Microbial ecologists continue to seek a greater understanding of the factors that govern the ecological significance of microbial community structure. Changes in community structure have been shown to have functional significance for processes that are mediated by a narrow spectrum of organisms, such as nitrification and denitrification, but in some cases, functional redundancy in the community seems to buffer microbial ecosystem processes. The functional significance of microbial community structure is frequently obscured by environmental variation and is hard to detect in short-term experiments. We examine the functional significance of free-living diazotrophs in a replicated long-term tillage experiment in which extraneous variation is minimized and N-fixation rates can be related to soil characteristics and diazotroph community structure. Soil characteristics were found to be primarily impacted by tillage management, whereas N-fixation rates and diazotroph community structure were impacted by both biomass management practices and interactions between tillage and biomass management. The data suggest that the variation in diazotroph community structure has a greater impact on N-fixation rates than do soil characteristics at the site. N-fixation rates displayed a saturating response to increases in diazotroph community diversity. These results show that the changes in the community structure of free-living diazotrophs in soils can have ecological significance and suggest that this response is related to a change in community diversity.

Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15 N, nearly all applications of this technique to date have used 13 C. Successful application of SIP using 15 N-DNA ( 15 N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml ؊1 for 15 N-labeled DNA, relative to 0.036 g ml ؊1 for 13 C-labeled DNA. In contrast, variation in genome G؉C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml ؊1 . Thus, natural variation in genome G؉C content in complex communities prevents the effective separation of 15

No 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.

No abstract