Abteilung für Mikrobiologie und Molekularbiologie
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For centuries, peatlands have received little attention or recognition due to their perceived insignificance, invisibility, and negative associations. However, they are now receiving increasing attention due to the ecosystem services they provide as carbon and water reservoirs, as well as their rich biodiversity of rare and endangered animals and plants. In the discussion of greenhouse gas (GHG) emissions and their impact on the climate, peatlands are also recognized as being of significant importance. To comprehend the source of these GHG, it is necessary to examine the microbiome of peatlands and the effects of lowering (draining) and raising (rewetting) the water level. Peatlands store large amounts of soil organic carbon (SOC) in form of peat. Dead plant material is preserved in the water-saturated soils and the carbon dioxide (CO2) absorbed from the atmosphere through photosynthesis is stored as peat. However, when the water table in peatlands is lowered, atmospheric oxygen enters the soil, and the soil microbiome changes, leading to the microbial decomposition of SOC and the release of large quantities of CO2. Rewetting can halt aerobic decomposition processes by blocking oxygen. Anaerobic conditions are not conducive to aerobic organisms. Complex plant polymers, such as lignin, can no longer be broken down, preserving the peat body and allowing the peatland to once again serve as a long-term carbon sink. Under these anaerobic conditions, methanogenic archaea produce methane from metabolic products of fermentation processes. Methane is emitted in much smaller quantities, but has about 30 times the warming potential of CO2, making it the second most important GHG after CO2. To gain a better understanding of the processes involved in methanogenesis, it is essential to conduct a detailed investigation of the individual groups of archaea involved. The metabolic pathways of hydrogenotrophic methanogenesis from CO2 and hydrogen, as well as acetoclastic methanogenesis from acetate cleavage, have already been extensively researched and are considered the most significant pathways of methanogenesis. In recent years, additional organisms have been discovered that carry out methylotrophic methanogenesis from methanol and methylated compounds. The methylotrophic order of Methanomassiliicoccales was first described in 2012, but only one pure culture, Methanomassiliicoccus luminyensis, has been isolated. Further research into Methanomassiliicoccales, commonly found in mires, can enhance our understanding of methanogenesis not only in mires but also in other habitats, such as the gastrointestinal tracts (GIT) of animals or water body sediments. This study investigates the peat soil microbiomes, with a focus on methanogenic Archaea of the order Methanomassiliicoccales, at the six study sites of the WETSCAPES project. The sites are located in the three important fen types of Mecklenburg-Western Pomerania (alder forest, percolation fen, and coastal flooded fen), each in drained and rewetted areas. The first article investigates the microbial composition of prokaryotes and eukaryotes in the sites using Illumina MiSeq Amplicon sequencing of 16S rRNA and 18S rRNA genes. The aerobic microorganisms responsible for decomposing plant biomass are present in drained peatlands. In rewetted sites, the relative abundance of these organisms was significantly lower, while other groups, such as fermenting bacteria and methanogenic archaea, occurred in greater numbers. Quantitative polymerase chain reaction (qPCR) was used to determine the number of methanogenic archaea per gram soil. The results indicate that methanogens are at least 10 times more abundant in rewetted peatlands than in drained sites, suggesting increased methanogenesis in rewetted peatlands. These findings were confirmed by GHG measurements. The second article presents an overview of the interactions between water transport, soil chemistry, primary production, peat formation, material conversion and transport, microorganisms, and GHG exchange. The findings are based on state-of-the-art methods in the relevant research areas and combine initial results from all project partners of the WETSCAPES project. The third article focuses on an application-oriented approach and tests different rewetting strategies. The objective of this study was to decrease methane emissions by removing the topsoil and additional planting of peat mosses. The results of the study, which included measurements of GHG emissions and qPCR analyses of methanogenic archaea, showed a significant reduction in methane emissions after the top 30 cm of soil were removed compared to the untreated area. The fourth article deals with the methylotrophic order Methanomassiliicoccales, which belongs to the group of methanogenic archaea. Over a period of 2.5 years, at 12 time points, at three soil depths, 16S rRNA gene sequencing was used to produce a seasonal and spatial analysis of the Methanomassiliicoccales phylotypes present. The results revealed a distribution of the phylotypes based on soil depth, redox potential, and season, which were related to their physiological characteristics. In the fifth article, the 1.52 Mbp genome of "Ca. Methanogranum gryphiswaldense U3.2.1", a Methanomassiliicoccales strain, enriched to > 80% from peat soil of the rewetted percolation fen, was presented. The sixth article presented the microscopic, physiological, and genomic studies of Ca. Methanogranum gryphiswaldense U3.2.1. This hydrogen-dependent, methylotrophic strain from the family Methanomethylophilaceae, uses methanol and trimethylamine as electron acceptors. Despite various attempts, a pure culture has not yet been achieved. The study demonstrates the impact of rewetting on the soil microbiome, revealing its complexity. The development of the microbiome was influenced by various biotic and abiotic factors, depending on the location and water level. The abundance of methanogenic archaea was significantly higher in the rewetted sites compared to the drained ones. The Methanomassiliicoccales were found to be a significant contributor to the methanogenic archaea in the investigated sites. It remains to be examinated whether they also significantly contribute to methane formation. Enrichment cultures and genome analyses were used to investigate the substrate spectrum of Ca. Methanogranum gryphiswaldense U3.2.1 obtained from peat soil. The extraction of a pure culture from peat soils has not yet been successful and therefore remains a goal of future investigations.
