Phenotypic and genotypic richness of denitrifiers revealed by a novel isolation strategy

Present-day knowledge on the regulatory biology of denitrification is based on studies of selected model organisms. These show large variations in their potential contribution to NO₂^-, NO, and N₂O accumulation, attributed to lack of genes coding for denitrification reductases, but also to variations in their transcriptional regulation, as well as to post-transcriptional phenomena. To validate the relevance of these observations, there is a need to study a wider range of denitrifiers. We designed an isolation protocol that identifies all possible combinations of truncated denitrification chains (NO₃^-/NO₂^-/NO/N₂O/N₂). Of 176 isolates from two soils (pH 3.7 and 7.4), 30 were denitrifiers sensu stricto, reducing NO₂^- to gas, and five capable of N₂O reduction only. Altogether, 70 isolates performed at least one reduction step, including two DNRA isolates. Gas kinetics and electron flow calculations revealed that several features with potential impact on N₂O production, reported from model organisms, also exist in these novel isolates, including denitrification bet-hedging and control of NO₂^-/NO/N₂O accumulation. Whole genome sequencing confirmed most truncations but also showed that phenotypes cannot be predicted solely from genetic potential. Interestingly, and opposed to the commonly observed inability to reduce N₂O under acidic conditions, one isolate identified as Rhodanobacter reduced N₂O only at low pH.