Norges forskningsråd: 231282
Denitrifying bacteria accumulate NO 2 − , NO, and N 2 O, the amounts depending on transcriptional regulation of core denitrification genes in response to O 2 -limiting conditions. The genes include nar , nir , nor and nosZ , encoding NO 3 − -, NO 2 − -, NO- and N 2 O reductase, respectively. We previously constructed a dynamic model to simulate growth and respiration in batch cultures of Paracoccus denitrificans . The observed denitrification kinetics were adequately simulated by assuming a stochastic initiation of nir -transcription in each cell with an extremely low probability (0.5% h -1 ), leading to product- and substrate-induced transcription of nir and nor , respectively, via NO. Thus, the model predicted cell diversification: after O 2 depletion, only a small fraction was able to grow by reducing NO 2 − . Here we have extended the model to simulate batch cultivation with NO 3 − , i.e., NO 2 − , NO, N 2 O, and N 2 kinetics, measured in a novel experiment including frequent measurements of NO 2 − . Pa . denitrificans reduced practically all NO 3 − to NO 2 − before initiating gas production. The NO 2 − production is adequately simulated by assuming stochastic nar -transcription, as that for nirS , but with a higher probability (0.035 h -1 ) and initiating at a higher O 2 concentration. Our model assumes that all cells express nosZ , thus predicting that a majority of cells have only N 2 O-reductase (A), while a minority (B) has NO 2 − -, NO- and N 2 O-reductase. Population B has a higher cell-specific respiration rate than A because the latter can only use N 2 O produced by B. Thus, the ratio B A is low immediately after O 2 depletion, but increases throughout the anoxic phase because B grows faster than A. As a result, the model predicts initially low but gradually increasing N 2 O concentration throughout the anoxic phase, as observed. The modelled cell diversification neatly explains the observed denitrification kinetics and transient intermediate accumulations. The result has major implications for understanding the relationship between genotype and phenotype in denitrification research. Author Summary Denitrifiers generally respire O 2 , but if O 2 becomes limiting, they may switch to anaerobic respiration (denitrification) by producing NO 3 − -, NO 2 − -, NO- and/or N 2 O reductase, encoded by nar , nir , nor , and nosZ genes, respectively. Denitrification causes transient accumulation of NO 2 − and NO/N 2 O emissions, depending on the activity of the four reductases. Denitrifiers lacking nosZ produce ~100% N 2 O, whereas organisms with only nosZ are net consumers of N 2 O. Full-fledged denitrifiers are equipped with all four reductases, genetic regulation of which determines NO 2 − accumulation and NO/N 2 O emissions. Paracoccus denitrificans is a full-fledged denitrifying bacterium, and here we present a modelling approach to understand its gene regulation. We found that the observed transient accumulation of NO 2 − and N 2 O can be neatly explained by assuming cell diversification: all cells expressing nosZ , while a minority expressing nar and nir + nor . Thus, the model predicts that in a batch culture of this organism, only a minor sub-population is full-fledged denitrifier. The cell diversification is a plausible outcome of stochastic initiation of nar- and nir transcription, which then becomes autocatalytic by NO 2 − and NO, respectively. The findings are important for understanding the regulation of denitrification in bacteria: product-induced transcription of denitrification genes is common, and we surmise that diversification in response to anoxia is widespread.