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The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5′-untranslated region (5′-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.
Accurate determination of the evolutionary relationships between genes is a foundational challenge in biology. Homology—evolutionary relatedness—is in many cases readily determined based on sequence similarity analysis. By contrast, whether or not two genes directly descended from a common ancestor by a speciation event (orthologs) or duplication event (paralogs) is more challenging, yet provides critical information on the history of a gene. Since 2009, this task has been the focus of the Quest for Orthologs (QFO) Consortium. The sixth QFO meeting took place in Okazaki, Japan in conjunction with the 67th National Institute for Basic Biology conference. Here, we report recent advances, applications, and oncoming challenges that were discussed during the conference. Steady progress has been made toward standardization and scalability of new and existing tools. A feature of the conference was the presentation of a panel of accessible tools for phylogenetic profiling and several developments to bring orthology beyond the gene unit—from domains to networks. This meeting brought into light several challenges to come: leveraging orthology computations to get the most of the incoming avalanche of genomic data, integrating orthology from domain to biological network levels, building better gene models, and adapting orthology approaches to the broad evolutionary and genomic diversity recognized in different forms of life and viruses.
Weltweit werden etwa 17% aller Infektionskrankheiten von Vektoren auf den Menschen übertragen. Dabei dienen meist blutsaugende Arthropoden wie Stechmücken, Zecken oder Sandfliegen als Überträger von Bakterien, Viren oder einzelligen Parasiten. Zur letzteren Gruppe gehört auch der protozoische Erreger der Chagas-Krankheit Trypanosoma cruzi. Er wird von hämatophagen Triatominae, einer Unterfamilie der Raubwanzen (Hemiptera: Reduviidae) während der Blutmahlzeit an einem infizierten Säugerwirt aufgenommen, durchläuft komplexe Entwicklungsschritte im intestinalen Trakt der triatominen Insekten und wird anschließend über den Fäzes und Urin der Wanzen abgegeben. Die Infektion des nächsten Wirts erfolgt dann durch das versehentliche Einreiben der Erreger in die Stichwunde oder auf Schleimhäute. Auch eine Infektion über die orale Aufnahme von kontaminierter Nahrung, Mutter-Kind-Infektionen und die Übertragung durch Blutkonserven und Organtransplantate sind möglich. Die Chagas‑Krankheit, oder auch Amerikanische Trypanosomiasis, ist insbesondere in Mittel- und Südamerika verbreitet und betrifft nach Schätzungen der WHO 6 bis 7 Millionen Menschen. Infolge von globaler Immigration und erhöhtem Reiseverkehr treten jedoch in den letzten Jahrzehnten auch vermehrt Fälle in Europa, den USA, Kanada und den westlichen Pazifikstaaten auf. Da dort bislang geeignete Vektoren fehlen, kommt es außerhalb des lateinamerikanischen Kontinents nicht zu vektorübertragenen Infektionen. Dies könnte sich jedoch im Zuge des Klimawandels und einer voranschreitenden Globalisierung ändern, sollte der Ausbreitung der Chagas-Krankheit eine Ausbreitung ihrer triatominen Vektoren folgen.
