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Appl. Environ. Microbiol. doi:10.1128/AEM.02610-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Stable isotope probing with ¹⁵N₂ reveals novel non-cultivated diazotrophs in soil

Department of Crop and Soil Sciences, Cornell University, Ithaca, NY 14853

^* To whom correspondence should be addressed. Email: dhb28{at}cornell.edu.

	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 in a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of non-cultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel ¹⁵N₂-DNA-stable isotope probing (SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from ¹⁵N-labeled DNA provides 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 Beta Proteobacteria and Actinobacteria respectively. Analysis of nifH genes from ¹⁵N-labeled DNA also revealed three microbial groups, one of which was associated with Alpha Proteobacteria, 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 ¹⁵N₂-DNA-stable isotope probing (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.