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See detailCondensing the omics fog of microbial communities
Muller, Emilie UL; Glaab, Enrico UL; May, Patrick UL et al

in Trends in Microbiology (2013), 21(7), 325333

Natural microbial communities are ubiquitous, complex, heterogeneous and dynamic. Here, we argue that the future standard for their study will require systematic omic measurements of spatially and ... [more ▼]

Natural microbial communities are ubiquitous, complex, heterogeneous and dynamic. Here, we argue that the future standard for their study will require systematic omic measurements of spatially and temporally resolved unique samples in line with a discovery-driven planning approach. Resulting datasets will allow the generation of solid hypotheses about causal relationships and, thereby, will facilitate the discovery of previously unknown traits of specific microbial community members. However, to achieve this, solid wet-lab, bioinformatic and statistical methodologies are required to have the promises of the emerging field of Eco-Systems Biology come to fruition. [less ▲]

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See detailSequential isolation of metabolites, RNA, DNA, and proteins from the same unique sample
Roume, Hugo UL; Heintz-Buschart, Anna; Muller, Emilie UL et al

in Methods in Enzymology (2013), 531

In microbial ecology, high-resolution molecular approaches are essential for characterizing the vast organismal and functional diversity and understanding the interaction of microbial communities with ... [more ▼]

In microbial ecology, high-resolution molecular approaches are essential for characterizing the vast organismal and functional diversity and understanding the interaction of microbial communities with biotic and abiotic environmental factors. Integrated omics, comprising genomics, transcriptomics, proteomics, and metabolomics allows conclusive links to be drawn between genetic potential and function. However, this requires truly systematic measurements. In this chapter, we first assess the levels of heterogeneity within mixed microbial communities, thereby demonstrating the need for analyzing biomolecular fractions obtained from a single and undivided sample to facilitate multi-omic analysis and meaningful data integration. Further, we describe a methodological workflow for the reproducible isolation of concomitant metabolites, RNA (optionally split into large and small RNA fractions), DNA, and proteins. Depending on the nature of the sample, the methodology comprises different (pre)processing and preservation steps. If possible, extracellular polar and nonpolar metabolites may first be extracted from cell supernatants using organic solvents. Cells are homogenized by cryomilling before small molecules are extracted with organic solvents. After cell lysis, nucleic acids and protein fractions are sequentially isolated using chromatographic spin columns. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological (biological wastewater treatment biomass), environmental (freshwater planktonic communities), and biomedical (human fecal sample) research interest. The methodological framework should be applicable to other microbial communities as well as other biological samples with a minimum of tailoring and represents an important first step in standardization for the emerging field of Molecular Eco-Systems Biology. [less ▲]

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See detailA biomolecular isolation framework for eco-systems biology
Roume, Hugo UL; Muller, Emilie UL; Cordes, Thekla UL et al

in ISME Journal (The) (2013), 7(1), 110-121

Mixed microbial communities are complex, dynamic and heterogeneous. It is therefore essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single samples to ... [more ▼]

Mixed microbial communities are complex, dynamic and heterogeneous. It is therefore essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single samples to facilitate meaningful data integration, analysis and modeling. We have developed a new methodological framework for the reproducible isolation of high-quality genomic DNA, large and small RNA, proteins, and polar and non-polar metabolites from single unique mixed microbial community samples. The methodology is based around reproducible cryogenic sample preservation and cell lysis. Metabolites are extracted first using organic solvents, followed by the sequential isolation of nucleic acids and proteins using chromatographic spin columns. The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and biomedical research interest. The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future. [less ▲]

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See detailCell culture apparatus and culture methods using same
Wilmes, Paul UL; Zenhausern, Frederic; Estes, Matt et al

Patent (2013)

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See detailLinking mixed microbial community phenotype to individual genotypes
Muller, Emilie UL; Pinel, Nicolás; May, Patrick UL et al

Poster (2013)

Biological wastewater treatment is arguably the most widely used biotechnological process on Earth. Wastewater also represents a valuable energy commodity that is currently not being harnessed ... [more ▼]