Inwieweit Triatominae unter heutigen Bedingungen klimatisch geeignete Habitate außerhalb des amerikanischen Kontinents finden, wurde innerhalb des ersten Projekts der vorliegenden Dissertation untersucht. Dazu wurde mit Hilfe der ökologischen Nischenmodellierung und Vorkommensdaten verschiedener vektorkompetenter Raubwanzenarten sowie klimatischer Umweltvariablen die klimatische Eignung verschiedenster Lebensräume modelliert und global projiziert. Es zeigte sich, dass insbesondere tropische und subtropische Gebiete Afrikas sowie Ost- und Südostasiens zwischen 21° nördlicher Breite und 24° südlicher Breite für viele triatomine Vektorarten geeignete Bedingungen aufweisen. Auffällig ist dabei insbesondere die Art Triatoma rubrofasciata, welche nachweislich bereits in Südchina, Vietnam und weiteren Ländern Afrikas und Asiens gefunden wurde. Die Modellierung
offenbarte, dass weitere ausgedehnte Teile der Küstenregionen Afrikas und Südostasiens als für T. rubrofasciata klimatisch geeignet angesehen werden müssen. Eine weitere Ausbreitung dieser Art ist demnach äußerst wahrscheinlich und stellt bislang das größte Risiko autochthon übertragener Chagas-Infektionen außerhalb des amerikanischen Kontinents dar. Es konnten außerdem zwei triatomine Arten identifiziert werden, namentlich T. infestans und T. sordida, welche in gemäßigten Klimazonen geeignete Habitate finden. Zu diesen gehören beispielsweise Neuseeland und Teile Australiens, aber auch südeuropäische Länder wie Spanien, Italien, Griechenland und Portugal. Da mit einer Ausweitung der klimatisch geeigneten Gebiete infolge des sich verändernden Klimas zu rechnen ist, wäre ein Monitoring der Vektoren, wie es bereits in Südchina etabliert ist, aber insbesondere die Einführung der Meldepflicht für Amerikanische Trypanosomiasis in diesen Regionen sinnvoll. Die Ergebnisse der Studie zeigen deutlich, dass die bisher vernachlässigte Tropenkrankheit Chagas nicht allein ein Problem des lateinamerikanischen Kontinents ist, sondern deren Erforschung vielmehr weltweit Beachtung finden sollte.
So konzentrierten sich die folgenden Forschungsprojekte der Promotion verstärkt auf die Mechanismen, welche die Entwicklung und Transmission des Parasiten und die Interaktion mit seinen Vektoren betreffen. Von besonderem Interesse waren dabei die ökologischen Prozesse, welche bei der Kolonisation des Darmtrakts der Vektoren durch T. cruzi ablaufen und essentiell für die Proliferation und damit die Übertragung des Parasiten sind. Eine entscheidende Rolle spielen dabei die mit dem Vektor assoziierten Mikroorganismen und ihre funktionellen Fähigkeiten – zusammengefasst als Mikrobiom bezeichnet. Dieses erfüllt wichtige physiologische Funktionen des Insekts und kann beispielsweise das Immunsystem und die Detoxifikation beeinflussen. Um die Veränderungen der organismischen Zusammensetzung und der funktionellen Kapazitäten, welche die Infektion mit dem Pathogen im Darmtrakt der Vektoren auslösen, zu untersuchen, wurde ein metagenomischer Shotgun Sequenzierungsansatz gewählt. Die daraus resultierenden Datensätze wurden anschließend bioinformatisch ausgewertet und auf ihre mikrobielle Zusammensetzung und metabolischen Fähigkeiten hin untersucht. Es zeigte sich zunächst, dass das Bakterium Rhodococcus rhodnii, welches lange als alleiniger echter Symbiont des untersuchten Vektors Rhodnius prolixus galt, in seiner Funktionalität nicht einzigartig im Mikrobiom des Insekts ist. ...
Peronospora belbahrii is one of the most destructive downy mildew diseases that has emerged throughout the past two decades. Due to the lack of quarantine regulations and its possible seed-borne nature, it has spread globally and is now present in most areas in which basil is produced. While most obligate biotrophic, plant parasitic oomycetes are highly host-specific, there are a few that have a wider host range, e.g. Albugo candida, Bremia tulasnei, and Pseudoperonospora cubensis. Recently, it was shown that Peronospora belbahrii is able to infect Rosmarinus, Nepetia, and Micromeria in Israel in cross-infection trials, hinting an extended host range for also this pathogen. In this study, a newly occurring downy mildew pathogen on lavender was investigated with respect to its morphology and phylogeny, and it is shown that it belongs to Peronospora belbahrii as well. Thus, it seems that Peronospora belbahrii is currently extending its host range to additional members of the tribe Mentheae and Ocimeae. Therefore, it seems advisable to scrutinise all commonly used members of these tribes in order to avoid further spread of virulent genotypes.
