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See detailIntegrated time-resolved multi-omics for understanding microbial niche ecology
Herold, Malte UL; Narayanasamy, Shaman UL; Martinez Arbas, Susana UL et al

Poster (2018, August)

Microbial communities are strongly shaped by the niche breadths of their constituent populations. However, a detailed understanding of microbial niche ecology is typically lacking. Integrated multi-omic ... [more ▼]

Microbial communities are strongly shaped by the niche breadths of their constituent populations. However, a detailed understanding of microbial niche ecology is typically lacking. Integrated multi-omic analyses of host- or environment-derived samples offer the prospect of resolving fundamental and realised niches in situ. In turn, this is considered a prerequisite for niche engineering in order to drive an individual population or a community towards a specific phenotype, e.g., improvement of a biotechnological process. Here, we sampled floating islets on the surface of an activated sludge tank in a time-series spanning 51 time-points over 14 months. Multi-omics datasets (metagenomics, metatranscriptomics, metaproteomics, and (meta-)metabolomics) were generated for all time-points. Leveraging nucleotide sequencing data, we analyzed the community structure and reconstructed genomes for specific populations of interest. Moreover, based on their metabolic potential, three major groups emerged, serving as proxies for their respective fundamental niches . Time-resolved linkage of the proteomic and transcriptomic data to the reconstructed genomes revealed a fine-grained picture of niche realization. In particular, environmental factors (temperature, metabolites, oxygen) were significantly associated with gene expression of individual populations. Furthermore, we subjected the community to controlled oxygen conditions (stable or dynamic) in a bioreactor experiment and measured the transcriptomic response. Our results suggest short-term adaptations of populations of interest with respect to lipid metabolism, among other pathways. In conclusion, our work demonstrates how longitudinal multi-omic datasets can be integrated in order to further our understanding of microbial niche ecology within a biotechnological process with potential applications beyond waste water treatment. [less ▲]

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See detailComparative integrated omics: identification of key functionalities in microbial community-wide metabolic networks
Roume, Hugo UL; Buschart, Anna UL; Muller, Emilie UL et al

in Biofilms and Microbiomes (2015), 1(15007),

BACKGROUND: Mixed microbial communities underpin important biotechnological processes such as biological wastewater treatment (BWWT). A detailed knowledge of community structure and function relationships ... [more ▼]

BACKGROUND: Mixed microbial communities underpin important biotechnological processes such as biological wastewater treatment (BWWT). A detailed knowledge of community structure and function relationships is essential for ultimately driving these systems towards desired outcomes, e.g., the enrichment in organisms capable of accumulating valuable resources during BWWT. METHODS: A comparative integrated omic analysis including metagenomics, metatranscriptomics and metaproteomics was carried out to elucidate functional differences between seasonally distinct oleaginous mixed microbial communities (OMMCs) sampled from an anoxic BWWT tank. A computational framework for the reconstruction of community-wide metabolic networks from multi-omic data was developed. These provide an overview of the functional capabilities by incorporating gene copy, transcript and protein abundances. To identify functional genes, which have a disproportionately important role in community function, we define a high relative gene expression and a high betweenness centrality relative to node degree as gene-centric and network topological features, respectively. RESULTS: Genes exhibiting high expression relative to gene copy abundance include genes involved in glycerolipid metabolism, particularly triacylglycerol lipase, encoded by known lipid accumulating populations, e.g., Candidatus Microthrix parvicella. Genes with a high relative gene expression and topologically important positions in the network include genes involved in nitrogen metabolism and fatty acid biosynthesis, encoded by Nitrosomonas spp. and Rhodococcus spp. Such genes may be regarded as ‘keystone genes’ as they are likely to be encoded by keystone species. CONCLUSION: The linking of key functionalities to community members through integrated omics opens up exciting possibilities for devising prediction and control strategies for microbial communities in the future. [less ▲]

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See detailCommunity-integrated omics links dominance of a microbial generalist to fine-tuned resource usage
Muller, Emilie UL; Pinel, Nicolas; Laczny, Cedric Christian UL et al

in Nature Communications (2014)

Microbial communities are complex and dynamic systems that are primarily structured according to their members’ ecological niches. To investigate how niche breadth (generalist versus specialist lifestyle ... [more ▼]

