title: Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies.
abstract: Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or "who does what, where and when?" Metabolic labeling with C1 compounds substituted with (13)C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of (13)C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter.
authors.affiliation: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.|Institute for Ecology, Friedrich Schiller University Jena, Dornburger Strasse 159, 07743, Jena, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Taubert
authors.affiliation: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.
authors.forename: Carolina
authors.initials: C
authors.lastname: Grob
authors.affiliation: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Howat
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Burns
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK.
authors.forename: Yin
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada, N2L 3G1.
authors.forename: Josh D
authors.initials: JD
authors.lastname: Neufeld
authors.affiliation: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK. j.c.murrell@uea.ac.uk.
authors.forename: J Colin
authors.initials: JC
authors.lastname: Murrell
mesh_terms: D017554:Carbon Compounds, Inorganic / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D017753:Ecosystem; D004783:Environmental Microbiology; D007553:Isotope Labeling / Q000379:methods*; D056186:Metagenomics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D017554:Carbon Compounds, Inorganic; D002247:Carbon Isotopes; D015342:DNA Probes; D004269:DNA, Bacterial
keywords: Carbon-13; DNA stable isotope probing; DNA-SIP; High-throughput sequencing; Isotopic labeling; Metagenomics; Methylotrophy; One-carbon compounds