In Zeiten der globalen Klimaerwärmung und des Klimawandels werden Strategien zur Vermeidung, Reduzierung oder Wiederverwertung von CO2-Emissionen sowie die Abkehr von fossilen Energieträgern immer wichtiger. Aus diesem Grund finden Technologien zur Bindung, Speicherung und Wiederverwertung von CO2 immer größere Aufmerksamkeit und diverse chemische als auch biologische Ansätze werden verfolgt. Eine dieser Möglichkeiten umfasst die Reduktion von CO2 mit Hilfe von molekularem Wasserstoff. Im Prozess der direkten Hydrogenierung von CO2 zu Ameisensäure bzw. Formiat wird nicht nur CO2 gebunden, sondern ebenfalls H2 in flüssiger Form gespeichert. Die Ameisensäure weist gegenüber dem hochflüchtigen Wasserstoffgas verschiedene Vorteile auf und zählt zu der Gruppe der flüssigen, organischen Wasserstoffspeicherverbindungen. Daneben ist das Einsatzgebiet von Ameisensäure als Ausgangstoff für Chemikalien oder als mikrobielle Kohlenstoffquelle sehr vielseitig und die Verbindung erfreut sich zunehmenden Interesses.
Die Natur hält biologische Katalysatoren (Enzyme) für die Reduktion von CO2 bereit. Die Gruppe der obligat anaeroben, acetogenen Bakterien verwendet so genannte Formiatdehydrogenasen als CO2-Reduktasen, um CO2 im Wood-Ljungdahl-Weg (WLP) der Bakterien fixieren zu können. Diese Enzyme katalysieren die reversible 2-Elektronen Reduktion von CO2 zu Ameisensäure. Kürzlich konnte aus den beiden Vertretern A. woodii (mesophil) und T. kivui (thermophil) ein neuartiger, cytoplasmatischer Enzymkomplex isoliert werden. Dieser Enzymkomplex koppelt die Reduktion von CO2 direkt an die Oxidation von H2 und wird deshalb als Wasserstoff-abhängige CO2-Reduktase bezeichnet (engl. hydrogen-dependent CO2 reductase, HDCR). Die HDCR katalysiert dabei die reversible Hydrogenierung von CO2 zu Formiat mit annähernd gleicher Kinetik und gleichen Umsatzraten. Die bei der CO2 Reduktion erreichten Umsatzraten übertrafen dabei bisherige chemische als auch biologische Katalysatoren um mehre Größenordnungen.
Im Hinblick auf die besonderen katalytischen Eigenschaften der HDCRs wurde in dieser Arbeit die biotechnologische Anwendbarkeit der Enzyme als Biokatalysatoren zur Speicherung und Sequestrierung von H2 und CO2 in Form von Ameisensäure untersucht. Im Speziellen wurde ein HDCR-basiertes Ganz-Zell-System für das thermophile Bakterium T. kivui entwickelt. Um eine Ganz-Zell basierte Umwandlung von H2 und CO2 zu Formiat zu gewährleisten, wurde zuvor die Weiterverwertung des Formiats zu Acetat im WLP gestoppt. Durch eine Reduktion des zellulären ATP-Gehalts konnte eine weitere Prozessierung des aus der HDCR-Reaktion gebildeten Formiats im Zellstoffwechsel des Bakteriums unterbunden werden. Die Formiatbildung aus H2 und CO2 wurde in Zellsuspensionen von T. kivui untersucht und charakterisiert. Hier zeigten T. kivui Zellen die höchste spezifische Formiatbildungsrate, die bis dato in der Literatur genannt wurde. Ebenfalls wurde in dieser Arbeit die Umwandlung von Synthesegas (H2 + CO2 und CO) und CO zu Formiat geprüft. Bioenergetisch entkoppelte und auf CO-adaptierte T. kivui Zellen konnten in der Tat Synthesegas exklusiv zu Formiat umsetzen. Um die CO-Verwertung zu Acetat und Formiat im Stoffwechsel der Rnf- (A. woodii) und Ech-Acetogenen (T. kivui) verstehen zu können, wurden Mutanten von Δhdcr, ΔcooS, ΔhydBA, Δrnf and Δech2 von A. woodii und T. kivui zur Hilfe genommen. In beiden Organismen war die CO-basierte Formiatbildung vom Vorhandensein eines funktionalen HDCR-Enzymkomplexes abhängig.