Microbial communities are complex and dynamic systems that are primarily structured according to their members’ ecological niches. To investigate how niche breadth (generalist versus specialist lifestyle strategies) relates to ecological success, we develop and apply an integrative workflow for the multi-omic analysis of oleaginous mixed microbial communities from a biological wastewater treatment plant. Time- and space-resolved coupled metabolomic and taxonomic analyses demonstrate that the community-wide lipid accumulation phenotype is associated with the dominance of the generalist bacterium Candidatus Microthrix spp. By integrating population-level genomic reconstructions (reflecting fundamental niches) with transcriptomic and proteomic data (realised niches), we identify finely tuned gene expression governing resource usage by Candidatus Microthrix parvicella over time. Moreover, our results indicate that the fluctuating environmental conditions constrain the accumulation of genetic variation in Candidatus Microthrix parvicella likely due to fitness trade-offs. Based on our observations, niche breadth has to be considered as an important factor for understanding the evolutionary processes governing (microbial) population sizes and structures in situ. [less ▲]

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See detailSystematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia
Hugerth, Luisa; Muller, Emilie UL; Hu, Yue et al

in PLoS ONE (2014)

High-throughput sequencing of ribosomal RNA gene (rDNA) amplicons has opened up the door to large-scale comparative studies of microbial community structures. The short reads currently produced by ... [more ▼]

High-throughput sequencing of ribosomal RNA gene (rDNA) amplicons has opened up the door to large-scale comparative studies of microbial community structures. The short reads currently produced by massively parallel sequencing technologies make the choice of sequencing region crucial for accurate phylogenetic assignments. While for 16S rDNA, relevant regions have been well described, no truly systematic design of 18S rDNA primers aimed at resolving eukaryotic diversity has yet been reported. Here we used 31,862 18S rDNA sequences to design a set of broad-taxonomic range degenerate PCR primers. We simulated the phylogenetic information that each candidate primer pair would retrieve using paired- or single-end reads of various lengths, representing different sequencing technologies. Primer pairs targeting the V4 region performed best, allowing discrimination with paired-end reads as short as 150 bp (with 75% accuracy at genus level). The conditions for PCR amplification were optimised for one of these primer pairs and this was used to amplify 18S rDNA sequences from isolates as well as from a range of environmental samples which were then Illumina sequenced and analysed, revealing good concordance between expected and observed results. In summary, the reported primer sets will allow minimally biased assessment of eukaryotic diversity in different microbial ecosystems. [less ▲]

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See detailCommunity integrated omics links the dominance of a microbial generalist to fine-tuned resource usage
Muller, Emilie UL; Pinel, Nicolás; Laczny, Cedric Christian UL et al

Scientific Conference (2014)

Microbial communities are complex and dynamic systems that are influenced by stochastic-neutral processes but are mainly structured by resource availability and usage. High-resolution “meta-omics” offer ... [more ▼]

Microbial communities are complex and dynamic systems that are influenced by stochastic-neutral processes but are mainly structured by resource availability and usage. High-resolution “meta-omics” offer exciting prospects to investigate microbial populations in their native environment. In particular, integrated meta-omics, by allowing simultaneous resolution of fundamental niches (genomics) and realised niches (transcriptomics, proteomics and metabolomics), can resolve microbial lifestyles (generalist versus specialist lifestyle strategies) in situ. We have recently developed the necessary wet- and dry-lab methodologies to carry out systematic molecular measurements of microbial consortia over space and time, and to integrate and analyse the resulting data at the population-level. We applied these methods to oleaginous mixed microbial communities located on the surface of anoxic biological wastewater treatment tanks to investigate how niche breadth (generalist versus specialist lifestyle strategies) relates to community-level phenotypes and ecological success (i.e. population size). Coupled metabolomics and 16S rRNA gene-based deep sequencing demonstrate that the community-wide lipid accumulation phenotype is associated with the dominance of Candidatus Microthrix parvicella. By integrating population-level genomic reconstructions with transcriptomic and proteomic data, we found that the dominance of this microbial generalist population results from finely tuned resource usage and optimal foraging behaviour. Moreover, the fluctuating environmental conditions constrain the accumulation of variations, leading to a genetically homogeneous population likely due to fitness trade-offs. By integrating metagenomic, metatranscriptomic, metaproteomic and metabolomic information, we demonstrate that natural microbial population sizes and structures are intricately linked to resource usage and that differing microbial lifestyle strategies may explain the varying degrees of within-population genetic heterogeneity observed in metagenomic datasets. Elucidating the exact mechanism driving fitness trade-offs, e.g., antagonistic pleiotropy or others, will require additional integrated omic datasets to be generated from samples taken over space and time. Based on our observations, niche breadth and lifestyle strategies (generalists versus specialists) have to be considered as important factors for understanding the evolutionary processes governing microbial population sizes and structures in situ. [less ▲]

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See detailCommunity integrated omics links the dominance of a microbial generalist to fine-tuned resource usage
Muller, Emilie UL; Pinel, Nicolás; Laczny, Cedric Christian UL et al

