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

Abstract

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 N2 O 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/N2 O/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 N2 O 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 N2 O production, reported from model organisms, also exist in these novel isolates, including denitrification bet-hedging and control of NO2-/NO/N2 O 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 N2 O under acidic conditions, one isolate identified as Rhodanobacter reduced N2 O only at low pH.