Für eine mögliche biotechnologische Anwendung wurde die Maßstabsvergrößerung des Ganz-Zell-Systems angestrebt und hin zum Bioreaktormaßstab mit kontrollierten Prozessbedingungen skaliert. Diese Arbeit demonstriert die effiziente Umwandlung von H2 und CO2 zu Formiat und vice versa unter Verwendung eines Rührkesselreaktors. Der Prozess zeigte eine Effizienz von 100% für die Umwandlung von CO2 zu Formiat und spezifische Raten von 48.3 mmol g-1 h-1 wurden von A. woodii Zellen erreicht. Die spezifische H2-Produktionsrate (qH2) aus der Ameisensäureoxidation betrug 27.6 mmol g-1 h-1 und mehr als 2.12 M Ameisensäure konnte über einen Zeitraum von 195 h oxidiert werden. Wichtige Parameter der Enzymkatalyse wie Wechselzahl (engl. turnover frequency, TOF) und katalytische Produktivität (engl. turnover number, TON) wurden ebenfalls im Versuch bestimmt. Basierend auf dem generierten Prozessverständnis und der effizienten Reversibilität der katalysierten Reaktionen wurde abschließend ein Ganz-Zell-basierter Bioreaktoraufbau gewählt, der die vielfache Speicherung und Freisetzung von H2 in einem einzigen Rührkesselreaktor und unter Verwendung des gleichen Katalysators ermöglicht. Über eine Prozesszeit von 2 Wochen und 15 CO2 Reduktions-/Formiat Oxidations-Zyklen konnte so im Mittel 330 mM Formiat produziert und oxidiert werden.
Zusammenfassend thematisiert diese Arbeit die biotechnologische Anwendbarkeit eines Ganz-Zell-Systems zur Speicherung und Sequestrierung von H2 und CO2 in Form von Formiat und vice versa. Die katalytische Aktivität der betrachteten Organismen fußt dabei auf der Aktivität eines neuartigen Enzymkomplexes, der erstmals in der Gruppe der acetogenen Bakterien entdeckt wurde. Der als Wasserstoff-abhängige CO2-Reduktase bezeichnete Enzymkomplex könnte die zukünftige Konzipierung Enzym-inspirierter und effizienter chemischer Katalysatoren vorantreiben. Auch der Einsatz des Enzyms/der Zellen in so genannten Hydrogelen oder die Etablierung elektrochemischer Prozesse sind vorstellbar. Diese Arbeit stellt somit eine Basis für mögliche zukünftige Anwendungen des etablierten Ganz-Zell-Systems von A. woodii und T. kivui im Bereich der Wasserstoffökonomie dar.
The increasing demand of the high value ω-3 fatty acids due to its beneficial role for human health, explains the huge need for alternative production ways of ω-3 fatty acids. The oleaginous alga Phaeodactylum tricornutum is a prominent candidate and has been investigated as biofactory for ω-3 fatty acids, e.g. the synthesis of eicosapentaenoic acid (EPA). In general, the growth and the lipid content of diatoms can be enhanced by genetic engineering or are influenced by environmental factors, e.g. nutrients, light or temperature.
