2012 Annual Report 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: New primer sets for important N-cycling genes (nosZ and nrfA) have been developed and validated for use in reference strains and soils. Results are the first to demonstrate significant presence of microbial populations with ability to reduce nitrate and nitrite to ammonia (DNRA) in soil, and the diversity of nrfA is so far expanded beyond what has been previously well-studied only in gut microbial species. New bacterial isolates harboring atypical nosZ, including strains that are not traditional denitrifiers or possess a complete denitrification pathway, and previously understudied DNRA populations have been isolated from two agricultural soils with contrasting texture and drainage characteristics. Combined with the genetic results, these populations represent active bacteria within agricultural soil systems that aid in processes of N-retention. Two publications have been prepared and in review; approximately 100 new nrfA gene sequences will be deposited in the national databases Genbank and the Functional Gene Pipeline used for genomics studies.