Poster (2014)

Microbial communities are complex and dynamic systems that are influenced by stochastic-neutral processes but are mainly structured by resource availability and usage. High-resolution “meta-omics” offer ... [more ▼]

Microbial communities are complex and dynamic systems that are influenced by stochastic-neutral processes but are mainly structured by resource availability and usage. High-resolution “meta-omics” offer exciting prospects to investigate microbial populations in their native environment. In particular, integrated meta-omics, by allowing simultaneous resolution of fundamental niches (genomics) and realised niches (transcriptomics, proteomics and metabolomics), can resolve microbial lifestyles strategies (generalist versus specialist) in situ. We have recently developed the necessary wet- and dry-lab methodologies to carry out systematic molecular measurements of microbial consortia over space and time, and to integrate and analyse the resulting data at the population-level. We applied these methods to oleaginous mixed microbial communities located on the surface of anoxic biological wastewater treatment tanks to investigate how niche breadth (generalist versus specialist strategies) relates to community-level phenotypes and ecological success (i.e. population size). Coupled metabolomics and 16S rRNA gene-based deep sequencing demonstrate that the community-wide lipid accumulation phenotype is associated with the dominance of Candidatus Microthrix parvicella. By integrating population-level genomic reconstructions with transcriptomic and proteomic data, we found that the dominance of this microbial generalist population results from finely tuned resource usage and optimal foraging behaviour. Moreover, the fluctuating environmental conditions constrain the accumulation of variations, leading to a genetically homogeneous population likely due to fitness trade-offs. By integrating metagenomic, metatranscriptomic, metaproteomic and metabolomic information, we demonstrate that natural microbial population sizes and structures are intricately linked to resource usage and that differing microbial lifestyle strategies may explain the varying degrees of within-population genetic heterogeneity observed in metagenomic datasets. Elucidating the exact mechanism driving fitness trade-offs, e.g., antagonistic pleiotropy or others, will require additional integrated omic datasets to be generated from samples taken over space and time. Based on our observations, niche breadth and lifestyle strategies (generalists versus specialists) have to be considered as important factors for understanding the evolutionary processes governing microbial population sizes and structures in situ. [less ▲]

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See detailThe sequential isolation of metabolites, RNA, DNA, and proteins from a single, undivided mixed microbial community sample
Muller, Emilie UL; Buschart, Anna UL; Roume, Hugo UL et al

in Protocol Exchange (2014)

Integrated omics of microbial consortia, comprising systematized metagenomic, metatranscriptomic, metaproteomic and meta-metabolomic analyses, allows in-depth characterization of organismal and functional ... [more ▼]

Integrated omics of microbial consortia, comprising systematized metagenomic, metatranscriptomic, metaproteomic and meta-metabolomic analyses, allows in-depth characterization of organismal and functional diversity in situ. To allow meaningful meta-omic data integration, truly systematic measurements of the typically heterogeneous sample biomass is required. Therefore, there is a need for analyzing biomolecular fractions obtained from single, undivided samples. Here, we share a methodological workflow for the reproducible isolation of concomitant polar and non-polar metabolites, RNA, DNA and proteins from samples obtained from a biological wastewater treatment plant. The methodological framework is applicable to other biological samples [1,2], is compatible with different kits for biomacromolecular isolation [1,2] with minimal tailoring, and represents an important first step in standardization for the emerging field of Molecular Eco-Systems Biology. [less ▲]

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See detailMolecular Eco-Systems Biology of Lipid Accumulating Microbial Communities in Biological Wastewater Treatment Plants
Roume, Hugo UL

Doctoral thesis (2013)

Biological wastewater treatment is based on the use of microorganisms capable of intense metabolic activity that results in the removal of a large proportion of organic and inorganic contaminants. Given ... [more ▼]

