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.