In this study, the potential of P. tricornutum as biofactory was improved by heterologously expressing the hexose uptake protein 1 (HUP1) from the Chlorophyte Chlorella kessleri.
An in situ localization study revealed that only the full length HUP1 protein fused to eGFP was correctly targeted to the plasma membrane, whereas the N-terminal sequence of the protein is only sufficient to enter the ER. Protein and gene expression data displayed that the gene-promoter combination was relevant for the expression level of HUP1, while only cells expressing the protein under the light-inducible fcpA promoter showed a significant expression. In these mutants an efficient glucose uptake was detectable under mixotrophic growth condition, low light intensities and low glucose concentrations leading to an increased cell dry weight.
In a second approach, the growth and lipid content of wildtype cells were analyzed in a small 1l photobioreactor. Here, a commercial F/2 medium and a common culture medium, ASP and modified versions were compared. There was neither a significant impact on the growth and lipid content in P. tricornutum cells due to the supplemention of trace elements nor due to elevated salt concentrations in the media. In a modified version of ASP medium, with adapted nitrate and phosphate concentration a constantly high biomass productivity was achieved, yielding the highest value of 82 mg l-1 d-1 during the first three days. This was achieved even though light intensity was reduced by 40%. The differences in biomass productivity as well as the lipid content and the lipid composition underlined the importance of the choice of culture medium and the harvest time for enhanced growth and EPA yields in P. tricornutum.
Non-technical summary: There has been a long history of conflicts, studies, and debate over how to both protect rivers and develop them sustainably. With a pause in new developments caused by the global pandemic, anticipated further implementation of the Paris Agreement and high-level global climate and biodiversity meetings in 2021, now is an opportune moment to consider the current trajectory of development and policy options for reconciling dams with freshwater system health. Technical summary: We calculate potential loss of free-flowing rivers (FFRs) if proposed hydropower projects are built globally. Over 260,000 km of rivers, including Amazon, Congo, Irrawaddy, and Salween mainstem rivers, would lose free-flowing status if all dams were built. We propose a set of tested and proven solutions to navigate trade-offs associated with river conservation and dam development. These solution pathways are framed within the mitigation hierarchy and include (1) avoidance through either formal river protection or through exploration of alternative development options; (2) minimization of impacts through strategic or system-scale planning or re-regulation of downstream flows; (3) restoration of rivers through dam removal; and (4) mitigation of dam impacts through biodiversity offsets that include restoration and protection of FFRs. A series of examples illustrate how avoiding or reducing impacts on rivers is possible – particularly when implemented at a system scale – and can be achieved while maintaining or expanding benefits for climate resilience, water, food, and energy security. Social media summary: Policy solutions and development pathways exist to navigate trade-offs to meet climate resilience, water, food, and energy security goals while safeguarding FFRs.
Wie Zootiere kommunizieren
(2022)
Die Kirschen in Nachbars Garten sind vermeintlich süßer – und Männer mit festen Partnerinnen für andere Frauen oft attraktiver. Ehering-Effekt nennen das die Psychologen. Dahinter steckt ein uraltes Gesetz der Biologie. Vereinfacht ausgedrückt: Nicht alle guten Männer sind vergeben, doch diejenigen Männer, die vergeben sind, sind gut. Ähnlich ist es auch im Tierreich: So haben Forscher der Goethe-Universität herausgefunden, dass bei Fischen, genauer bei Atlantikkärpflingen, die Weibchen Partner bevorzugen, die zuvor bereits mit anderen Partnern zusammen waren. Interessant ist allerdings, dass es den Weibchen egal war, ob der Partner vorher hetero- oder homosexuelles Verhalten gezeigt hatte. Bei den Fischen ist sexuelle Aktivität an sich offenbar ein Qualitätsmerkmal, das gesunde von kranken und unterernährten Partnern unterscheidet. Bisexualität erhöht also den Fortpflanzungserfolg.
