Improved primers and a pyrosequencing approach recover wide range of nitrogen reductase genes from peatlands

Oligonucleotides used for the amplification of nitrogen reductase (nifH like) gene fragments from environmental samples were re-examined for use in a project designed to survey microbial communities in Canada's boreal ecosystem. A wide diversity of nitrogen reductase gene sequences were aligned, revealing that commonly used primers for the amplification of nifH gene fragments are inadequate for targeting the full range of sequence variants in the database. Conserved regions were used to design improved redundant primers which were synthesized with bar codes and sequencing adaptors attached. The PCR products from 16 peatland samples were mixed with other amplicons representing a range of phylogenetic and functional gene targets, and sequenced on a Roche FLX sequencing platform. Products from different gene targets and samples were separated after sequencing based on string recognition programs. Nitrogen reductase gene sequences were analyzed and compared to others in the database. Our primers target all known nifH, vnfH and anfH like sequences from each of the previously defined clusters I, II , III, and IV. We recovered sequences from peat samples that belong to clusters I, III, IV, and many intermediate sequences that call into question the integrity of the previously defined clusters. We also amplified a high diversity of protochlorophyllide reductase gene (frxC like) sequences as these genes share most of the conserved protein motif of the nitrogenase reductases. The study demonstrated the utility of pyrosequencing for multiple barcoded amplicons from multiple samples.