We are interested in the characterisation of complex microbial communities with cultivation-independent molecular genomic approaches to disclose the ecological function of uncultured archaea and bacteria. Meta-genomic technologies have been used for many years for this purpose and as a logical next step we have recently established meta-transcriptomic approaches in our laboratory.
Meta-transcriptomics offers the opportunity to reach beyond the community’s genomic potential as assessed in DNA-based methods, towards its in situ activity. In addition, the analysis of the RNA pool of a community links its taxonomic structure and function, as it is naturally enriched not only in functionally but also taxonomically relevant molecules, i.e. mRNA and rRNA, respectively. In addition rRNA can be used for PCR-independent community profiling of all three domains of life.
We have established appropriate experimental and analytical procedures (the “double-RNA” approach) for in-depth characterization of microbial communities by studying mRNA and rRNA molecules simultaneously from the same sample.
Long Term Carbon Storage in Cryoturbated Arctic Soils - CryoCarb
The overarching goal of CryoCARB is to advance organic carbon estimates for cryoturbated soils, focusing on the Eurasian Arctic and to understand the vulnerability of these carbon stocks in a future climate. Our vision is that one can build on this knowledge to improve existing models to better predict the responses of cryoturbated soils to future climate conditions. The constraints to our understanding of carbon dynamics in cryogenic soils are currently manifold. First. due to cryoturbation, organic matter is unevenly distributed within the soil, making SOC estimation very difficult. There is evidence that the North American arctic carbon stock is bigger than previously thought, also because of underestimation of carbon stored in distorted, broken and warped horizons. Second, most studies dealing with SOC in arctic soils fail to account for carbon stored in the upper permafrost, although the latter is directly under threat in a rapidly warming Arctic. Thawing of the upper permafrost will also mobilize old, geogenic C, which is rarely addressed. Third, the mechanisms of carbon stabilization are largely unknown thus hampering the prediction of c1imate-C02 feedbacks. Knowledge of the chemical composition of organic matter and the processes on how carbon is stabilized is necessary to predict the magnitude and the time-scale at which SOC will get remobilized from thawing permafrost under climate change.
The molecular basis of cell division is well studied only in model microorganisms. Yet, the vast majority of these are not cultivable and their reproduction modes are unexplored. In the course of my dissertation I will focus on environmental Gammaproteobacteria that coat the surface of two marine nematodes Laxus oneistus and Robbea sp.3. Both of these rod-shaped microbial symbionts display extraordinarily reproductive strategies as they grow in width and set their constricting rings longitudinally. We want to understand the molecular and cell biological mechanisms by which these two nematode symbionts grow and reproduce. Do longitudinally dividing bacteria share the same cell division molecules with model Gammaproteobacteria or do, instead, utilize different ones? Which are the core septum positioning mechanisms and molecules conserved in all Gammaproteobacteria? We will address these questions by using a wide palette of microscopy-based techniques (e.g. negative stain electron microscopy and confocal laser scanning microscopy), biochemical approaches such as peptidoglycan composition analysis and in vitro reconstitution of bacterial cytoskeletal components, as well as ectopic expression of symbiont cell division proteins in E. coli and fission yeast. In order to possess the complete repertoire of cell division proteins of the aforementioned symbionts, their complete genomes will be sequenced. This will also allow us to gain insights about their evolution and ecology.
Stilbonematids (Desmodoridae, Chromadoria) are marine nematodes coated with sulfur-oxidizing bacteria. They are the only known marine metazoans capable of establishing monospecific ectosymbioses. Hundreds of highly specialized hypodermal glandular sensory organs (GSOs) appear to play a fundamental role in symbiosis establishment and maintenance: they produce the mucus the symbionts are embedded in.
In the course of our ongoing research project, we want to study abundantly expressed stilbonematid genes discovered by pyrosequencing-based transcriptome analysis. Among these, some are secreted by the GSOs onto the worm's surface and might play a role in symbiosis. In order to understand their function, we will analyze their expression pattern within the GSO and try to silence them by RNA interference.
Concomitantly, we will start to explore how the microbial partners manage to divide without loosing physical contact with their hosts. This requires a highly unusual division mode in which the fission plan is set longitudinally to the symbiont long axis.
The study of relatively simple, naturally occurring symbioses may be instrumental in understanding how beneficial and pathogenic microbes interact with the mucosal surfaces of higher vertebrates.