For decades, researchers have focused on containing terrestrial oil pollution. The heterogeneity of soils, with immense microbial diversity, inspires them to transform pollutants and find cost-effective bioremediation methods. In this study, the mycoremediation potentials of five filamentous fungi isolated from polluted soils in Kazakhstan were investigated for their degradability of n-alkanes and branched-chain alkanes as sole carbon and energy sources. Dry weight estimation and gas chromatography–mass spectrometry (GC-MS) monitored the growth and the changes in the metabolic profile during degradation, respectively. Penicillium javanicum SBUG-M1741 and SBUG-M1742 oxidized medium-chain alkanes almost completely through mono- and di-terminal degradation. Pristane degradation by P. javanicum SBUG-M1741 was 95%, while its degradation with Purpureocillium lilacinum SBUG-M1751 was 90%. P. lilacinum SBUG-M1751 also exhibited the visible degradation potential of tetradecane and phytane, whereby in the transformation of phytane, both the mono- and di-terminal degradation pathways as well as α- and ß-oxidation steps could be described. Scedosporium boydii SBUG-M1749 used both mono- and di-terminal degradation pathways for n-alkanes, but with poor growth. Degradation of pristane by Fusarium oxysporum SBUG-M1747 followed the di-terminal oxidation mechanism, resulting in one dicarboxylic acid. These findings highlight the role of filamentous fungi in containing oil pollution and suggest possible degradation pathways.
The proteasome is one of the major cellular protein degradation systems. The respective substrates include cell cycle regulators, kinases, transcription factors, antigens and enzymes. As such the proteasome plays a major role in cell function, survival and proliferation. Another key target of the proteasome are damaged and misfolded proteins as well as those proteins that are no loner required. Therefore, the proteasome is key in maintaining proteostasis, which describes the balance between the synthesis of new proteins and the removal of damaged, old and misfolded ones. Several factors such as ageing, disease and certain medications can impair proteasome function and thus disturb cellular proteostasis. The current work focused on novel genetic mutations in the various subunits constituting the proteasome. The proteasomal mutations were found to cause two very distinct disease phenotypes, PRAAS (an autoinflammatory disorder) and NDD (neurodevelopmental delay). The novel mutations discussed in this work were found to impair proteasome function through a variety of mechanisms and lead to decreased proteolytic capacity in the cells of the patients, resulting in a disturbance of cellular proteostasis and disease manifestation.
Understanding cellular mechanisms of stress management relies on omics data as a valuable resource. However, the lack of absolute quantitative data on protein abundances remains a significant limitation, particularly when comparing protein abundances across different cell compartments. In this study, we aimed to gain deeper insights into the proteomic responses of the Gram-positive model bacterium Bacillus subtilis to disulfide stress. We determined proteome-wide absolute abundances, focusing on different sub-cellular locations (cytosol and membrane) as well as the extracellular medium, and combined these data with redox state determination. To quantify secreted proteins in the culture medium, we developed a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria. We concentrated extracellular proteins, which are highly diluted in the medium, using StrataClean beads along with a set of standard proteins to determine the extent of the concentration step. The resulting data set provides new insights into protein abundances in different sub-cellular compartments and the extracellular medium, along with a comprehensive proteome-wide redox state determination. Our study offers a quantitative understanding of disulfide stress management, protein production, and secretion in B. subtilis.
