Stable Isotope Probing with 15 N 2 Reveals Novel Noncultivated Diazotrophs in Soil

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Abstract
Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel 15 N 2 -DNA stable isotope probing ( 5 N 2 -DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from 15 N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria , respectively. Analysis of nifH genes from 15 N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that 15 N 2 -DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales .

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