Denitrification response patterns during the transition to anoxic respiration and posttranscriptional effects of suboptimal pH on nitrous [corrected] oxide reductase in Paracoccus denitrificans

Abstract

Denitrification in soil is a major source of atmospheric N(2)O. Soil pH appears to exert a strong control on the N(2)O/N(2) product ratio (high ratios at low pH), but the reasons for this are not well understood. To explore the possible mechanisms involved, we conducted an in-depth investigation of the regulation of denitrification in the model organism Paracoccus denitrificans during transition to anoxia both at pH 7 and when challenged with pHs ranging from 6 to 7.5. The kinetics of gas transformations (O(2), NO, N(2)O, and N(2)) were monitored using a robotic incubation system. Combined with quantification of gene transcription, this yields high-resolution data for direct response patterns to single factors. P. denitrificans demonstrated robustly balanced transitions from O(2) to nitric oxide-based respiration, with NO concentrations in the low nanomolar range and marginal N(2)O production at an optimal pH of 7. Transcription of nosZ (encoding N(2)O reductase) preceded that of nirS and norB (encoding nitrite and NO reductase, respectively) by 5 to 7 h, which was confirmed by observed reduction of externally supplied N(2)O. Reduction of N(2)O was severely inhibited by suboptimal pH. The relative transcription rates of nosZ versus nirS and norB were unaffected by pH, and low pH had a moderate effect on the N(2)O reductase activity in cells with a denitrification proteome assembled at pH 7. We thus concluded that the inhibition occurred during protein synthesis/assembly rather than transcription. The study shed new light on the regulation of the environmentally essential N(2)O reductase and the important role of pH in N(2)O emission.

FIG. 1.

Kinetics of oxygen depletion and denitrification in P. denitrificans when grown in sealed 120-ml flasks with 7% initial O₂ in headspace and 0.2, 1, and 2 mM nitrite in 50 ml Sistrom's medium. (A) Oxygen concentration in the liquid (μM), estimated from measured rate of O₂ depletion in headspace (20); (B) nitrite concentration (μM) calculated from accumulation of N₂ and NO; (C) NO concentration in liquid (nM) assuming equilibrium between headspace and liquid; (D) measured N₂ (μmol flask⁻¹) corrected for 3.4% loss for each sampling. N₂O was monitored as well, but only traces (<2 nmol flask⁻¹) were detectable for a brief period (not shown).

FIG. 2.

Effect of pH on the reduction of N-oxides. Aerobic cultures in Sistrom's medium with 2 mM NO₃⁻ and pHs of 6, 6.25, 6.5, 7, and 7.5 were made anoxic by He washing (time zero). (A) Concentration of nitrite (μM; pHs of 6 and 7 only); (B) concentration of NO (nM in liquid); (C) accumulated N₂O (nmol flask⁻¹); (D) accumulated N₂ (μmol flask⁻¹).

FIG. 3.

Expression of the genes encoding NO₂⁻ reductase (nirS), NO reductase (norB), and N₂O reductase (nosZ) after anaerobization (by He washing at time zero) of a culture in Sistroms' medium with 2 mM NO₃⁻ at pH 7 (left panel) and pH 6 (right panel). The numbers of transcripts per cell (mRNA cell⁻¹) are given, assuming that all the cells present are transcribing. Ratios were calculated based on the four maximal expression levels for the respective genes.

FIG. 4.

Reduction of externally supplied N₂O prior to expression of nirS. Aerobic cultures in Sistrom's medium with 2 mM NO₃⁻ at pH 6 (lower panel) and pH 7 (upper panel) were made anoxic (time zero) and supplied with N₂O in the headspace. The figure shows the measured concentration of N₂O (μmol flask⁻¹), which should be stable if no N₂O reduction is taking place (left axis, corrected for the 3.4% dilution by each sampling), and NO accumulation (nM in liquid; right axis).

FIG. 5.

Effect of pH on N₂O reductase activity in cells in which the denitrification proteome was assembled at pH 7. Actively denitrifying cultures grown in weakly buffered medium at pH 7 were transferred to strongly buffered media at different pH levels and supplied with 6 μmol pure N₂O in headspace. The figure shows the reduction rate of N₂O (μmol flask⁻¹ h⁻¹) in basal Sistroms' medium for single flasks, plotted against pH.