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United States Department of Agriculture

Agricultural Research Service

Title: 15n-Dna Stable Isotope Probing (Sip) for the Analysis of Contaminant-Degrading Microorganisms: Assessment of Feasibility and Limitations

Authors
item Cupples, Alison
item Shaffer, Elizabeth - UNIV OF ILLINOIS
item CHEE SANFORD, JOANNE
item Sims, Gerald

Submitted to: Society of Environmental Toxicology and Chemistry Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: August 8, 2005
Publication Date: November 13, 2005
Citation: Cupples, A.M., Shaffer, E.A., Chee Sanford, J.C., Sims, G.K. 2005. 15n-dna stable isotope probing (sip) for the analysis of contaminant-degrading microorganisms: assessment of feasibility and limitations [abstract]. Society of Environmental Toxicology and Chemistry.

Technical Abstract: Stable isotope probing (SIP) is a novel method allowing for the separation and subsequent identification of organisms actively assimilating isotopically labeled compounds. Thus far the method has focused on the incorporation of 13C-labeled substrates into nucleic acids or other biomarkers, advantageous with DNA-SIP because of the high carbon content and the taxonomic information that can subsequently be obtained. Here, we examine the feasibility of 15N-DNA SIP, an approach that could ultimately be used to identify microorganism responsible for in situ degradation of organic contaminants containing nitrogen (such as triazines or explosives) or those involved in nitrogen cycling. To do this, the change in DNA buoyant density (BD) resulting from 15N incorporation by Escherichia coli, a low guanine and cytosine (GC) organism (~50%), and Micrococcus luteus, a high GC organism (~70%), was determined through isopycnic density gradient centrifugation of light (14N) and heavy (15N) DNA, followed by fractionation, and DNA quantification in each fraction. The differential change in DNA BD was 0.0148 ± 0.0022 g mL-1 (mean ± standard deviation, n = 3) for E. coli and 0.0127 ± 0.002 g mL-1(n = 3) for M. luteus. These buoyant density differentials were less than that obtained with 13C labeled (0.0380 g mL-1) or with combined 13C and 15N-labeled E. coli DNA (0.0445 g mL-1). These results indicate that retrieval of target heavy DNA fractions cannot be as efficiently achieved with 15N-label as with 13C -label, particularly in mixed microbial populations varying in GC content. However, isolation and molecular community characterization of multiple fractions of the density gradient will allow active and non-active microbial populations to be distinguished. The high sensitivity of 15N-DNA SIP coupled with molecular community profiling makes it a promising technology for use in bioremediation studies with defined cultures and potentially environmental samples.

Last Modified: 7/25/2014
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