1a. Objectives (from AD-416):
Objective 1) determine the factors that control DNRA versus denitrification pathways in Shewanella loihica strain PV4; Objective 2) explore whether pathway regulators that control DNRA and denitrification in a pure culture apply universally and govern regulatory networks in nitrate/nitrite reducing mixed cultures and complex soil communities; Objective 3) use (meta)genomic and (meta)transcriptomic approaches to characterize the diversity of non-denitrifier nosZ gene sequences and their regulatory controls under different environmental conditions; Objective 4) measure N flux and associated C turnover in soil mesocosms under a variety of controlled environmental conditions (e.g., pH, temperature, soil moisture) using two relevant soil types.
1b. Approach (from AD-416):
The project is designed for 3 years. Shewanella loihica strain PV4 and Anaeromyxobacter spp. will be used as models to explore the physiological controls and regulatory networks that govern nitrate reduction pathways (e.g., DNRA versus denitrification) and associated C metabolism. Tools for quantitative evaluation of pathway-specific molecular biomarkers (i.e., genes, transcripts, protein) will be designed, tested and applied to assess the prevalence of DNRA vs. denitrification pathways and determine the environmental variables (e.g., C:N ratios, pH, temperature, soil moisture, soil type) that control activity of these pathways. Cultivation-based studies, genetic approaches, tag-encoded pyrosequencing, deep sequencing, metagenomics followed by a metatranscriptome analysis, and proteomic workflows for key biomarkers (e.g., nrfA, nosZ) will be used (U Tennessee, GaTech). N flux and associated C turnover will be quantitatively assessed using mass balance approaches, which will include 13C- and 15N-labeled compounds in mesocosm studies combined with nucleic acid- and protein-targeted approaches (ARS, U Illinois).
3. Progress Report:
Sampling from the two field sites associated with the parent project was nearly completed for year two of the project. Community profiles were completed through June 2013 and will continue into year three of the parent project. Installment of passive gas samplers and surface gas flux measurements were installed. New gene sequences for nosZ and nrfA obtained from the field sites were collected and deposited in the National Center for Biotechnology Information Genbank database and used for development of reference libraries for metagenomic sequencing efforts and quantitative gene measurements in conjunction with Georgia Technological University and University of Tennessee, respectively. One manuscript was published and three manuscripts are currently under review or in preparation from this year’s work.