Stable Isotope Probing with 15N2 as a Tool to Uncover the Functional Significance of Non-Cultivated Diazotrophs in Soil.
Daniel H. Buckley, Varisa Haungyutitham, Shi-Fang Hsu, and Tyrrell Nelson. Cornell University, Department of Crop and Soil Sciences, Ithaca, NY 14853
The ecological mechanisms that link microbial diversity and community function in soils can be revealed by focusing on individual functional groups, such as free-living diazotrophs, that mediate well defined ecological processes. A remarkable diversity of non-cultivated diazotrophs exists in soils suggesting that current models of N-fixation, based on cultivated strains, may not adequately explain the activity of these organisms in natural communities. Stable Isotope Probing (SIP) with 15N2 represents a powerful new tool that can be used to identify the functional capacities of non-cultivated diazotrophs in microbial communities. SIP promises to open new avenues of exploration for organisms that resist cultivation in the laboratory because it allows for direct linkage of molecular markers to specific microbial processes as they occur under in situ conditions or in controlled microcosm experiments. The basic principle of SIP is that an isotopically-labeled compound is added to the soil and used by microorganisms which then incorporate the label into their nucleic acids. The labeled heavy-DNA can then be isolated from the unlabeled light-DNA by isopycnic centrifugation in order to identify the organisms that incorporated the labeled compound under in situ conditions. The major concern when using 15N as an isotopic label instead of 13C is that the density difference between 13C-labeled DNA and unlabeled DNA is ≤ 0.036 g cm-3, while that between 15N-labeled DNA and 14N DNA is only ≤ 0.016 g cm-3. We have determined that it is possible to reproducibly characterize the density of DNA to ± 0.002 g cm-3 after ultracentrifugation in CsCl gradients and thus SIP can be used to identify DNA molecules that have greater than 30% incorporation of a 15N-label. However, the native density of DNA can vary by more than 0.039 g cm-3 as a result of differences in its G + C content. Thus, it is important to control for natural variation in genome G + C content any time SIP is used by performing experiments in parallel in the presence of either 14N2 or 15N2, fractionating gradients, and then using Terminal-Restriction Length Fragment Polymorphism analysis of 16S rRNA genes to examine gradient fractions and to identify those specific fractions that contain 15N-labeled DNA. The labeled-DNA in these fractions is then purified from unlabeled DNA by a secondary centrifugation in CsCl gradients containing bisbenzimide. This method has been used to examine the diazotrophic community composition of soils incubated in the presence of 15N2 under different experimental conditions. Significant fixation of 15N2 was observed in soil incubated in the presence of atmospheric levels of oxygen and was observed to increase significantly in response to additions of CH4 and H2. Fixation of 15N2 into soil was less when soil was wetted or when oxygen was excluded. This 15N-SIP method was used successfully on soils that had a total d15N enrichment of 21.2‰ over controls corresponding to approximately 59 ng of 15N-labeled DNA g-1 soil demonstrating that 15N-SIP can be achieved even with samples that have low rates of N-fixation. Analyses of nifH and 16S rRNA genes from gradient fractions demonstrate the effect of microcosm conditions on diazotroph community composition and reveal the activity of non-cultivated diazotrophs in soil.