Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification

Dissimilatory nitrate reduction to ammonium (DNRA) is now known to be a more prevalent process in terrestrial ecosystems than previously thought. The key enzyme, a pentaheme cytochrome c nitrite reductase NrfA associated with respiratory nitrite ammonification, is encoded by the nrfA gene in a broad phylogeny of bacteria. The lack of reliable and comprehensive molecular tools to detect diverse nrfA from environmental samples has hampered efforts to meaningfully characterize the genetic potential for DNRA in environmental systems. In this study, modifications were made to optimize the amplification efficiency of previously-designed PCR primers, targeting the diagnostic region of NrfA between the conserved third- and fourth heme binding domains, and to increase coverage to include detection of environmentally relevant Geobacteraceae-like nrfA. Using an alignment of the primers to >270 bacterial nrfA genes affiliated with 18 distinct clades, modifications to the primer sequences improved coverage, minimized amplification artifacts, and yielded the predicted product sizes from reference-, soil-, and groundwater DNA. Illumina sequencing of amplicons showed the successful recovery of nrfA gene fragments from environmental DNA based on alignments of the translated sequences. The new primers developed in this study are more efficient in PCR reactions, although gene targets with high GC content affect efficiency. Furthermore, the primers have a broader spectrum of detection and were validated rigorously for use in detecting nrfA from natural environments. These are suitable for conventional PCR, qPCR, and use in PCR access array technologies that allow multiplex gene amplification for downstream high throughput sequencing platforms.