Biological wastewater treatment is arguably the most widely used biotechnological process on Earth. Wastewater also represents a valuable energy commodity that is currently not being harnessed comprehensively. Mixed microbial communities that naturally occur at the air-water interface of certain biological wastewater treatment systems accumulate excess long chain fatty acids intracellularly. This phenotypic trait may potentially be exploited for the transformation of lipid-rich wastewater into biodiesel (fatty acid methyl esters). Using a molecular Eco-Systems Biology approach, we are studying which genes contribute to the lipid accumulation phenotype and, thus, overall community function. We first compared the lipid accumulation phenotype to the structure of lipid accumulating communities from a local wastewater treatment plant by coupled deep sequencing of the 16S rRNA locus, metagenome sequencing and metabolomic analysis of 4 biological replicates sampled at 4 different time points. Based on the results of these analyses and in order to obtain a detailed view of the structure and function of the respective microbial communities, metagenomic, metatranscriptomic, metaproteomic and (meta-)metabolomic analyses were completed for a single representative biological sample of highest interest. In order to facilitate meaningful data integration of this highly heterogeneous consortium, biomolecular fractions used for the omic analyses were extracted from a unique single sample using a recently developed biomolecular isolation protocol. The coupled survey and the comparative metagenomic analysis demonstrate that the communities change significantly from dates with warm water temperatures to cold water temperatures while alpha diversity remains stable. In the winter period, this switch results in a strong enrichment of a bacterial genus well known to accumulate intracellular lipids, namely Microthrix spp., a representative genome of which has recently been sequenced by us. Correlation networks based on microorganism and concomitant intra- and extra-cellular metabolite abundances provides an overview of organisms potentially involved in the community-wide lipid accumulating phenotype. A sample with the highest abundance of Microthrix spp. was subsequently chosen for the construction of a community-wide metabolic model using metagenomic, metatranscriptomic, metaproteomic and (meta-)metabolomics data. Based on these omic datasets, expressed enzyme variants linked to the lipid accumulation phenotype have been identified and are currently undergoing in vitro characterization. Meta-omic analyses offer exciting prospects for elucidating the genetic blueprints and the functional relevance of specific populations within microbial communities. Consequently, connecting the overall community phenotype to specific genotypes will allow much needed fundamental ecological understanding of microbial community and population dynamics, particularly in relation to environment-driven demography changes leading to tipping points and catastrophic bifurcations. [less ▲]

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See detailA model microbial community for Eco-Systems Biology
Muller, Emilie UL; Roume, Hugo UL; Buschart, Anna UL et al

Poster (2013)

Objective Microbial communities (MCs) play crucial roles in human health and disease. In-depth characterization of the vast organismal and functional diversity of MCs is now facilitated by high-resolution ... [more ▼]

Objective Microbial communities (MCs) play crucial roles in human health and disease. In-depth characterization of the vast organismal and functional diversity of MCs is now facilitated by high-resolution molecular approaches. Systematic measurements are key for meaningful data integration, analysis and modeling. Based on a model MC from a biological wastewater treatment plant, we have developed a new framework based on wet- and dry-lab methods for the integrated analyses of MCs at the population- as well as at the community-level. Methods The overall methodological framework first relies on a standardised wet-lab procedure for the isolation of concomitant biomolecules, i.e., DNA, RNA, proteins and metabolites, from single undivided samples. Purified biomolecular fractions then are subjected to high-resolution omic analyses including metagenomics, metatranscriptomics, metaproteomics and (meta-) metabolomics. The resulting data form the input for integrated bioinformatic analyses. Population-level integrated omic analyses rely on a newly developed binning and re-assembly method, which yields near-complete genome reconstructions for dominant populations. Community-level analyses involve the reconstruction of community-wide metabolic networks. Functional omic data is then mapped onto these reconstructions and contextualized. Results Application of the population-centric workflow has allowed us to reconstruct and identify 10 major populations within the model MC and has led to the identification of a key generalist population, Candidatus Microthrix spp., within the community. Analysis of the community-wide metabolic networks has allowed the identification of keystone genes involved in lipid and nitrogen metabolism within the MC. Conclusions Our new methodological framework offers exciting new prospects for elucidating the functional relevance of specific populations and genes within MCs. The established workflows are now being applied to samples of biomedical research interest such as human gastrointestinal tract-derived samples. [less ▲]

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See detailSystematic molecular measurements reveal key microbial populations driving community-wide phenotype
Muller, Emilie UL; Pinel, Nicolás; May, Patrick UL et al

Poster (2013)

Natural microbial communities are heterogeneous and dynamic. Therefore, a major consideration for multiple omic data studies is the sample-to-sample heterogeneity, which can lead to inconsistent results ... [more ▼]

