Quantitative microbial ecology through stable isotope probing

Bacteria grow and transform elements at different rates, yet quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to Stable Isotope Probing (SIP) that enables determining the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon specific density curves are produced for labeled and non-labeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon’s density shift relative to that taxon’s density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative Stable Isotope Probing (qSIP). We demonstrate qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variation in 18 O and 13 C composition after exposure to 18 O-H 2 O or 13 C-glucose. Addition of glucose increased assimilation of 18 O into DNA from 18 O-H 2 O. However, the increase in 18 O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because glucose addition indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.