Although present in very large numbers, very little is known about the physiology of ammonia oxidizing archaea. Their chemolithoautotrophic growth mode has so far been shown only for a single cultivated isolate from a marine aquarium and for two enrichments from hot environments. Therefore, the physiology of ammonia oxidizing archaea in particular of those from soil has remained elusive and their contribution to nitrification has been debated. We have recently isolated a chemolithoautotrophic ammonia oxidizing archaeon from a garden soil in Vienna that is stably growing in laboratory cultures for three years now. The overall goal of this project is to get a deeper insight into the physiology, general activities, evolution and genomic potential of Candidatus Nitrososphaera viennensis and thus to develop it into a model organism for ammonia oxidizing archaea from soil. For this purpose we will perform detailed physiological characterisations, as well as genomic and functional genomic studies. In total we expect to get a deeper insight into this widely distributed and potentially ecologically significant group of archaea.
Sequence analyses of complete bacterial and archaeal genomes have led to the discovery of Clustered Regularly Interspaced Short Palindromic Repeats (in short CRISPR). The potential function of these repeats and their intervening short spacer sequences as well as the function of their associated (Cas-) proteins as constituents of an immune defense system against viruses and other genetic elements, has only recently been recognized. Although CRISPR/Cas systems are found widespread in bacterial and archaeal genomes and exhibit considerable diversity, little insights into the action of most of the CRISPR modules have been obtained in particular in Archaea due to the lack of suitable in vivo test systems. We have recently demonstrated CRISPR/Cas-based immune defense in the hyperthermophilic archaeon Sulfolobus solfataricus. Recombinant variants of the SSV1 virus containing a gene of the conjugative plasmid pNOB8 that represents a target for a corresponding CRISPR spacer in the chromosome were tested in transfection experiments. Almost 100% immunity against the recombinant virus was observed when the chromosomal CRISPR spacer matched perfectly to the protospacer. Different from bacterial systems immunity was still detected, albeit at decreased levels, when mutations distinguished target and spacer. CRISPR/Cas targeting was independent of the transcription of the target gene. Furthermore, a mini CRISPR locus introduced on the viral DNA with spacers targeting the (non-essential) chromosomal beta-galactosidase gene was unstable in host cells and triggered recombination with the indigenous CRISPR locus. Our experiments demonstrate in vivo activity of CRISPR/Cas in archaea for the first time and suggest that – unlike the recently demonstrated in vitro cleavage of RNA in Pyrococcus - DNA is targeted in this archaeon.
The functioning of Arctic soil ecosystems is crucially important for the global climate. Permafrost soils contain nearly twice as much carbon as the atmosphere and it is assumend that large quantities of carbon are lost (in the form of methane and carbon dioxide) when these soils thaw. Understanding the composition and functioning of the microbial communities in arctic soils is therefore crucial in order to be able to predict their vulnerability and reactions in a changing climate. Nitrification is considered important for ecosystem functioning in the arctic, because the availability of nitrogen, the major limiting nutrient in the system, is directly dependent on it. But the major drivers of nitrification in the arctic are currently unknown. We have recently measured different gross situand potential nitrification rates in arctic soils that were dominated by distinct phylogenetic clades of ammonia oxidizing archaea (AOA) suggesting differences in the activities of various clades and also dominance of AOA over ammonia oxidizing bacteria (AOB) in most soils or even their exclusive presence in some. Furthermore, an enrichment of an arctic AOA of an uncharacterized lineage was obtained that is abundant in the European arctic tundra. overall goal of this proposal is to get insight into the distribution and activity of AOA in arctic ecosystems. To achieve this we will follow two experimental paths: One involves the study of AOA in diverse arctic ecosystems and the second the characterization of Nitrosovradea arctica, an organism representing one of the abundant lineages in arctic soils. will study the distribution and abundance of the six major AOA clades in various arctic soils, using deep sequencing and a clade-specific quantitative PCR assay and will link these to environmental parameters and gross nitrification rates. Samples will be obtained in the frame of two international projects studying terrestrial arctic ecosystems, in which we participate (CryoCarb, ESF) or collaborate (CryoN/PAGE21, EU). The direct and indirect contribution of AOA to NO production and nitrification will be studied in incubation experiments with various inhibitors. Metatranscriptomics will be employed in order to investigate their activity in soils at situand increased temperatures. In the second part of the project, the enrichment culture of the arctic strain will be used to study growth characteristics, inhibitors, and NO emission. Furthermore, the genomic sequence will be determined and transcription studies will be performed in order to analyse adaptations and physiological characteristics. Both project parts will be closely interlinked.total, we will contribute to a better understanding of arctic ammonia oxidizers including their direct or indirect influence on nitrification and NO production and we will contribute to a better understanding of the physiology of ammonia oxidizing archaea in general.
