An evaluation of primers for detecting denitrifiers via their functional genes

Abstract

Summary Microbial populations provide nitrogen cycling ecosystem services at the nexus of agriculture, environmental quality and climate change. Denitrification, in particular, impacts socio‐environmental systems in both positive and negative ways, through reduction of aquatic and atmospheric nitrogen pollution, but also reduction of soil fertility and production of greenhouse gases. However, denitrification rates are quite variable in time and space, and therefore difficult to model. Microbial ecology is working to improve the predictive ecology of denitrifiers by quantifying and describing the diversity of microbial functional groups. However, metagenomic sequencing has revealed previously undescribed diversity within these functional groups, and highlighted a need to reevaluate coverage of existing DNA primers for denitrification functional genes. We provide here a comprehensive in silico evaluation of primer sets that target diagnostic genes in the denitrification pathway. This analysis makes use of current DNA sequence data available for each functional gene. It contributes a comparative analysis of the strengths and limitations of each primer set for describing denitrifier functional groups. This analysis identifies genes for which development of new tools is needed, and aids in interpretation of existing datasets, both of which will facilitate application of molecular methods to further develop the predictive ecology of denitrifiers.

Phylogenomics of Rhodocyclales and its distribution in wastewater treatment systems

“…Surprisingly, no comprehensive PCR-based survey on the diversity of denitrifiers in AS has been performed. A recent study performed by Ma et al 75 updated the primers for detecting environmental denitrifiers, which would facilitate the study of denitrifier diversity in AS samples together with the metagenomic approach, as applied by the present study. Further combination of transcriptional and biochemical studies on the denitrifying process in full-scale AS is important to understand the true functional contribution and operational parameters related to their functions.…”

Targeted metagenomics reveals extensive diversity of the denitrifying community in partial nitritation anammox and activated sludge systems

“…However, for all denitrifying genes, several primer pairs have been published and continuously reassessed, especially for nitrite reductase ( nirK and nirS ; Braker & Tiedje, 2003; Casciotti & Ward, 2001; Throback et al, 2004). Ma et al (2019) evaluated the coverage of existing primer pairs in silico based on a metagenomic study. The results pointed out that the existing nirK, nirS , and norB primers have low coverage over the entire gene pool, and in consequences are hardly suitable for molecular methods to investigate denitrifiers.…”

Distinct Community Composition of Previously Uncharacterized Denitrifying Bacteria and Fungi across Different Land-Use Types

“…We used cluster-specific primers because of their high detectability of diverse nirK and nirS genes. These primers represent the best potential for detecting these genes (Ma et al, 2019), but do not identify genes from archaea (Bonilla-Rosso et al, 2016;Kobayashi et al, 2018) or potentially those from some bacterial or fungal groups (Ma et al, 2019). In addition, the results on taxonomic assignment depend on the fullness of the database.…”