The ORCID iDs are missing for the second, fifth, and sixth authors. Please see the authors’ respective ORCID iDs here:
Author Christine Hertler’s ORCID iD is: 0000-0002-8252-9674 (https://orcid.org/0000-0002-8252-9674).
Author Jan Ole Berndt’s ORCID iD is: 0000-0001-7241-3291 (https://orcid.org/0000-0001-7241-3291).
Author Ingo J. Timm’s ORCID iD is: 0000-0002-3369-813X (https://orcid.org/0000-0002-3369-813X).
Neuro-vascular communication is essential to synchronize central nervous system development. Here, we identify angiopoietin/Tie2 as a neuro-vascular signaling axis involved in regulating dendritic morphogenesis of Purkinje cells (PCs). We show that in the developing cerebellum Tie2 expression is not restricted to blood vessels, but it is also present in PCs. Its ligands angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are expressed in neural cells and endothelial cells (ECs), respectively. PC-specific deletion of Tie2 results in reduced dendritic arborization, which is recapitulated in neural-specific Ang1-knockout and Ang2 full-knockout mice. Mechanistically, RNA sequencing reveals that Tie2-deficient PCs present alterations in gene expression of multiple genes involved in cytoskeleton organization, dendritic formation, growth, and branching. Functionally, mice with deletion of Tie2 in PCs present alterations in PC network functionality. Altogether, our data propose Ang/Tie2 signaling as a mediator of intercellular communication between neural cells, ECs, and PCs, required for proper PC dendritic morphogenesis and function.
Aim: Knowledge concerning species distribution is important for biodiversity conservation and environmental management. Fungi form a large and diverse group of species and play a key role in nutrient cycling and carbon storage. However, our understanding of fungal diversity and distribution remains limited, particularly at large spatial scales. Here, we predicted the diversity and distribution of ectomycorrhizal and saprotrophic macrofungi at relatively fine spatial resolution at a continental scale and examined the importance of variables that affect the distribution of these two functional groups. Location: Europe. Time period: 1990–2018. Major taxa studied: Macrofungi. Methods: From observations of 1,845 macrofungal species, we predicted the diversity and distribution of two functional groups of macrofungi at a resolution of 5 km across eight European countries based on 25 environmental variables using the MAXENT model. We determined the importance of variables that affect the distribution of these two functional groups of macrofungi using the built-in jackknife test in the model. Results: Analysis of the modelling results showed that eastern Denmark and southern Sweden are biodiversity hotspots for both functional groups of macrofungal species. Tree species and human disturbance (i.e., the human footprint index) were found to be the two most important predictor variables explaining the distribution of ectomycorrhizal and saprotrophic macrofungi. Main conclusions: Overall, our study demonstrates that tree species and human disturbance have played a more important role than climatic factors in determining the diversity and distribution of macrofungi at the continental scale. Our study suggests that fungal diversity and distribution might change considerably if the strongest predictors (i.e., tree species) were to be affected by climate change and/or human activity. Changes in fungal diversity might, in turn, influence other processes, because fungi are important in driving ecosystem processes, such as nutrient and carbon cycling.
An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
With 5-10 newly diagnosed patients per 100,000 people every year, glioblastoma is the most common malignant primary brain tumor. Despite extensive research activity in the last decades, clinical effectiveness of the currently available therapy standard of surgery, radiochemotherapy and tumor-treating fields is still limited and mean survival rates in unselected collectives are only about one year. Accordingly, there is an urgent need to explore new therapeutic options. The current standard of care includes surgery followed by radiation therapy in combination with the alkylating chemotherapeutic agent Temozolomide. Even with successful initial therapy, tumor recurrence is still inevitable. Currently, there are no defined recommendations for clinical management of the disease in the event of tumor recurrence. Only 20-30% of patients qualify for a second surgical resection, while other options include retreatment with Temozolomide, CCNU (Lomustine) or Regorafenib and enrollment in a clinical trial.
