|Shaffer, Elizabeth - UNIV OF ILLINOIS|
|Cupples, Alison - PREV. USDA POST DOC|
|Chee Sanford, Joanne|
Submitted to: Microbial Ecology International Symposium
Publication Type: Abstract Only
Publication Acceptance Date: May 8, 2006
Publication Date: August 20, 2006
Citation: Sims, G.K., Shaffer, E.A., Cupples, A.M., Chee Sanford, J.C. 2006. Examining the ecology of heterocyclic N utilization [abstract]. International Society for Microbial Ecology Paper. 2668. Technical Abstract: Recently, we observed that N deprivation may enhance degradation of the atrazine ring and the pyrimidine moiety of cloransulam-methyl, with little effect on degradation of the homocyclic ring for the latter. To extend this work to the organism level, we coupled DNA-based stable isotope probing (SIP), adapted for use with 15N-labeled substrates. Assimilation of 15N by pure cultures produced limited change in buoyant density (BD), ranging from 0.013 g mL-1 (M. luteus, GC = 72%) to 0.015 g mL-1 (E. coli, GC = 50%), however, using gradient fractionation, followed by comparing T-RFLP from fractions of labeled and control treatments increased sensitivity for detecting 15N-enriched DNA. This approach enabled us to detect 9 OTU fragments when 15NH4SO4 treatment was compared to unlabeled NH4SO4 in soil, and an increase in BD (>0.0035 g mL-1) of 16S rRNA upon exposure of Pseudaminobacter sp. strain C147 to side chain 15N-atrazine (25 mg L-1) in culture. Using soil from an atrazine spill site, we observed nearly 60% mineralization 14C-ring atrazine over a 25-day course. Comparisons of T-RFLP data between samples of this soil treated (maximal field application rate) with 15N-labeled and unlabeled atrazine revealed upward shifts in BD associated with specific fragment sizes, suggesting label incorporation by these particular organisms. Our results demonstrate utilization of atrazine side chain N by a subset of the total soil population, and provide an approach for examining regulation of that process by combining molecular tools with process level measurements.