Denitrification allows sustained respiratory metabolism during periods of anoxia, an advantage in soils with frequent anoxic spells. However, the gains may be more than evened out by the energy cost of producing the denitrification machinery, particularly if the anoxic spell is short. This dilemma could explain the evolution of different regulatory phenotypes observed in model strains, such as sequential expression of the four denitrification genes needed for a complete reduction of nitrate to N2, or a "bet hedging" strategy where all four genes are expressed only in a fraction of the cells. In complex environments such strategies would translate into progressive onset of transcription by the members of the denitrifying community. We exposed soil microcosms to anoxia, sampled for amplicon sequencing of napA/narG, nirK/nirS, and nosZ genes and transcripts after 1, 2 and 4 h, and monitored the kinetics of NO, N2O, and N2. The cDNA libraries revealed a succession of transcribed genes from active denitrifier populations, which probably reflects various regulatory phenotypes in combination with cross-talks via intermediates ( , NO) produced by the "early onset" denitrifying populations. This suggests that the regulatory strategies observed in individual isolates are also displayed in complex communities, and pinpoint the importance for successive sampling when identifying active key player organisms.
Keywords: OTU clustering; amplicon sequencing; denitrification; denitrifying genes; transcription.