The development of immunotherapies for glioblastoma, in particular, has been the focus of intense preclinical and clinical efforts. However, low numbers of mutations and a highly immunosuppressive tumor microenvironment result in glioblastoma being considered an immunologically “cold” tumor. Strategies successfully established in mutagen-induced tumors with antibodies directed against the PD-1, PD-L1 or CTLA-A4 immune checkpoints have therefore failed in glioblastoma.
Cellular immunotherapies based on chimeric antigen receptor (CAR)-technology have emerged as an alternative powerful option to tackle immunologically “cold” tumors. Several CAR-T cell products targeting glioma antigens have been developed and some evidence of clinical activity has been demonstrated. Natural killer (NK) cells as carriers of CAR constructs have several advantages over T cells, including a much lower risk of neurotoxicity and better interaction with immune cells in the microenvironment. Based on the human NK cell line NK-92, a clinical-grade product, suitable as an off-the-shelf therapeutic, has been developed. The NK-92/5.28.z clone (CAR-NK) expresses a CAR based on the HER2-specific antibody FRP5 in addition to signal-enhancing CD28 and CD3ζ domains. Similar to several other tumor entities, overexpression of the growth factor receptor HER2 is often found in glioblastoma patients. Because of its substantial role in the regulation of cell proliferation, survival, differentiation, angiogenesis and invasion, this receptor is classified as an oncogene. HER2 overexpression plays a major role in the malignant transformation of cells and its oncogenic potential has been studied in detail in breast cancer. However, HER2 expression was also found in up to 80% of glioblastomas, which correlates with an impaired probability of survival. Under physiological conditions, HER2 is not expressed in the adult central nervous system, making it a promising target antigen for glioblastoma immunotherapy.
In previous projects, it has already been shown that these CAR-NK cells exhibit a high and specific lytic activity towards HER2+ glioblastoma cells. While repetitive intratumoral injections of CAR-NK cells already significantly extended symptom-free survival in murine orthotopic xenograft models, CAR-NK cell therapy in immunocompetent mice promotes an endogenous anti-tumor immune response which improves tumor control and provides persisting anti-tumor immunity after therapy of early-stage tumors. However, in more advanced tumor models, efficacy is limited and induction of the checkpoint-molecule PD-L1 in response to CAR-NK-cell therapy was identified as a key mechanism of therapy resistance.
Immunotherapy employing the intravenous administration of checkpoint inhibitors has already revolutionized the treatment of various malignant diseases such as melanoma or lung cancer. In particular, the approach of cancer immunotherapy has focused on the systemic administration of antibodies directed against immune checkpoints such as PD-1, PD-L1 and CTLA-4. In glioblastoma, both tumor cells and microglia, the brain-resident macrophages, express PD-L1, which hinders the activation of CD8+ and CD4+ T cells. Therefore, immunotherapy directed against the PD-1/PD-L1 axis represents a promising approach for the treatment of glioblastoma. One problem, however, is the severe toxicity caused by the systemic effects of checkpoint inhibitors, since the immune response is stimulated not only in tumor tissue but also in healthy organs. Serious side effects such as colitis, hepatitis, pancreatitis or hypophysitis, including numerous deaths, have been reported.
This study aimed to improve the efficacy of CAR-NK cell therapy by combining it with adeno-associated virus (AAV)-mediated transfer of anti-PD-1 antibodies as a strategy to enable local combination therapy to control intracranial tumors.
AAVs carrying a payload coding for an anti-PD-1 immunoadhesin (aPD-1) retargeted to HER2-expressing cells by fusion of so-called Designed Ankyrin Repeat Proteins (DARPins) with a viral capsid protein were employed for this to focus checkpoint inhibitor therapy to the tumor area, resulting in high intratumoral and low systemic drug concentrations. ...
We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.
