Regulation of denitrification at the cellular level: a clue to the understanding of N2O emissions from soils

Denitrifying prokaryotes use NOx as terminal electron acceptors in response to oxygen depletion. The process emits a mixture of NO, N2O and N2, depending on the relative activity of the enzymes catalysing the stepwise reduction of NO3− to N2O and finally to N2. Cultured denitrifying prokaryotes show characteristic transient accumulation of NO2−, NO and N2O during transition from oxic to anoxic respiration, when tested under standardized conditions, but this character appears unrelated to phylogeny. Thus, although the denitrifying community of soils may differ in their propensity to emit N2O, it may be difficult to predict such characteristics by analysis of the community composition. A common feature of strains tested in our laboratory is that the relative amounts of N2O produced (N2O/(N2+N2O) product ratio) is correlated with acidity, apparently owing to interference with the assembly of the enzyme N2O reductase. The same phenomenon was demonstrated for soils and microbial communities extracted from soils. Liming could be a way to reduce N2O emissions, but needs verification by field experiments. More sophisticated ways to reduce emissions may emerge in the future as we learn more about the regulation of denitrification at the cellular level

Biochemical properties of Paracoccus denitrificans FnrP:Reactions with molecular oxygen and nitric oxide

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~6-fold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers

Kellermann et al 2022_Preparation for denitrification at the cusp of anoxia

Supporting information to Kellermann et al, submitted to AEM June 2022; five separate files named supplemental item 1-5. Title of paper:Preparation for denitrification and phenotypic diversification at the cusp of anoxia; a purpose for N2O reductase vis a vis multiple roles of O2 Supplemental item 1 is a description and qualification of the glucose oxidase-catalase (GOX) approach used for the removal of residual oxygen in experimental vials before inoculation. In supplemental item 2, we display the gas- and flow cytometry data in the O2 spiking experiment, including the data shown in Figure 2 and 3 in the paper. In supplemental item 3, we show the gas kinetics and microscopy analyses from the N2O spiking experiment, which in the paper is summarized in Fig 4. We also show the lack of positive effect from N2O addition on Nir expression in a NosZ deficient mutant. In supplemental item 4, we describe the steps taken to estimate apparent specific growth rates in single vials and cell yield per mol electron to N-oxides. In supplemental item 5, we describe a simple experiment where nitrite reducing cultures of Paracoccus denitrificans was spiked with N2O and O2 and the subsequent rate of nitrite reduction was assessed.Movies 1-6:Red (mCherry-NirS expression) and green (FITC, growth) fluorescence in single cells captured by flow cytometry (Movie 1-4; O2-spiking experiment) or fluorescence microscopy and image analyses (Movie 5-6; N2O spiking experiment). Movie 1: vial 1.2; Movie 2: vial 1.3; Movie 3: vial 1.8; Movie 4: vial 1.9; Movie 5: vial 2.3 and 2.4; Movie 6: vial 2.7 and 2.11.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

Expression of nitrous oxide reductase in Paracoccus denitrificans is regulated by oxygen and nitric oxide through FnrP and NNR.

The reductases performing the four steps of denitrification are controlled by a network of transcriptional regulators and ancillary factors responding to intra- and extracellular signals, amongst which are oxygen and N oxides (NO and N

Comparison of measured and simulated data assuming stochastic initiation of <i>nirS</i> transcription.

<p>Each panel compares the measured <math><mrow><msubsup><mi>NO</mi><mn>2</mn><mo>−</mo></msubsup></mrow></math> depletion (sub-panel) and N₂ accumulation (main panel; n = 3–4) with simulations. The simulations are carried out with an optimised specific-probability of <i>nirS</i> transcriptional initiation (average r_Ni = 0.004 h^-1, Eqs <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004621#pcbi.1004621.e080" target="_blank">4</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004621#pcbi.1004621.e081" target="_blank">5</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004621#pcbi.1004621.e085" target="_blank">6</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004621#pcbi.1004621.e086" target="_blank">7</a>), allowing 7.7–22.1% of the population to produce NirS + <i>c</i>Nor (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004621#pcbi.1004621.e087" target="_blank">Eq 8</a>) during the available time-window (= 19.5–47.3 h).</p

Comparison of the measured N₂O with that simulated.

<p>Each main panel (A–D) compares the measured N₂O (single vial results) with the default simulation using the parameter values given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004621#pcbi.1004621.t002" target="_blank">Table 2</a>, i.e., <math><mrow><msub>K<mrow><mi>m</mi><msub>N<mn>2</mn></msub>O</mrow></msub></mrow></math> = 0.6 μM (estimated through optimisation) and <math><mrow><mi>v</mi><msubsup>e<mrow><mi>m</mi><mi>a</mi><mi>x</mi><msub>N<mn>2</mn></msub>O</mrow><mo>−</mo></msubsup></mrow></math> = 5.5×10⁻¹⁵ mol e^- cell^-1 h^-1 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004621#pcbi.1004621.ref024" target="_blank">24</a>]. In contrast, each inserted panel shows the simulated N₂O assuming 1) N₂O consumption only by the cells producing N₂O (Z^NaNi + Z^Ni), and 2) the literature value for <math><mrow><msub>K<mrow><mi>m</mi><msub>N<mn>2</mn></msub>O</mrow></msub></mrow></math> = 5 μM [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004621#pcbi.1004621.ref042" target="_blank">42</a>]. The results show that the default simulation best explains the measured N₂O kinetics, assuming its production by a small fraction (Z^NaNi + Z^Ni) and consumption by the entire population (Z⁻ + Z^Na+ Z^NaNi + Z^Ni).</p

Initial values for the state variables.

<p>Initial values for the state variables.</p