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United States Department of Agriculture

Agricultural Research Service

Title: DNA Buoyant Density Shifts During 15n DNA Stable Isotope Probing.

Authors
item Cupples, Alison
item Shaffer, Elizabeth - UNIV OF ILLINOIS
item Chee Sanford, Joanne
item Sims, Gerald

Submitted to: Microbiological Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 1, 2006
Publication Date: March 24, 2006
Citation: Cupples, A.M., Shaffer, E.A., Chee Sanford, J.C., Sims, G.K. 2006. DNA buoyant density shifts during 15N DNA stable isotope probing. Microbiological Research. 161. Available: http://www.sciencedirect.com.

Interpretive Summary: Nucleic acid based stable isotope probing (DNA SIP) is a novel method that holds much promise for studying in situ (on site) microbial processes, such as herbicide degradation or nutrient cycling. The method involves adding a labeled (15N or 13C label) compound of interest to soil or water, extracting DNA, separating heavy from light DNA using an ultracentrifuge (heavy DNA has incorporated the label), then identifying the organisms that have taken up the label using molecular techniques. Here, we examine the limitations and feasibility of a SIP method that targets nitrogen containing compounds (15N DNA SIP). To do this, we determined the change in DNA weight (buoyant density) from the bacteria, Escherichia coli and Micrococcus luteus following uptake of 15NH4Cl. Additionally, we found that this increase was much less than that provided by 13C label incorporation, therefore previously developed methods for 13C DNA SIP will not be appropriate for 15N DNA SIP. Also, we explored the effectiveness of different molecular techniques for identifying the separation of light and heavy DNA. In summary, we developed a viable method for 15N DNA SIP that can now be applied to environmental samples to better understand in situ processes. The impact of this research is that, for the first time, we can examine organisms degrading herbicides in situ, rather than under laboratory conditions. These findings can be used to provide a better understanding of nitrogen cycling and herbicide degradation in agricultural settings, enabling better management strategies for avoiding herbicide persistence and preventing nitrogen loss.

Technical Abstract: Successful use of 13C stable isotope probing (SIP) to investigate microbial function in natural environments has stimulated interest in SIP technology using other isotopes. 15N-SIP is effective in buoyant density (BD) gradient separation of isotopically-enriched DNA, however, the resulting change in BD is limited, challenging the ability to discern active populations against a background of mixed (GC content) taxa. Based on this study, we conclude that more effective use of 15N-SIP requires characterization of BD, DNA concentration, and molecular analysis of the community over the full spectrum of BD gradient, rather than examining selective fractions. We also demonstrate enhancement of SIP technology with dual isotope labeled DNA. These modifications will benefit the use of 15N-SIP in natural environments, where DNA quantities may be low and populations likely to be complex.

Last Modified: 8/21/2014
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