Regulation of the Emissions of the Greenhouse Gas Nitrous Oxide by the Soybean Endosymbiont Bradyrhizobium diazoefficiens

Abstract

The greenhouse gas nitrous oxide (N₂O) has strong potential to drive climate change. Soils are a major source of N₂O, with microbial nitrification and denitrification being the primary processes involved in such emissions. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model microorganism to study denitrification, a process that depends on a set of reductases, encoded by the napEDABC, nirK, norCBQD, and nosRZDYFLX genes, which sequentially reduce nitrate (NO₃^-) to nitrite (NO₂^-), nitric oxide (NO), N₂O, and dinitrogen (N₂). In this bacterium, the regulatory network and environmental cues governing the expression of denitrification genes rely on the FixK₂ and NnrR transcriptional regulators. To understand the role of FixK₂ and NnrR proteins in N₂O turnover, we monitored real-time kinetics of NO₃^-, NO₂^-, NO, N₂O, N₂, and oxygen (O₂) in a fixK₂ and nnrR mutant using a robotized incubation system. We confirmed that FixK₂ and NnrR are regulatory determinants essential for NO₃^- respiration and N₂O reduction. Furthermore, we demonstrated that N₂O reduction by B. diazoefficiens is independent of canonical inducers of denitrification, such as the nitrogen oxide NO₃^-, and it is negatively affected by acidic and alkaline conditions. These findings advance the understanding of how specific environmental conditions and two single regulators modulate N₂O turnover in B. diazoefficiens.

Keywords: denitrification; dinitrogen; gene expression; nitric oxide; nitrous oxide reductase.

Figure 1

Schematic representation of the denitrification process and its regulation in Bradyrhizobium diazoefficiens. B. diazoefficiens can reduce nitrate (NO₃⁻) to nitrite (NO₂⁻), nitric oxide (NO), nitrous oxide (N₂O), and dinitrogen (N₂) by the periplasmic nitrate reductase (Nap), copper-containing nitrite reductase (NirK), nitric oxide reductase type c (cNor), and nitrous oxide reductase (N₂OR) enzymes, respectively. In B. diazoefficiens, expression of denitrification enzymes is tightly regulated by the FixLJ, FixK₂, and NnrR regulatory proteins (see Introduction for further details). However, despite the coordinated activation of each reductase, environmental unfriendly gases such as NO and N₂O can leak from denitrification and be released to the atmosphere.

Figure 2

Denitrification phenotypes of the parental strain B. diazoefficiens 110spc4 (A) and the two mutant strains ∆fixK₂ (B) and ∆nnrR (C). (A–C) measurement of O₂ and NO₃⁻ respiration, concentrations of denitrifying intermediaries (NO₂⁻, NO, N₂O, N₂), and bacterial growth (OD₆₀₀) yielded from such dynamics. (D–F) electron flow rates to O₂ and nitrogen oxides (NO_x). Cells were incubated with 2% O₂ and 10 mM NO₃⁻ as oxic and anoxic respiratory substrates, respectively. O₂, NOx concentrations, and bacterial growth were monitored by automatic sampling from headspace and liquid phase. See Figure S1 to visualize individual gases’ dynamics from (A,C). Data are the means and standard deviations of at least three different cultures.

Figure 3

Impact of FixK₂ and NnrR inactivation on N₂O consumption. Measurement of O₂ and N₂O respiration and concentrations of NO and N₂. B. diazoefficiens 110spc4 parental strain (A,B) and fixK₂ (C,D) and nnrR (E,F) mutant strains were incubated in vials containing 0.5% O₂ and 5% N₂O as oxic respiratory and anoxic respiratory substrates, respectively. In addition, a second set of vials were also supplemented with 10 mM NO₃⁻ (B,D,F) as anoxic respiratory substrate. The gradual decline in N₂O concentration in (C,D,E) corresponds to dilution of headspace gases due to sampling. Data are the means and standard deviations of at least three different cultures.

Figure 4

Impact of C-source and pH on N₂O consumption. Measurement of O₂ and N₂O respiration and concentrations of NO and N₂. B. diazoefficiens 110spc4 parental strain was incubated in vials containing 0.5% O₂, 5% N₂O, and 10 mM NO₃⁻ as substrates for aerobic and anaerobic respiration, respectively. C-sources (A,B) and pH (C–F) of the growth medium were modified as shown on the graphs (see Material and Methods for further details). O₂ and NOx concentrations were monitored by automatic sampling from headspace phase. Data are the means and standard deviations of at least three different cultures.