Virale Erreger können bei Mensch und Tier schwere bis fatale Enzephalitiden auslösen. Jedoch wird in nur ca. 50 % der Fälle das ätiologische Agens identifiziert. In dieser Arbeit wurde das Potential der Next Generation Sequencing-basierten Metagenomdiagnostik (mNGS) zur Identifizierung neuer Erreger bei infektiösen Enzephalitiden von Mensch und Tier untersucht. Ein weiteres Ziel war es, die Endemiegebiete des Virus der Borna’schen Krankheit 1 (BoDV-1) in Deutschland, Österreich, der Schweiz und Liechtenstein mittels phylogeographischer Analysen genauer zu definieren.
Es konnte im Rahmen dieser Arbeit ein wahrscheinlichkeitsbasiertes mathematisches Modell entwickelt und validiert werden, das die individuellen Nachweisgrenzen von mNGS-Analysen und die minimal benötigte Datensatzgröße zur Detektion mindestens eines Virus-Reads in Abhängigkeit des Virus-Wirts-Verhältnisses und der Datensatzgröße berechnet (Ebinger et al. (2020), Comput Struct Biotechnol J).
Des Weiteren wurde mNGS bei fatalen Enzephalitiden von Zoo- und Haussäugetieren angewendet, was einerseits zur Identifizierung von Rustrela-Virus (RusV) als ätiologischem Agens führte, einem Virus, das mit dem im Menschen vorkommenden Rötelnvirus verwandt ist. RusV wurde zudem in Gelbhalsmäusen (Apodemus flavicollis) detektiert, die aufgrund der phylogenetischen Nähe des Virus und den fehlenden Anzeichen einer Entzündung als Reservoirwirt identifiziert wurden (Bennett, Paskey, Ebinger et al. (2020), Nature). Andererseits wurde ein bislang unbekanntes ovines Enterovirus in Lämmern bei einem akuten Ausbruchsgeschehen auf einem österreichischen Milchschafbetrieb als Ursache von fatalen Enzephalitiden identifiziert (Weissenböck, Ebinger et al. (2021), Transbound Emerg Dis).
Die phylogeographische Analyse von insgesamt 246 BoDV-1-Infektionen in Mensch, Haussäugetieren und in den als Reservoirtier geltenden Feldspitzmäusen (Crocidura leucodon) ergaben eine umfassende und detaillierte Definition der BoDV-1-Endemiegebiete. Es konnte belegt werden, dass sich die verschiedenen speziesübergreifenden phylogenetischen Cluster in kaum überlappende regionale Subkladen aufteilen und die meisten zoonotischen Spillover-Infektionen in der Nähe der Wohn- oder Haltungsorte der jeweiligen Fälle auftraten (Ebinger et al. (2023), eingereicht).
Insgesamt tragen die Ergebnisse zu einem deutlich verbesserten Verständnis der Sensitivität und Interpretierbarkeit von mNGS-Analysen im klinischen und wissenschaftlichen Kontext bei und geben wichtige Hinweise zum Vorkommen, Verbreitung, Wirtsspektrum und genetischer Diversität von Rubiviren, ovinen Enteroviren und BoDV-1.
Respiratory infections are associated with high morbidity and mortality rates worldwide and represent a large burden for healthcare systems. Every year, millions of people die from diseases that are associated with bacterial or viral infections, such as pneumonia. The prevention and treatment of these respiratory infectious diseases is thus a major challenge for our time. Recent research has revealed strong links between the gastrointestinal microbiome and a variety of diseases. While this body of work suggests that a host’s microbiome plays an important role in protection against pathogenic agents, maintenance of immune homeostasis, and acquiring nutrients, our understanding of how infections affect the taxonomic and especially functional composition of the microbiome, remains in its infancy.
The aim of this dissertation was to characterize the influence of monocausal respiratory infections on the structure and function of the gastrointestinal microbiome using Metaproteomics. This was done in two different biomedical models. First, infection experiments were performed in swine, a relevant natural pathogen-host system, and second, in an experimental murine infection model. Each animal model has specific advantages that allow to address different concerns. The porcine model allowed individual longitudinal characterization of the gastrointestinal tract microbiome during Influenza A virus (IAV) infection over a 30-day period (paper II), while the identification of pathogen-specific signatures during pneumonia could be performed in the murine model (paper III). As a starting point, a robust multi-omics pipeline for fecal samples that allowed standardized and reproducible analysis of porcine and murine samples was established (Paper I). Of major importance was the contact-free homogenization step for pulverization of permanently frozen fecal samples. This process helped to reduce local effects and increase the comparability of the samples, as the exact same homogenizedmaterial could now be used for each omics technique. The omics analyses were subsequently optimized with protocols designed for the extraction of specific target molecules. This significantly reduced the amount of sample required per analysis, without compromising the quality of the results.
