 vermittelter Mutagenese, und zwar für masp1, masp2, cfd, c1s, c4b, c5 und c9. Die larvale Rumpfregeneration war in diesen Mutanten unauffällig. Allerdings zeigten c4b Mutanten eine verringerte Kardiomyozytenproliferation und eine differenzielle Expression von einigen Markergenen, einschließlich einer erhöhten Expression von inflammatorischen Zytokinen.
Meine Studien führten zu neuen Einblicken in das Komplementsystem im Kontext der Regeneration. Ich fand heraus, dass mehrere komplementkomponentenkodierenden Gene in regenerierendem Zebrabärblinggewebe exprimiert werden, und zwar im Herzgewebe, im larvalen Rumpf und in der adulten Flosse. Darüber hinaus zeige ich, dass die verletzungsinduzierte Expression von komplementkodierenden Genen in regenerierendem Gewebe dem Regenerationsmasterregulator il11ra nachgeschaltet ist. Speziell c3a.1, c4b und c7a wurden durch il11/ stat3 reguliert...
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.
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 all 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, chemicals that induced proliferation, growth, and triglyceride accumulation in 3T3-L1 adipocytes were found in one third of the products. Since the majority did not target peroxisome proliferator-activated receptor γ, the effects are 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.
Teaser Plastics contain a potent mixture of chemicals promoting adipogenesis, a key process in developing obesity.
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.
Differential derepression of the genome of potato tuber cells can be initiated by slicing the tissue into disks. The consequence of this procedure on the cells of the wound surface is dedifferentiation and cell division followed by redifferentiation to a suberized phellem cell. The drift of glucose-, glucose-1-phosphate-, glucose-6-phosphate-, fructose-6-phosphate- and 6-phospho-gluconatelevels has been determined in the derepressed tissue. With the exception of 6-phospho-gluconate all intermediates so far investigated showed a rise in concentration after derepression.
This is interpreted as a consequence of altered enzymic activities which were estimated for phosphoglucomutase, hexokinase, phosphoglucoisomerase, gluco-6-phosphate- and 6-phosphogluconatedehydrogenase. The two dehydrogenases were activated after derepression, the activation represented a de-novo-synthesis, as was demonstrated with the inhibitors Actidione (translation) and p-Fluorophenyl-alanine (protein synthesis in general). Hexokinase and phosphoglucoisomerase were not severely affected by cutting the tissue. Phosphoglucomutase was degrated rapidly, the degradation being dependent on protein synthesis. The importance of an enhanced activity of the pentose phosphate shunt for the stressed cell is emphasized and the possibility of an alteration in the osmotic pressure within the cell and especially in the nucleus — a primary consequence of wounding — as a cause of derepression in potato tuber cells is discussed.
The toxicity of microplastics on Daphnia magna as a 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 effects on overall population size and structure, 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 28–42% 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 impacts 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 be 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.
The toxicity of microplastics on Daphnia magna as a 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 particles mL-1) and recorded the effects on overall population size and structure, the size of the individual animals, and resting egg production. Particle exposure adversely affected the population size and structure and induced resting egg production. The terminal population size was 28–42% 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 impacts 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 be 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.
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.
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.
Thermoanaerobacter kivui is an acetogenic model organism that reduces CO2 with electrons derived from H2 or CO, or from organic substrates in the Wood–Ljugdahl pathway (WLP). For the calculation of ATP yields, it is necessary to know the electron carriers involved in coupling of the oxidative and reductive parts of metabolism. Analyses of key catabolic oxidoreductases in cell-free extract (CFE) or with purified enzymes revealed the physiological electron carriers involved. The glyceraldehyde-3-phosphate dehydrogenase (GA3P-DH) assayed in CFE was NAD+-specific, NADP+ was used with less than 4% and ferredoxin (Fd) was not used. The methylene-THF dehydrogenase was NADP+-specific, NAD+ or Fd were not used. A Nfn-type transhydrogenase that catalyzes reduced Fd-dependent reduction of NADP+ with NADH as electron donor was also identified in CFE. The electron carriers used by the potential electron-bifurcating hydrogenase (HydABC) could not be unambiguously determined in CFE for technical reasons. Therefore, the enzyme was produced homologously in T. kivui and purified by affinity chromatography. HydABC contained 33.9 ± 4.5 mol Fe/mol of protein and FMN; it reduced NADP+ but not NAD+. The methylene-THF reductase (MetFV) was also produced homologously in T. kivui and purified by affinity chromatography. MetFV contained 7.2 ± 0.4 mol Fe/mol of protein and FMN; the complex did neither use NADPH nor NADH as reductant but only reduced Fd. In sum, these analysis allowed us to propose a scheme for entire electron flow and bioenergetics in T. kivui.
Streams and rivers are characterised by the presence of various chemicals of emerging concern (CECs), including pesticides, pharmaceuticals, personal care products, and industrial chemicals. While these chemicals are found usually only in low (ng/L) concentrations, they might still harm aquatic life and disrupt the ecological balance of aquatic ecosystems due to their high ecotoxicological potency. Environmental risk assessments that account for the complexity of exposures are needed in order to evaluate the toxic pressure of these chemicals, which also provide suggestions for risk mitigation and management, if necessary. Currently, most studies on the co-occurrence and environmental impacts of CECs are conducted in countries of the Global North, leaving massive knowledge gaps in countries of the Global South.
In this study, we implement a multi-scenario risk assessment strategy to improve the assessment of both the exposure and hazard components in the chemical risk assessment process. Our strategy incorporates a systematic consideration and weighting of CECs that were not detected, as well as an evaluation of the uncertainties associated with Quantitative Structure-Activity Relationships (QSARs) predictions for chronic ecotoxicity. Furthermore, we present a novel approach to identifying mixture risk drivers. To expand our knowledge beyond well-studied aquatic ecosystems, we applied this multi-scenario strategy to the River Aconcagua basin of Central Chile. The analysis revealed that the concentrations of CECs exceeded acceptable risk thresholds for selected organism groups and the most vulnerable taxonomic groups. Streams flowing through agricultural areas and sites near the river mouth exhibited the highest risks. Notably, the eight risk drivers among the 153 co-occurring chemicals accounted for 66-92% of the observed risks in the river basin. Six of them are pesticides and pharmaceuticals, chemical classes known for their high biological activity in specific target organisms.