Ecological Patterns of nifH Genes in Four Terrestrial Climatic Zones Explored with Targeted Metagenomics Using FrameBot, a New Informatics Tool

Biological nitrogen fixation is an important component of sustainable soil fertility and a key component of the nitrogen cycle. We used targeted metagenomics to study the nitrogen fixation-capable terrestrial bacterial community by targeting the gene for nitrogenase reductase (nifH). We obtained 1.1 million nifH 454 amplicon sequences from 222 soil samples collected from 4 National Ecological Observatory Network (NEON) sites in Alaska, Hawaii, Utah, and Florida. To accurately detect and correct frameshifts caused by indel sequencing errors, we developed FrameBot, a tool for frameshift correction and nearest-neighbor classification, and compared its accuracy to that of two other rapid frameshift correction tools. We found FrameBot was, in general, more accurate as long as a reference protein sequence with 80% or greater identity to a query was available, as was the case for virtually all nifH reads for the 4 NEON sites. Frameshifts were present in 12.7% of the reads. Those nifH sequences related to the Proteobacteria phylum were most abundant, followed by those for Cyanobacteria in the Alaska and Utah sites. Predominant genera with nifH sequences similar to reads included Azospirillum, Bradyrhizobium, and Rhizobium, the latter two without obvious plant hosts at the sites. Surprisingly, 80% of the sequences had greater than 95% amino acid identity to known nifH gene sequences. These samples were grouped by site and correlated with soil environmental factors, especially drainage, light intensity, mean annual temperature, and mean annual precipitation. FrameBot was tested successfully on three ecofunctional genes but should be applicable to any.