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

Authors: CANNON, J - University Of Illinois; SANFORD, R - University Of Illinois; Connor, Lynn; YANG, WENDY - University Of Illinois; Chee Sanford, Joanne

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/20/2019
Publication Date: 3/21/2019
Citation: Cannon, J.C., Sanford, R.A., Connor, L.M., Yang, W.H., Chee Sanford, J.C. 2019. Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification. Applied and Environmental Microbiology. 160:49-59. https://doi.org/10.1016/j.mimet.2019.03.020.
DOI: https://doi.org/10.1016/j.mimet.2019.03.020

Interpretive Summary: Dissimilatory nitrate reduction to ammonium (DNRA) has increasingly been revealed over recent years to occur more in upland soils than previously thought. In environments like agricultural soils, there is high interest in the fate of nitrogen fertilizers to improve use efficiency in crop management and mitigation of N-loss (e.g. via denitrification), especially in the form of gaseous N2O, a potent greenhouse gas. DNRA has been long considered a N-retaining process by bacteria in natural environments, with N2O also produced as a side product of the process, yet we still know very little about DNRA activity in soils, the microbial populations that mediate the process, and the controlling factors. Suitable molecular tools are needed to study DNRA more effectively. The key enzyme is nitrite reductase (NrfA) encoded by the nrfA gene found in diverse bacteria as we previously reported (Welsh et al., 2014). In this study, we made substantial improvements to PCR primers that we previously designed but were now optimized for more efficient amplification of diverse nrfA from reference strains and soil. NrfA sequence analysis of >270 known bacterial nrfA genes from reference strains and from nrfA genes detected from soil metagenomic analysis allowed modifications to primer pairs that would allow broad detection of nrfA using PCR conditions that were better optimized for coverage over most of the diversity of nrfA that could be expected. Further, a primer set primarily specific to members of the Geobacteriaceae, a common group of bacteria in many soils, was newly designed. Validation of the primer sets were made using ten reference genomes spanning diverse NrfA clades. PCR product sizes varied by host (236-278 bp), enabling amplified fragment length polymorphisms (AFLP) to be used as a relatively rapid and easy tool to assess nrfA community patterns. The primers were demonstrated to amplify the expected product sizes from reference strains and while efficiencies of amplification differed among host genes, all were detected as expected. The Geobacteriacea-specific primers amplified nrfA from a representative member of this group, Geobacter bemedjiensis, where the broad range primer set did not. We also applied the primers to soil DNA and effectively showed the utility of the modified primers as an improvement in detection over the previously published ones. The impact of these results are the imporved effectiveness in PCR detection of nrfA from natural environments, including Geobacteriacea, and enable more effective tools to assess DNRA in agricultural soils.

Technical Abstract: Dissimilatory nitrate reduction to ammonium (DNRA) has increasingly been revealed over recent years to be a more prevalent process in terrestrial ecosystems than previously thought. The key enzyme, a pentaheme nitrite reductase NrfA associated with respiratory nitrite-ammonification, is encoded by the nrfA gene found in a broad phylogeny of bacteria as we previously reported (Welsh et al., 2014). Until recently, detection of diverse nrfA from environments had been hampered by the lack of reliable and comprehensive molecular tools. In this study, modifications were made to previously-designed PCR primers that targeted the region of NrfA between the conserved diagnostic third and fourth heme binding domains, but had lacked detection for Geobacteriaceae nrfA that are commonly found in soil environments. The primers were optimized to improve the compatibility of the primer pairs and applicability of a single annealing temperature for more efficient amplification of diverse nrfA from reference strains and soil. Using the primer target regions identified by Welsh et al. (2014), alignments to >270 known bacterial nrfA genes from reference genomes and nrfA gene contigs assembled from soil metagenomes, modifications to the forward- and reverse primer sequences were determined by in silico analysis that would allow broad detection of nrfA using PCR conditions that were better optimized for coverage over the wide range of GC content (34-67%) that could be expected. Further, a primer set primarily specific to members of the Geobacteriaceae was newly designed. Validation of the primer sets were made using ten reference genomes spanning diverse NrfA clades. PCR product sizes varied by host (236-278 bp), enabling amplified fragment length polymorphisms (AFLP) to be used as a relatively rapid and easy tool to assess nrfA community patterns. The primers were demonstrated to amplify the expected product sizes from reference strains and amplification from an assembled pool of reference genomes revealed differential amplification efficiencies likely due to %GC content of the host. The Geobacteriacea-specific primers amplified nrfA from Geobacter bemedjiensis as expected, where the broad range primer set did not. Application of the primers to soil DNA resulted in products that separated into a range of product sizes expected for nrfA as revealed by AFLP. This study yields effective new primer pairs for use in detection of nrfA from natural environments, including Geobacteriacea, and enable use for conventional PCR, qPCR, and allow suitable conditions for use of new PCR access array technologies that allow multiplex gene detection for downstream high throughput sequencing platforms.