Role of norEF in denitrification, elucidated by physiological experiments with Rhodobacter sphaeroides

Abstract

Many denitrifying organisms contain the norEF gene cluster, which codes for two proteins that are thought to be involved in denitrification because they are expressed during the reduction of nitrite and nitric oxide. The products of both genes are predicted to be membrane associated, and the norE product is a member of the cytochrome c oxidase subunit III family. However, the specific role of norEF is unknown. The denitrification phenotypes of Rhodobacter sphaeroides strains with and without norEF genes were studied, and it was found that loss of norEF lowered the rate of denitrification from nitrate and resulted in accumulation of micromolar concentrations of nitric oxide during denitrification from nitrite. norEF appears to have no direct role in the reduction of nitric oxide; however, since deletion of norEF in the wild-type 2.4.3 strain had essentially no influence on the kinetics of potential nitric oxide reduction (Vmax and Ks), as measured by monitoring the depletion of a bolus of nitric oxide injected into anoxic cultures without any other electron acceptors. However, norEF-deficient cells that had undergone a more chronic exposure to micromolar concentrations of nitric oxide showed an ∼50% reduction in Vmax but no change in apparent Ks. These results can explain the occurrence of norEF in the 2.4.3 strain of R. sphaeroides, which can reduce nitrate to nitrous oxide, and their absence from strains such as 2.4.1, which likely use nitric oxide reductase to mitigate stress due to episodic exposure to nitric oxide from exogenous sources.

FIG 1

Respiration profiles during the switch from oxic to anoxic conditions in batch cultures of 2.4.3 with norEF intact (A and C) and 2.4.3 ΔnorEF (B and D) with 1% initial O₂ and 1 mM nitrate (A and B) or nitrite (C and D). The main graphs show O₂ reduction (μM in liquid), NO (nM in liquid), and N₂O (μmol vial⁻¹) accumulation. The insets show electron flow (μmol e⁻ vial⁻¹ h⁻¹) to O₂ or NO_x assuming no or negligible nitrite accumulation in nitrate-treated cultures.

FIG 2

O₂ depletion, NO and N₂O accumulation, and electron flow to O₂ and NO_x in 2.4.1 pAK1 and 2.4.1 pAK1 + norEF when treated with 1% initial O₂ and 1 or 2 mM nitrite, respectively. (A) Arrest in denitrification and accumulation of μM concentrations of NO in 2.4.1 pAK1. (B) Restoration of Nor function (as seen by the 100% recovery of added nitrite as N₂O) in 2.4.1 pAK1 upon the inclusion of 2.4.3-derived norEF.

FIG 3

Taxis response of wild-type 2.4.3, wild-type 2.4.1, 2.4.1 with nirK (pAK1), or 2.4.1 with nirK and norEF (pAK1 + norEF) to a source of nitrite. The nitrite plug is visible in the center of the plate. Plates had been incubated under hypoxic conditions for 18 h. The thick band in the 2.4.3 plate is an accumulation of cells which does not occur in the 2.4.1 strains.

FIG 4

Kinetics of NO reduction in the 2.4.3 wild type versus the ΔnorEF mutant. (A) Rates (mmol g⁻¹ cell dry weight h⁻¹) plotted against the NO concentration in liquid (nM), together with the model fitted to the entire data set (V_max = 4.2 mmol g⁻¹ cell dw h⁻¹, K_s = 577 nM). (B) Log-log plot of the same data to visualize the relationships between the strains and model at low NO concentrations.

FIG 5

Kinetics of NO reduction in norEF-deficient cells after exposure to high NO concentrations. Wild-type 2.4.3 and the ΔnorEF mutant were first cultured in medium with 1 mM NO₂⁻, leading to accumulation of μM concentrations of NO in the ΔnorEF mutant (as shown in Fig. 1D; also see Fig. S7 in the supplemental material) but not in wild-type 2.4.3. The cells were then harvested, washed once in sterile Sistrom's medium, and transferred to anoxic vials with fresh medium without NO₂⁻ and NO₃⁻ for measuring rates of NO reduction. (A) Rates (mmol g⁻¹ cell dry weight h⁻¹) plotted against NO concentration in the liquid. The line shows the predictions by the Michaelis-Menten model (V_max = 4.2 mmol g⁻¹ h⁻¹, K_s = 577 nM) (Fig. 4). (B) Log-log plot for inspection of data at low concentrations.

FIG 6

O₂ depletion and NO and N₂O accumulation (A and B) and electron flow to O₂ and NO_x (C and D) in batch cultures of 2.4.3 Δnap-β with 1% initial O₂ and 1 mM nitrate (A) or 0.25 mM nitrite (B). The e⁻ flow rate in panel C assumes negligible accumulation of nitrite. In the culture depicted in panel B, a second pulse of 1 mM nitrite was added after 23 h and all of the available nitrite was reduced to N₂O.