Taxonomic characterization of the microbiome from healthy and infected animals revealed commonalities as well as differences across model organisms’ intestinal microbiome compositions. One of the most prominent similarities was the dysbiosis of the gastrointestinal microbiome induced by respiratory infection. This dysbiosis was evident in an alteration of the Firmicutes/Bacteroidetes ratio in both the porcine and murine model. Longitudinal characterization of the porcine intestinal microbiome demonstrated that animals exhibited low interindividual variance and that the gastrointestinal microbiome was subject to natural dynamics. The low interindividual variance enabled the identification of consistent infection-related changes in the longitudinal development of the microbiome (paper II). Thus, IAV-induced dysbiosis of the microbial community was reflected at the taxonomic level in decreased abundance of Lachnospiraceae, Clostridiaceae, Veillonellaceae, and Selenomonadaceae, with concomitant increases in Prevotellaceae and Bacteroidaceae. In addition, the Lactobacillaceae family showed an opposite trend over time when comparing healthy and IAV-infected swine. Complementing the longitudinal data from the porcine model, we used the mouse model to characterize the effects of bacterial and viral induced pneumonia on the intestinal microbiome (paper III). Independent of the pathogen, we detected increased abundance of Desulfovibrionaceae and Odoribacteraceae during pneumonia. In contrast, the abundance of Prevotellaceae, Tannerellaceae, and Eubacteriaceae declined during bacterial and viral infection. Furthermore, the identification of pathogen specific signatures was possible, which was evident in pathogen-dependent differentiation of the intestinal microbiomes. Thus, during pneumococcal pneumonia, increased abundance of Akkermansiaceae and Spirocheataceae was detected with simultaneously reduced Clostridiaceae, whereas IAV infection resulted in an increased abundance of Staphylococcaceae.
In addition to taxonomic profiling, the impact of respiratory infections on the functional composition of the intestinal microbiome of swine and mice was investigated. Despite variation in their influence on the taxonomic composition at family level, similar effects of respiratory infections on functional composition were found in both models. In mouse and swine, IAV infection resulted in increased expression of protein groups involved in the synthesis of short-chain fatty acids. This commonality across models suggests that short-chain fatty acids play an important role during respiratory infection and recovery. Analysis of the data obtained from the porcine experiments revealed that the functional composition of the healthy intestinal microbiome exhibited a largely steady state despite some temporal taxonomic dynamics. This result indicated functional redundancy within the microbiome of healthy pigs. Nevertheless, IAV infection resulted in dysbiosis of this stable state. This was reflected by a significant increase in the expression of proteins involved in the transport and metabolism of amino acids and carbohydrates, as well as proteins involved in the production of short-chain fatty acids. Finally, the influence of bacterial and viral infections on the functional composition of the murine intestinal microbiome was also investigated (paper III). Based on the pathogen-specific profiles observed with metaproteomics, it was possible to distinguish between viral and bacterial infections. For example, bacterial colonization and infection resulted in similar functional profiles, with comparable infection driven effects on the intestinal microbiome. However, the alterations in the expression profile in the microbiome of mice colonized with pneumococci were less pronounced than during acute bacterial pneumonia. In contrast, viral infection caused a significantly different functional profile. An exclusive feature of bacterial pneumonia was the decreased expression of proteins associated with energy metabolism (e.g., ATPases) with concomitant increased abundance of transporters or secretory channels (e.g., OmpA, TonB, TolA). In contrast, increased expression of proteins assigned to the ATPase complex were characteristic of viral infections.
The data generated in this work provided new insights into the impact of monocausal infections on the taxonomic and functional composition of the microbial community of the gastrointestinal tract. These results may serve as a basis for future research on co-infections. Furthermore, the identification of pathogen specific signatures represents a promising observation for clinical setup.