Natural microbial communities are heterogeneous and dynamic. Therefore, a major consideration for multiple omic data studies is the sample-to-sample heterogeneity, which can lead to inconsistent results if the different biomolecular fractions are obtained from distinct sub-samples. Conversely, systematic omic measurements, i.e. the standardised, reproducible and simultaneous measurement of multiple features from a single undivided sample, result in fully integrable datasets. Objective In order to prove the feasibility and benefits of such systematic measurements in the study of the respective contributions of different populations to the community-wide phenotype, we purified and analysed all biomolecular fractions, i.e. DNA, RNA, proteins and metabolites, obtained from a unique undivided sample of lipid accumulating microbial community (LAMC) from wastewater treatment plant and integrate the resulting datasets. Methods One time point of particular interest was first selected out of 4 LAMC samples for its high diversity and strong lipid accumulation phenotype. Then, the systematic measurement strategy was applied to the selected undivided LAMC sample and the purified biomolecules were analysed by high-throughput techniques. DNA and RNA sequencing reads were assembled at the population-level using different binning strategies. A database, containing predicted proteins, was constructed to identify the detected peptides. Finally, all biomolecular information was mapped onto the assembled composite genomes to identify the precise roles of the different populations in the community-wide lipid accumulation phenotype. Results Metabolomics and 16S diversity analyses were used to select the sample of highest interest for detailed analysis. The systematic measurements of the selected sample followed by data integration have allowed us to probe the functional relevance of the population-level composite genomes, leading to the identification of the LAMC key players. Conclusion As community phenotype is not the sum of the different partner phenotypes, understanding a microbial community system requires more than the study of isolated organisms. Even if both approaches are complementary, top-down systematic approached only provides a holistic perspective of micro-ecological processes. [less ▲]

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See detailEco-Systems Biology of the Human Microbiome
Wilmes, Paul UL

Scientific Conference (2012, November)

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See detailEco-Systems Biology of natural microbial communities
Wilmes, Paul UL

Scientific Conference (2012, October)

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See detailEco-Systems Biology: from wastewater plant to the human gastrointestinal tract
Wilmes, Paul UL

Scientific Conference (2012, June)

Detailed reference viewed: 13 (3 UL)
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See detailGenome-based and functional differentiation: hallmarks of microbial adaptation, divergence and speciation?
Wilmes, Paul UL

in Ogilvie, L.A.; Hirsch, P.R (Eds.) Microbial Ecological Theory: Current Perspectives (2012)

The recent application of high-throughput molecular biology methods to natural microbial communities is profoundly changing our view on the microbial world. In particular, our understanding of microbial ... [more ▼]

The recent application of high-throughput molecular biology methods to natural microbial communities is profoundly changing our view on the microbial world. In particular, our understanding of microbial population-level differentiation for ecological adaptation that leads to microbial divergence and speciation has been profoundly altered. Numerous processes that underlie microbial differentiation have been identified but determining the relative significance of these processes remains challenging. For example, a major unresolved question is how much of observed genetic heterogeneity is due to neutral versus adaptive processes. Sequence-based and modelling analyses suggest that much of the observed variation is neutral but recent functional omic data suggest that at least some of it is functionally relevant and involved in adaption and divergence. From the limited amount of largely disjointed metagenomic and functional data obtained to date, extensive intra- and inter-system as well as extensive intra- and inter-population differences are apparent. Consequently, it is at present difficult to ascertain specific molecular patterns that define microbial groups that would be congruent with the definition of a microbial species. Concomitant analysis of community genomic complements, transcriptomes, proteomes and metabolomes over relevant spatial and temporal scales in the future will result in detailed molecular descriptions of distinct microbial entities. Such a system-level molecular organismal classification system will need to be solidly grounded in ecological theory, population genetic theory and evolutionary theory, and may be universally applicable to the three domains of life. [less ▲]

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See detailFrom gut to sludge: Molecular Eco-Systems Biology of Microbial Consortia
Muller, Emilie UL; Wilmes, Paul UL

Scientific Conference (2012)

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See detailA biomolecular isolation protocol for Eco-Systems Biology: How to isolate DNA, RNA, proteins and metabolites from a single unique sample
Roume, Hugo UL; Muller, Emilie UL; Shah, Pranjul UL et al

Poster (2012)

The Molecular Systems Biology approach based on the integration of omic datasets is hampered by the complexity, dynamic and heterogeneity of mixed microbial communities. In order to facilitate meaningful ... [more ▼]

The Molecular Systems Biology approach based on the integration of omic datasets is hampered by the complexity, dynamic and heterogeneity of mixed microbial communities. In order to facilitate meaningful data integration, individual biomolecular fractions need to be obtained from single unique samples. Our newly developed methodology allows for the isolation of high-quality genomic DNA, RNA, proteins and metabolites from single mixed microbial community samples (e.g. human faeces, freshwater filtrate), as well as from mammalian tissues. The framework lays the basis for standardized molecular (eco-)systematic studies on a range of different biological samples in the future. [less ▲]

Detailed reference viewed: 88 (6 UL)