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

Abstract

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 NO2(-)). Although many components of the regulatory network have been identified, there are gaps in our understanding of their role(s) in controlling the expression of the various reductases, in particular the environmentally important N(2)O reductase (N(2)OR). We investigated denitrification phenotypes of Paracoccus denitrificans mutants deficient in: (i) regulatory proteins (three FNR-type transcriptional regulators, NarR, NNR and FnrP, and NirI, which is involved in transcription activation of the structural nir cluster); (ii) functional enzymes (NO reductase and N(2)OR); or (iii) ancillary factors involved in N(2)O reduction (NirX and NosX). A robotized incubation system allowed us to closely monitor changes in concentrations of oxygen and all gaseous products during the transition from oxic to anoxic respiration. Strains deficient in NO reductase were able to grow during denitrification, despite reaching micromolar concentrations of NO, but were unable to return to oxic respiration. The FnrP mutant showed linear anoxic growth in a medium with nitrate as the sole NO(x), but exponential growth was restored by replacing nitrate with nitrite. We interpret this as nitrite limitation, suggesting dual transcriptional control of respiratory nitrate reductase (NAR) by FnrP and NarR. Mutations in either NirX or NosX did not affect the phenotype, but the double mutant lacked the potential to reduce N(2)O. Finally, we found that FnrP and NNR are alternative and equally effective inducers of N(2)OR.

Fig. 1.

Denitrification phenotypes of wild-type and FnrP-, NirI-, NarR- and NNR-deficient mutant strains when grown in Sistrom’s medium with 2 mM nitrate (KNO₃) and an initial O₂ concentration of 7 vol% in the headspace. Error bars indicate sd (n = 3). The N₂O concentration in the headspace remained below the detection limit (~0.5 µl l⁻¹, equivalent to 2 nmol N₂O per flask) in all cultures. The FnrP-deficient strains produced N₂ at constant rates when grown with nitrate (main panels), but when nitrate was replaced by nitrite (2 mM KNO₂) the rate of N₂ production increased exponentially (insert). The NarR-, NNR- and NirI-deficient strains produced traces of N₂ when grown with nitrate (17–20 nmol N₂ per flask h⁻¹). When supplied with nitrite, the NarR-deficient mutant produced N₂ at an exponentially increasing rate (insert). The oxic phases of the experiments with nitrite are not shown (inserts).

Fig. 2.

Denitrification phenotypes of NosZ-, NorB-, NorC-, NosX-, NirX- and NirX.NosX-deficient mutant strains when grown in Sistrom’s medium with 2 mM nitrate (KNO₃) and an initial O₂ concentration of 7 vol% in the headspace. Error bars indicate sd (n = 3). The results for the NorB-deficient strain were practically identical to those for the NorC-deficient strain (only one is shown). The same was the case for the NosX- and NirX-deficient strains. N₂O remained below the detection limit (~0.5 µl l⁻¹, equivalent to 2 nmol N₂O per flask) in cultures with the NorB-, NorC-, NosX- and NirX-deficient strains. Note that the NO concentrations for NorC/NorB-deficient mutants are plotted against the right-hand y axis, while the left-hand y axis is used for the NO concentrations in the other strains.