Simulation models predict that the oxygen content of the global ocean will decrease by 25% until the end of the century due to an increased stratification of the oceanic surface waters and a rise in temperature. This loss in oxygen will inevitably lead to an expansion of hypoxic and anoxic areas in the global ocean with major consequences for the oceanic carbon and nitrogen cycling. In this proposal, we assess the functional diversity of chemolithoautotrophic prokaryotic communities in two contrasting marine environments, the deep-water masses of the North Atlantic along a latitudinal gradient and around the redoxcline in the central Baltic Sea. Both environments have been shown previously to harbor highly active chemolithoautotrophic prokaryotic communities with dark carbon dioxide fixation rates approaching surface water phytoplankton activity. Specific focus is put on the functional diversity of prokaryotes in the carbon and nitrogen cycling in both systems, including the sulfur cycle in the central Baltic. Biogeochemical rate measurements are tightly linked to functional gene analyses using among other approaches metagenomics and metatranscriptomics. Information obtained from these analyses will guide the development of primers for QPCR to determine the abundance of genes indicative for geochemically relevant processes in the water column of the two systems. Incubation experiments using stable and radio-isotopes in combination with molecular techniques such as SIP-RNA analyses, single-cell analyses using Raman-FISH, NanoSIMS and MICRO-FISH will allow insights into the dynamics of the functional diversity of chemolithotrophic microbial communities in suboxic and anoxic marine planktonic systems. Field studies will be complemented by laboratory model systems with isolated key players in order to understand the adaptive capacity and performance of chemolithoautotrophs in response to different environmental conditions. The combination of these approaches will provide the base for a significant advancement in our understanding of planktonic chemolithoautotrophy in the dark ocean.
Der Darm und seine Bewohner
Der menschliche Darm beherbergt eine riesige Zahl von Mikroorganismen, die mit ihren verschiedenen Stoffwechselfunktionen für unsere Gesundheit unentbehrlich sind. Die meisten dieser Symbionten sind aber noch wenig studiert. Ihre Vielfalt wird auf mehrere tausend Spezies geschätzt. Allein die genomische Information, die in der Gesamtheit dieser Mikroben enthalten ist, das sogenannte Metagenom, ist weit größer, als unser eigenes Human-Genom. Gerät das Zusammenspiel des Immunsystems und der komplexen Mikrobiota im Darm aus dem Gleichgewicht, z.B. durch Fehlernährung, so können sich chronische entzündliche Darmerkrankungen (Inflammatory Bowel Disease = IBD) entwickeln.
Entstehung schwerer Darmkrankheiten
Zu diesen Krankheiten gehören Morbus Crohn und Colitis Ulcerosa, die sich insbesondere in hoch industrialisierten Ländern ausbreiten. Etwa 1,5 Millionen Menschen in Europa und mehr als 2 Millionen US-AmerikanerInnen sind von diesen schweren Krankheiten betroffen und diese Zahlen steigen. Da erkrankte Personen eine Veränderung in der Zusammensetzung ihrer Darm-Mikrobiota aufweisen, wird vermutet, dass Mikroorganismen entweder die Krankheit unmittelbar auslösen, oder aber, dass sie zumindest eine wichtige Rolle bei ihrer Entstehung oder ihrem Verlauf spielen.
Die Auslöser finden
Ziel dieses Forschungsprojektes ist, es anhand experimenteller Mausmodelle Mikroorganismen oder mikrobielle Aktivitäten zu identifizieren, die IBD auslösen können oder seine Ausbreitung unterstützen. Hierdurch sollen sowohl molekulare Marker für die Diagnose von IBD identifiziert werden als auch Grundlagen für die Entwicklung wirksamer Therapeutika geschaffen werden.
Für dieses interdisziplinäre Projekt haben sich ExpertInnen aus den Bereichen mikrobielle Ökologie, mikrobielle Genomik, Mausgenetik, Immunbiologie, Maus- und Humanpathologie zusammengeschlossen. Da die Darm-Mikrobiota enorm komplex ist und viele Spezies nicht im Labor kultiviert werden können, ist die Anwendung von neuen Sequenziertechnologien (Hochdurchsatzmethoden) für die Metagenomanalyse und die funktionelle Genomanalyse erforderlich.