•     Stable isotope probing with 15N-labeled compounds.
Stable Isotope Probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of non-cultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N-labeled compounds, nearly all applications of this technique to date have used 13C-labeled compounds. We have developed a method which makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of ‘heavy' DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high G + C content DNA. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications. We are currently using this method to explore nitrogen fixation in a range of soil systems from agricultural systems to desert soil crusts.

•    Targeted Environmental Genomics: Stable isotope probing of non-cultivated diazotrophs in soil.
All known forms of life require fixed nitrogen (N) for biosynthesis and microbial N-fixation provides the largest natural source of fixed N in the biosphere. Free-living diazotrophs in soils provide the dominant natural source of fixed N in many terrestrial systems, and yet we know remarkably little about the ecology and evolution of these microorganisms as the majority of microorganisms in soil resist cultivation in the laboratory. We are using a novel approach that combines Stable

Isotope Probing (SIP) with environmental genomics in order to target the genomes of non-cultivated diazotrophs. This targeted environmental genomics approach will allow access to genomic DNA from these non-cultivated microbes, providing a remarkable opportunity to link genomic data to a microbial process as it occurs in the environment. An unparalleled advantage of this paradigm is that it brings the possibility of experimental approaches into environmental genomics, a field previously based primarily on observation. This research will significantly advance our understanding of the environmental mechanisms that regulate the diversity and activity of free-living N-fixing microorganisms in soils providing an unprecedented understanding of the functional significance of a microbial process of central importance to the global N cycle. The genome information obtained will also help to shed light on the evolution of N-fixation and on genome evolution in general by revealing the complex structure of nitrogenase operons in previously uncharacterized microorganisms.