Biological wastewater treatment is based on the use of microorganisms capable of intense metabolic activity that results in the removal of a large proportion of organic and inorganic contaminants. Given copious amounts of energy-dense organic molecules such as lipids accumulated by the microbial biomass, chemical energy may be directly harnessed from this for biofuel production. Here, lipid accumulating organism (LAO)-enriched microbial communities were studied using a molecular eco-systems biology approach. This involved the development of necessary methodologies including a new comprehensive biomolecular extraction method, yielding high-quality DNA, RNA, proteins and metabolites, as well as bioinformatic approaches for integrating and analysing the derived high-throughput genomic, transcriptomic, proteomic and metabolomic data. At the inception of the project, a full-scale wastewater treatment plant (WWTP) system with a strong presence of LAOs especially during winter months, i.e. the Schifflange WWTP (Esch-sur-Alzette, Luxembourg), was identified and selected for further study. 16S rRNA amplicon sequencing highlighted the presence of ubiquitous lipid accumulating bacteria closely related to Candidatus Microthrix parvicella which increase in abundance from autumn to winter over other highly abundant community members belonging to Alkanindiges spp. and Acinetobacter spp. In order to elucidate compositional, genetic and functional differences between autumn and winter LAO communities, a comparative integrative omic analysis was carried out on rationally selected autumn and winter LAO community samples. The results from metabolomic/lipidomic analyses between intra- and extracellular compartments support previous models of uptake of unprocessed long chain fatty acids (LCFAs) from the wastewater environment and their storage as triacyglycerols within LAOs. Furthermore, a tailored computational framework for the integration of multi-omic datasets into reconstructed community-wide metabolic networks and models was developed. The resulting networks provide overviews of functional capacity of the sampled LAO communities by incorporating gene copy numbers, transcript levels and protein frequency across the two studied environmental conditions. The identification of genes overexpressed, strongly associated with a specific season and/or possessing a high clustering coefficient suggests the existence of keystone genes, analogous to keystone species in species interaction networks. Examples of such keystone genes in the context of the LAO communities include genes coding for proteins involved in nitrogen and glycerolipid metabolism. The existence of such keystone genes opens up exciting possibilities for prediction and control strategies of microbial communities at the dawn of the field of 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 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 detailEco-systems biology of microbial communities: integration of biomolecular information from unique samples
Muller, Emilie UL; Roume, Hugo UL; Wilmes, Paul UL

Poster (2013)

In microbial ecology, high-resolution molecular approaches are essential to characterize the vast organismal and functional diversity and to understand the interactions between environmental factors and ... [more ▼]

In microbial ecology, high-resolution molecular approaches are essential to characterize the vast organismal and functional diversity and to understand the interactions between environmental factors and microbial communities (MCs). Molecular eco-systems biology based on the integration of genomics and functional omics, allows conclusive links to be drawn between genetic potential and function. However, the field faced major challenges arising from the heterogeneity and dynamics of MCs. Hence, to facilitate meaningful data integration, analysis and modeling, it is crucial to obtain standardised, reproducible and simultaneous measurements of multiple features from a unique sample. We have developed a new methodological framework for the isolation of high-quality DNA, large and small RNA, proteins and metabolites fractions from undivided MC samples. The methodology is based on cryogenic sample preservation and cell lysis. Metabolites are first extracted using organic solvents, followed by sequential isolation of biomacromolecules using chromatographic spin column technology. The methodology was validated by comparison to commonly used dedicated methods and its broad applicability was demonstrated on MCs of biotechnological, environmental and medical interest. Applying this method to sewage plant MCs has allowed us to determine community-level keystone genes and to probe the functional relevance of the population-level composite genomes, leading to the identification of key players of the community. This methodological framework lays the foundation for standardized molecular eco-systems biology and offers exciting prospects for elucidating the genetic blueprints and the functional relevance of specific populations within MCs, particularly in relation to environment-driven demography changes leading to catastrophic bifurcations. [less ▲]

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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 detailA Biomolecular Isolation Framework for Molecular Eco-Systems Biology
Muller, Emilie UL; Roume, Hugo UL; Shah, Pranjul UL et al

Poster (2012)

With the advent of high-throughput omic technologies, powerful and sensitive methods are available for the analysis of nucleic acid, protein and small molecule complements obtained from biological samples ... [more ▼]

With the advent of high-throughput omic technologies, powerful and sensitive methods are available for the analysis of nucleic acid, protein and small molecule complements obtained from biological samples. Molecular eco-systems biology studies based on the integration of genomic, transcriptomic, proteomic and metabolomic data are faced with major challenges arising from the complexity, dynamics and heterogeneity of mixed microbial consortia. In order to facilitate meaningful data integration, analysis and modeling, it is essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single unique samples. 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 column technology. 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 medical interest. Importantly, the developed methodology will allow exploitation of the inherent heterogeneity and dynamics within microbial consortia through spatial and temporal sampling of biological systems to allow later deconvolution of community-wide, population-wide and individual-level processes using the generated omic data. This approach has the potential to identify associations between distinct biomolecules and which may provide pointers towards unravelling previously unknown metabolic processes. Finally, by providing a standardized workflow, the methodology lays the foundation for comparative eco-systematic studies of different natural microbial consortia in the future. [less ▲]

Detailed reference viewed: 67 (2 UL)
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: 78 (6 UL)