Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies
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 13C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of 13C 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.
Key wordsCarbon-13 DNA stable isotope probing DNA-SIP High-throughput sequencing Isotopic labeling Methylotrophy Metagenomics One-carbon compounds
This work was possible thanks to financial support from the Gordon and Betty Moore Foundation Marine Microbiology Initiative Grant GBMF3303 to J. Colin Murrell and Yin Chen and through the Earth and Life Systems Alliance, Norwich Research Park, Norwich, UK.
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