DNA-Based Stable Isotope Probing.

Microbiomes on Earth are often considered the most heterogeneous biological entities, but their vital roles in driving global biogeochemical cycles often remain elusive. DNA-based stable isotope probing (DNA-SIP) provides a powerful means to establish a direct link between biogeochemical processes and the taxonomic identities of active microorganisms involved in the processes. Combined with high-throughput sequencing, it significantly aids in deciphering ecophysiological functions of active microorganisms at the level of microbial communities. DNA-SIP relies solely on the propagation of targeted microbial communities, during which the entire genomes of daughter cells are synthesized and increasingly 13C-labeled. This growth on 13C-labeled substrate in association with cell division provides solid evidence for the functional importance and metabolic potential of targeted microorganisms. The essential prerequisite for a successful DNA-SIP experiment is the identification, with confidence, of isotopically enriched 13C-DNA, of which the amount is generally too low to allow for the direct measurement of 13C atomic percent of nucleic acid. The 13C labeling can be readily identified in the fractionated DNA by quantification of functional genes specific to the known targeted microorganisms, and by high-throughput sequencing of the total microbial communities via 16S rRNA genes without prior knowledge of which microorganisms are 13C-labeled (i.e., highly enriched in the heavy fractions relative to 12C (natural isotope abundance) control treatments). In this chapter, the protocol for obtaining DNA highly enriched in heavy isotope is presented using diazotrophic methanotrophs in a paddy soil as a case study.