A biomolecular isolation framework foreco-systems biology
Hugo Roume1,2, Emilie EL Muller1, Thekla Cordes1, Jenny Renaut2, Karsten Hiller1
and Paul Wilmes1,21Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg and2Department of Environment and Agro-Biotechnologies, Public Research Centre - Gabriel Lippmann,Belvaux, Luxembourg
Mixed microbial communities are complex, dynamic and heterogeneous. It is therefore essential thatbiomolecular fractions obtained for high-throughput omic analyses are representative of singlesamples to facilitate meaningful data integration, analysis and modeling. We have developed a newmethodological framework for the reproducible isolation of high-quality genomic DNA, large andsmall RNA, proteins, and polar and non-polar metabolites from single unique mixed microbialcommunity samples. The methodology is based around reproducible cryogenic sample preservationand cell lysis. Metabolites are extracted first using organic solvents, followed by the sequentialisolation of nucleic acids and proteins using chromatographic spin columns. The methodology wasvalidated by comparison to traditional dedicated and simultaneous biomolecular isolation methods.To prove the broad applicability of the methodology, we applied it to microbial consortiaof biotechnological, environmental and biomedical research interest. The developed methodologicalframework lays the foundation for standardized molecular eco-systematic studies on a range ofdifferent microbial communities in the future.The ISME Journal (2013) 7, 110121; doi:10.1038/ismej.2012.72; published online 5 July 2012Subject Category: integrated genomics and postgenomics approaches in microbial ecologyKeywords: biomolecules; eco-systems biology; extraction; isolation; metabolomics; sample heterogeneity
Natural microbial communities play fundamentalroles in the Earths biogeochemical cycles as well asin human health and disease, and provide essentialservices to mankind. They represent highly com-plex, dynamic and heterogeneous systems (Denefet al., 2010). The advent of high-resolutionmolecular biology methodologies, including geno-mics (Tyson et al., 2004), transcriptomics (Frias-Lopez et al., 2008), proteomics (Ram et al., 2005)and metabolomics (Li et al., 2008), is facilitatingunprecedented insights into the structure andfunction of microbial consortia in situ. Beyondisolated biomolecular characterization, integratedomics combined with relevant statistical analysesoffer the ability to unravel fundamental ecologicaland evolutionary relationships, which are indis-cernible from isolated omic data sets (Wilmes et al.,2010). Furthermore, space- and time-resolved
integrated omics have the potential to uncoverassociations between distinct biomolecules, whichallows for discovery of previously unknownbiochemical traits of specific microbial communitymembers (Fischer et al., 2011). Such linkages will,however, only be discernible if representativebiomolecular fractions are obtained. Thus, withinthe emerging field of molecular eco-systems biology(Raes and Bork, 2008), molecule-level systematiccharacterizations of microbial consortia will onlyfulfill their full potential if biomolecular fractions(DNA, RNA, proteins and small molecules) areobtained from single unique samples (Kitano,2001). Only then will the subsequent integration ofhigh-resolution omic data enable true systems-levelviews of community-wide, population-wide andindividual-level processes. To our knowledge,no methodology currently exists for the isolation ofall concomitant small molecules (metabolites) andbiomacromolecules (DNA, RNA and proteins) frombiological systems.
A well-established method for the isolation ofconcomitant biomacromolecules only is basedaround the simultaneous addition of a monophasicmixture of phenol and guanidine isothiocyanate,commercially available as TRIzol and TRI Reagent(TR), and chloroform to biological samples to obtain
Correspondence: P Wilmes, Luxembourg Centre for SystemsBiomedicine, University of Luxembourg, L-4362 Esch-sur-Alzette,Luxembourg.E-mail: firstname.lastname@example.orgReceived 8 February 2012; revised 25 May 2012; accepted 29 May2012; published online 5 July 2012
The ISME Journal (2013) 7, 110121& 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13
an aqueous phase containing primarily RNA, aninterphase containing DNA and an organic phasecontaining proteins (Chomczynski, 1993; Hummonet al., 2007; Chey et al., 2011; Xiong et al., 2011).Individual biomacromolecular fractions are purifiedfrom the respective phases and may be subjected tospecialized downstream analyses. Adaptation of thestandard TR protocol for the additional extraction ofsmall molecules was carried out on plant materialby Weckwerth et al. (2004). For this, a solventmixture of methanol, chloroform and water is firstused for the fractionation of small molecules intopolar and non-polar metabolites, and the precipita-tion of biological macromolecules. Polar and non-polar metabolites are retrieved from the aqueous andorganic phases, respectively. RNA and proteins areisolated from the remaining pellet following extrac-tion in dedicated buffers and phenol, respectively.However, no genomic DNA fraction was obtainedusing this method, a need that is particularlyimportant in microbial communities that exhibitextensive genetic heterogeneity (Wilmes et al., 2009)and for which genomic context is thus essential formeaningful interpretation of functional omic data.
