Assessing Nitrite Reductase (nirK) Gene Composition in Different Cropping Systems Using Targeted Pyrotag and Whole Metagenome Approaches.

We used metagenomics to assess the composition of nitrite reductase (nirK) genes across different cropping systems at the Kellogg Biological Station’s Long Term Ecological Research Station in Michigan, USA.  Denitrification is a critical step in the nitrogen (N) cycle both as a means to ‘close the loop’ by returning terrestrial N to the atmosphere, but also as a contributor to nitrous oxide (N₂O), emissions, a potent greenhouse gas.  Nitrite reduction via nirK occurs in both heterotrophic denitrifiers and autotrophic ammonia oxidizers and is a key link in the denitrification pathway.  Different cropping system managements alter the composition of organisms that carry out N cycling and we used barcoaded pyrotag sequencing for nirK to isolate how the composition of this gene changes based on cropping system.  Differences in nirK were sensitive to both cropping systems and patterns in denitrification across a range of crop management strategies.  Under main cropping system experiment, pattern in sequence data showed an increased proportion of dentirifier in sites managed for agriculture concominant with increased N2O flux. In a biodiversity trial experiment, targeted nirK analysis showed a higher proportion of ammonia oxidizers under cover crops – suggesting incomplete denitrification.  Lab testing showed more denitrification potential (complete denitrification) under cover crops.  Results show the need to use pyrosequence data in conjunction with targeted assays to link functional ecology and align sequencing results field-based measures.