In temperate regions, climate warming alters temperature and precipitation regimes. During winter, a decline in insulating snow cover changes the soil environment, where especially frost exposure can have severe implications for soil microorganisms and subsequently for soil nutrient dynamics. Here, we investigated winter climate change responses in European beech forests soil microbiome. Nine study sites with each three treatments (snow exclusion, insolation, and ambient) were investigated. Long-term adaptation to average climate was explored by comparing across sites. Triplicated treatment plots were used to evaluate short-term (one single winter) responses. Community profiles of bacteria, archaea and fungi were created using amplicon sequencing. Correlations between the microbiome, vegetation and soil physicochemical properties were found. We identify core members of the forest-microbiome and link them to key processes, for example, mycorrhizal symbiont and specialized beech wood degraders (fungi) and nitrogen cycling (bacteria, archaea). For bacteria, the shift of the microbiome composition due to short-term soil temperature manipulations in winter was similar to the community differences observed between long-term relatively cold to warm conditions. The results suggest a strong link between the changes in the microbiomes and changes in environmental processes, for example, nitrogen dynamics, driven by variations in winter climate.
Background: Methanogenic archaea represent a less investigated and likely underestimated part of the intestinal tract microbiome in swine.
Aims/Methods: This study aims to elucidate the archaeome structure and function in the porcine intestinal tract of healthy and H1N1 infected swine. We performed multi-omics analysis consisting of 16S rRNA gene profiling, metatranscriptomics and metaproteomics.
Results and discussion: We observed a significant increase from 0.48 to 4.50% of archaea in the intestinal tract microbiome along the ileum and colon, dominated by genera Methanobrevibacter and Methanosphaera. Furthermore, in feces of naïve and H1N1 infected swine, we observed significant but minor differences in the occurrence of archaeal phylotypes over the course of an infection experiment. Metatranscriptomic analysis of archaeal mRNAs revealed the major methanogenesis pathways of Methanobrevibacter and Methanosphaera to be hydrogenotrophic and methyl-reducing, respectively. Metaproteomics of archaeal peptides indicated some effects of the H1N1 infection on central metabolism of the gut archaea.
Conclusions/Take home message: Finally, this study provides the first multi-omics analysis and high-resolution insights into the structure and function of the porcine intestinal tract archaeome during a non-lethal Influenza A virus infection of the respiratory tract, demonstrating significant alterations in archaeal community composition and central metabolic functions.
The goal of our study was to examine the effects of low abundances of nylon fibers on feeding rates of calanoid copepods (Crustacea, Copepoda) and doliolids (Tunicata, Thaliacea) in the presence of diatoms at near environmental concentration levels. In addition, we examined microscopically the fecal pellets produced by copepods and doliolids in the presence of fibers. Adult females of the calanoid Eucalanus pileatus and early gonozooids of Dolioletta gegenbauri (both of similar dry weight) cleared the diatom Rhizosolenia alata at similar rates. Nylon fibers were cleared at higher rates by Dolioletta gegenbauri compared to Eucalanus pileatus. Examination of fecal pellets revealed that copepods and doliolids could ingest the about 300 µm long fibers. The latter also ingested the occasionally occurring fibers of > 1 mm length. It appears that in seawater fiber abundances of about seven fibers ml−1 did not have a negative effect on feeding of either E. pileatus or D. gegenbauri. As doliolids and copepods remove plastic fibers from seawater by packing them into their pellets, they might play a role in the reduction of microplastic pollution and the microplastic transfer from the water column to the seafloor. Calanoid copepods may limit ingesting fibers by not perceiving them, as compared to doliolids which do not seem to be able to avoid ingesting them.
Linking transcriptional dynamics of CH4-cycling grassland soil microbiomes to seasonal gas fluxes
(2022)
Soil CH4 fluxes are driven by CH4-producing and -consuming microorganisms that determine whether soils are sources or sinks of this potent greenhouse gas. To date, a comprehensive understanding of underlying microbiome dynamics has rarely been obtained in situ. Using quantitative metatranscriptomics, we aimed to link CH4-cycling microbiomes to net surface CH4 fluxes throughout a year in two grassland soils. CH4 fluxes were highly dynamic: both soils were net CH4 sources in autumn and winter and sinks in spring and summer, respectively. Correspondingly, methanogen mRNA abundances per gram soil correlated well with CH4 fluxes. Methanotroph to methanogen mRNA ratios were higher in spring and summer, when the soils acted as net CH4 sinks. CH4 uptake was associated with an increased proportion of USCα and γ pmoA and pmoA2 transcripts. We assume that methanogen transcript abundance may be useful to approximate changes in net surface CH4 emissions from grassland soils. High methanotroph to methanogen ratios would indicate CH4 sink properties. Our study links for the first time the seasonal transcriptional dynamics of CH4-cycling soil microbiomes to gas fluxes in situ. It suggests mRNA transcript abundances as promising indicators of dynamic ecosystem-level processes.