Owing to the hazardous chemicals involvedand the methods being unsuited for routine high-throughput laboratory use, chromatographic spincolumn-based procedures have been introduced forthe isolation of concomitant biomacromolecules(Morse et al., 2006; Tolosa et al., 2007; Radpouret al., 2009). These methods rely on the pH- and salt-concentration-dependent adsorption of nucleicacids and proteins to solid phases such as silica orglass. The solid phases are washed and the bioma-cromolecules of interest are sequentially eluted.Such methods are now available as commercial kitsfrom Qiagen (AllPrep DNA/RNA/Protein Mini kit,QA) and Norgen Biotek (All-in-One Purification kitfor large RNA, small/microRNA, total proteins andgenomic DNA; NA). The latter has the advantage ofoffering the ability to deplete the total RNA fractionof small RNA (o200 nt), which can be analyzedseparately. Chromatographic spin column-based bio-macromolecular isolation has yet to be combined withthe extraction of concomitant small molecules.
Here, we combine sample processing, samplecryopreservation, cell disruption by cryomilling,small molecule extraction and biomacromolecularisolation based on chromatographic spin columns toresult in a universal methodological framework thatallows the standardized isolation of extracellularand intracellular polar and non-polar metabolites,genomic DNA, RNA (divided into large and smallRNA fractions) and proteins from single microbialcommunity samples (Figure 1). We validatedthe performance of the methodology by comparisonto widely used exclusive and simultaneous biomo-lecular isolation methods. Furthermore, we provedits general applicability to diverse mixed microbialcommunities of biotechnological, environmentaland biomedical research interest, that is, lipid-
accumulating organisms (LAOs), that may beexploited for the reclamation of energy-rich lipidsfrom wastewater, river water filtrate and humanfeces. The framework may also find broader appealfor integrated omics on other heterogeneous and/orprecious biological samples.
Materials and methods
For more details see Supplementary Materials andmethods.
Sampling and sample processing
LAO-enriched mixed microbial community. FloatingLAO biomass was sampled from the airwater inter-face of the anoxic activated sludge tank at theSchifflange wastewater treatment plant (Esch-sur-Alzette, Luxembourg; 49130048.2900N; 61104.5300E).For each sampling date, four different islets(I1-I4, defined herein as biological replicates;Supplementary Figure 1) were sampled using a levycane of 500 ml. Samples were collected in 50 mlsterile Falcon tubes and then immediately snap-frozen by immersion in liquid nitrogen and storedat ! 80 1C.
For the determination of sample heterogeneityby metabolomics, four replicates of 200 mg ofLAO biomass (technical replicates) were obtained
ProteomicsProteins PGenomic DNA
Large RNAe RNA
Figure 1 The developed biomolecular isolation frameworkhighlighting the NA-based methodological workflow. Legend:( ) Sample processing and preservation: immediate snap-freezingby immersion in liquid nitrogen for LAO-enriched mixed microbialcommunities; concentration by tangential flow filtration followedby high-speed centrifugation, then snap-freezing of the resultingcell pellet for freshwater mixed microbial communities; homo-genization with RNAlater followed by centrifugation steps beforesnap-freezing for fresh human fecal samples (Materials andmethods). ( ) Cryomilling and metabolite extraction: cryomillingof cell pellets and solvent extraction of the intracellular polar andnon-polar metabolite fractions (extracellular fractions were onlyprepared for the LAO-enriched microbial communities; Materialsand methods). ( ) Physicochemical biomacromolecular isolation:use of sequential physicochemical separation based aroundchromatographic spin columns following bead-beating in themodified NA-lysis buffer, resulting in the iso