The ingestion of microplastics (MPs) is well documented for various animals and spherical MPs (beads) in many studies. However, the retention time and egestion of MPs have been examined less, especially for irregular MPs (fragments) which are predominantly found in the environment. Furthermore, the accumulation of such particles in the gastrointestinal tract is likely to determine whether adverse effects are induced. To address this, we investigated if the ingestion and egestion of beads are different to those of fragments in the freshwater shrimp Neocaridina palmata. Therefore, organisms were exposed to 20–20,000 particles L−1 of either polyethylene (PE) beads (41 μm and 87 μm) or polyvinyl chloride (PVC) fragments (<63 μm). Moreover, shrimps were exposed to 20,000 particles L−1 of either 41 μm PE and 11 μm polystyrene (PS) beads or the PVC fragments for 24 h, followed by a post-exposure period of 4 h to analyze the excretion of particles. To simulate natural conditions, an additional fragment ingestion study was performed in the presence of food. After each treatment, the shrimps were analyzed for retained or excreted particles. Our results demonstrate that the ingestion of beads and fragments were concentration-dependent. Shrimps egested 59% of beads and 18% of fragments within 4 h. Particle shape did not significantly affect MP ingestion or egestion, but size was a relevant factor. Medium- and small-sized beads were frequently ingested. Furthermore, fragment uptake decreased slightly when co-exposed to food, but was not significantly different to the treatments without food. Finally, the investigations highlight that the assessment of ingestion and egestion rates can help to clarify whether MPs remain in specific organisms and, thereby, become a potential health threat.
Growing amounts of genomic data and more efficient assembly tools advance organelle genomics at an unprecedented scale. Genomic resources are increasingly used for phylogenetic analyses of many plant species, but are less frequently used to investigate within-species variability and phylogeography. In this study, we investigated genetic diversity of Fagus sylvatica, an important broadleaved tree species of European forests, based on complete chloroplast genomes of 18 individuals sampled widely across the species distribution. Our results confirm the hypothesis of a low cpDNA diversity in European beech. The chloroplast genome size was remarkably stable (158,428 ± 37 bp). The polymorphic markers, 12 microsatellites (SSR), four SNPs and one indel, were found only in the single copy regions, while inverted repeat regions were monomorphic both in terms of length and sequence, suggesting highly efficient suppression of mutation. The within-individual analysis of polymorphisms showed >9k of markers which were proportionally present in gene and non-gene areas. However, an investigation of the frequency of alternate alleles revealed that the source of this diversity originated likely from nuclear-encoded plastome remnants (NUPTs). Phylogeographic and Mantel correlation analysis based on the complete chloroplast genomes exhibited clustering of individuals according to geographic distance in the first distance class, suggesting that the novel markers and in particular the cpSSRs could provide a more detailed picture of beech population structure in Central Europe.
The effect of the extreme summer drought and heatwave 2018 in Central Europe on wood properties of oaks at four sandy valley river sites (Quercus robur L.) and one south-exposed schist slope (Qu. petraea (Matt.) Liebl.) in the middle Rhine and lower Main valley were studied and compared to well-watered trees from a riparian stand. While properties of the 2018 tree rings mostly resembled those of the previous (wet) year, significant decreases in Δ13C, wood density and ring width occurred in 2019 at most drought-prone sites. In the sandy sites, ring widths correlated with previous-year precipitation from June to August over a 20-year period. In organs formed in 2018, in general, decreasing Δ13C values were obtained in the order leaves, twigs, wood and acorns, with the values from acorns often resembling those from 2019-year rings. The observed changes indicated an increased intrinsic water use efficiency and lack of starch reserve formation during the unprecedented hot and dry summer 2018. Qu. petraea revealed quite different values from Qu. robur (lower Δ13C, wider and denser year rings), but qualitatively showed the same reaction to the drought in 2018, except for an enhanced formation of tyloses in recent-year tree rings.
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.