Abstract
Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter we provide protocols for obtaining labeled DNA, RNA, and proteins that can be used for downstream omics-based analyses.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Handelsman J (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68:669–685
Lynch MD, Neufeld JD (2015) Ecology and exploration of the rare biosphere. Nat Rev Microbiol 13:217–229
Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S et al (2007) The sorcerer II global ocean sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol 5:e77
Quince C, Curtis TP, Sloan WT (2008) The rational exploration of microbial diversity. ISME J 2:997–1006
Shade A, Hogan CS, Klimowicz AK, Linske M, McManus PS, Handelsman J (2012) Culturing captures members of the soil rare biosphere. Environ Microbiol 14:2247–2252
Radajewski S, Ineson P, Parekh NR, Murrell JC (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403:646–649
Dumont MG, Murrell JC (2005) Stable isotope probing - linking microbial identity to function. Nat Rev Microbiol 3:499–504
Neufeld JD, Wagner M, Murrell JC (2007) Who eats what, where and when? Isotope-labelling experiments are coming of age. ISME J 1:103–110
Uhlik O, Leewis MC, Strejcek M, Musilova L, Mackova M, Leigh MB, Macek T (2013) Stable isotope probing in the metagenomics era: a bridge towards improved bioremediation. Biotechnol Adv 31:154–165
Grob C, Taubert M, Howat AM, Burns OJ, Chen Y, Murrell JC (2015) Generating enriched metagenomes from active microorganisms with DNA stable isotope probing. Hydrocarb Lipid Microbiol Protoc 10:1007
Friedrich MW (2006) Stable-isotope probing of DNA: insights into the function of uncultivated microorganisms from isotopically labeled metagenomes. Curr Opin Biotechnol 17:59–66
Neufeld JD, Dumont MG, Vohra J, Murrell JC (2007) Methodological considerations for the use of stable isotope probing in microbial ecology. Microb Ecol 53:435–442
Schloss PD, Handelsman J (2003) Biotechnological prospects from metagenomics. Curr Opin Biotechnol 14:303–310
Wellington EM, Berry A, Krsek M (2003) Resolving functional diversity in relation to microbial community structure in soil: exploiting genomics and stable isotope probing. Curr Opin Microbiol 6:295–301
Martineau C, Whyte LG, Greer CW (2010) Stable isotope probing analysis of the diversity and activity of methanotrophic bacteria in soils from the Canadian high Arctic. Appl Environ Microbiol 76:5773–5784
Bell TH, Yergeau E, Martineau C, Juck D, Whyte LG, Greer CW (2011) Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing. Appl Environ Microbiol 77:4163–4171
Eyice Ö, Namura M, Chen Y, Mead A, Samavedam S, Schäfer H (2015) SIP metagenomics identifies uncultivated Methylophilaceae as dimethylsulphide degrading bacteria in soil and lake sediment. ISME J 9:2336–2348
Grob C, Taubert M, Howat AM, Burns OJ, Dixon JL, Richnow HH et al (2015) Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph. Environ Microbiol 17:4007–4018
Dumont MG, Radajewski SM, Miguez CB, McDonald IR, Murrell JC (2006) Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis. Environ Microbiol 8:1240–1250
Coyotzi S, Pratscher J, Murrell JC, Neufeld JD (2016) Targeted metagenomics of active microbial populations with stable-isotope probing. Curr Opin Biotechnol 41:1–8
Albertsen M, Hugenholtz P, Skarshewski A, Nielsen KL, Tyson GW, Nielsen PH (2013) Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nat Biotechnol 31:533–538
Dumont MG, Pommerenke B, Casper P, Conrad R (2011) DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment. Environ Microbiol 13:1153–1167
Haichar FZ, Roncato MA, Achouak W (2012) Stable isotope probing of bacterial community structure and gene expression in the rhizosphere of Arabidopsis thaliana. FEMS Microbiol Ecol 81:291–302
Huang WE, Ferguson A, Singer AC, Lawson K, Thompson IP, Kalin RM et al (2009) Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization. Appl Environ Microbiol 75:234–241
Dumont MG, Pommerenke B, Casper P (2013) Using stable isotope probing to obtain a targeted metatranscriptome of aerobic methanotrophs in lake sediment. Environ Microbiol Rep 5:757–764
Jehmlich N, Schmidt F, Taubert M, Seifert J, Bastida F, von Bergen M et al (2010) Protein-based stable isotope probing. Nat Protoc 5:1957–1966
Seifert J, Taubert M, Jehmlich N, Schmidt F, Volker U, Vogt C et al (2012) Protein-based stable isotope probing (protein-SIP) in functional metaproteomics. Mass Spectrom Rev 31:683–697
