Stable Isotope Probing and Metagenomics

Two promising culture-independent approaches that have been employed to assess the function and metabolic potential of uncultivated microorganisms are stable isotope probing (SIP) and metagenomics. This chapter discusses the methodology of metagenomics within the context of DNA stable isotope probing (DNA-SIP), and provides a description of the possible limitations and how these limitations can be overcome, summarizes the combined DNA-SIP and meta-genomic studies to date, and highlights future directions. The chapter also focuses on metagenomics as it relates to SIP and highlights some of the methodological considerations for cloning and characterization of labeled DNA from active and uncultivated microorganisms. A study using SIP and metagenomics with increasingly low substrate concentrations to characterize marine methylotrophs involved in C1 cycling of surface seawater was a proof-of-concept approach that utilized multiple displacement amplification (MDA) for the first time in association with DNA-SIP and metagenomics. The study also demonstrated that DNA-SIP employing near-in situ substrate concentrations may be used because the resulting low yields of DNA are still amenable to metagenomic analysis through MDA amplification. The combination of SIP, MDA, and metagenomics provides powerful access to the genomes of active-but-uncultivated microorganisms. An alternative approach for combining SIP and metagenomics is to profile the purified 13C-labeled DNA with high-throughput sequencing of cloned DNA fragments. DNA-SIP paired with metagenomics is expected to yield invaluable insight into the uncultured microbial world as the techniques become increasingly commonplace, isotopes become increasingly available and affordable, and experiments become increasingly well designed.