von Bergen M, Jehmlich N, Taubert M, Vogt C, Bastida F, Herbst FA et al (2013) Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology. ISME J 7:1877–1885
Pan C, Fischer CR, Hyatt D, Bowen BP, Hettich RL, Banfield JF (2011) Quantitative tracking of isotope flows in proteomes of microbial communities. Mol Cell Proteomics 10(M110):006049
Taubert M, Vogt C, Wubet T, Kleinsteuber S, Tarkka MT, Harms H et al (2012) Protein-SIP enables time-resolved analysis of the carbon flux in a sulfate-reducing, benzene-degrading microbial consortium. ISME J 6:2291–2301
Lünsmann V, Kappelmeyer U, Benndorf R, Martinez-Lavanchy PM, Taubert A, Adrian L et al (2016) In situ protein-SIP highlights Burkholderiaceae as key players degrading toluene by para ring hydroxylation in a constructed wetland model. Environ Microbiol 18:1176
Herbst FA, Bahr A, Duarte M, Pieper DH, Richnow HH, von Bergen M et al (2013) Elucidation of in situ polycyclic aromatic hydrocarbon degradation by functional metaproteomics (protein-SIP). Proteomics 13:2910–2920
Taubert M, Baumann S, von Bergen M, Seifert J (2011) Exploring the limits of robust detection of incorporation of 13C by mass spectrometry in protein-based stable isotope probing (protein-SIP). Anal Bioanal Chem 401:1975–1982
Taubert M, von Bergen M, Seifert J (2013) Limitations in detection of 15N incorporation by mass spectrometry in protein-based stable isotope probing (protein-SIP). Anal Bioanal Chem 405:3989–3996
Jehmlich N, Kopinke FD, Lenhard S, Vogt C, Herbst FA, Seifert J et al (2012) Sulfur-36S stable isotope labeling of amino acids for quantification (SULAQ). Proteomics 12:37–42
Justice NB, Li Z, Wang Y, Spaudling SE, Mosier AC, Hettich RL et al (2014) 15N- and 2H proteomic stable isotope probing links nitrogen flow to archaeal heterotrophic activity. Environ Microbiol 16:3224–3237
Slysz GW, Steinke L, Ward DM, Klatt CG, Clauss TR, Purvine SO et al (2014) Automated data extraction from in situ protein-stable isotope probing studies. J Proteome Res 13:1200–1210
Sachsenberg T, Herbst FA, Taubert M, Kermer R, Jehmlich N, von Bergen M et al (2015) MetaProSIP: automated inference of stable isotope incorporation rates in proteins for functional metaproteomics. J Proteome Res 14:619–627
Neufeld JD, Vohra J, Dumont MG, Lueders T, Manefield M, Friedrich MW, Murrell JC (2007) DNA stable-isotope probing. Nat Protoc 2:860–866
Whiteley AS, Thomson B, Lueders T, Manefield M (2007) RNA stable-isotope probing. Nat Protoc 2:838–844
Bartram AK, Lynch MD, Stearns JC, Moreno-Hagelsieb G, Neufeld JD (2011) Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end illumina reads. Appl Environ Microbiol 77:3846–3852
Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N et al (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621–1624
Jünemann S, Sedlazeck FJ, Prior K, Albersmeier A, John U, Kalinowski J et al (2013) Updating benchtop sequencing performance comparison. Nat Biotechnol 31:294–296
Wood DE, Salzberg SL (2014) Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol 15:R46
Segata N, Waldron L, Ballarini A, Narasimhan V, Jousson O, Huttenhower C (2012) Metagenomic microbial community profiling using unique clade-specific marker genes. Nat Methods 9:811–814
Menzel P, Ng KL, and Krogh A (2015) Kaiju: fast and sensitive taxonomic classification for metagenomics. bioRxiv. doi: 10.1101/031229.
Meyer F, Paarmann D, D’Souza M, Olson R, Glass EM, Kubal M et al (2008) The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9:386
Bartram A, Poon C, Neufeld J (2009) Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant. Biotechniques 47:1019–1022
Acknowledgments
Yin Chen acknowledges funding from NERC grant NE/I027061/1, and Yin Chen and J. Colin Murrell both acknowledge funding from the Gordon and Betty Moore Foundation Marine Microbiology Initiative Grant GBMF3303 and the Earth and Life Systems Alliance, Norwich Research Park, Norwich, UK. Josh D. Neufeld acknowledges a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Jameson, E. et al. (2017). DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms. In: Streit, W., Daniel, R. (eds) Metagenomics. Methods in Molecular Biology, vol 1539. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6691-2_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6691-2_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6689-9
Online ISBN: 978-1-4939-6691-2
eBook Packages: Springer Protocols