Nitrosomonas europaea expresses a nitric oxide reductase during nitrification

Abstract

In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea. Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans. NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells. NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene. The findings demonstrate the presence of an alternative site of N2O production in N. europaea.

FIG. 1.

Schematic representation of the nor gene cluster in wild-type N. europaea and in the Nor-deficient strains. norC starts at genomic position 2163869, and norD ends at genomic position 2166533 (4). Nor-deficient strains were engineered by insertion of the suicide vectors pNORBsu and pNORQsu as indicated. Arrows indicate primers used for construction of the suicide vectors, verification of correct integration, and construction of the complementation vector.

FIG. 2.

Nitric oxide consumption by cells and membrane protein fractions measured under anoxic conditions with a Clark-type electrode with phenazine ethosulfate (PES) as a mediator of ascorbate-derived electrons. Additionally, horse heart cytochrome c was present when assaying membranes. Cells were harvested in the early stationary growth phase and stored at 0°C overnight. The disappearance of NO before the addition of cells or protein represents the background rate of NO consumption via chemical conversion. Arrowheads in a to d mark the addition of cells to a final OD₆₀₀ of 0.9 (50 μl). The arrowheads in e mark the addition of NorB-deficient cells, NO from a saturated solution, and wild-type cells, respectively. Equal amounts of cells of both strains were added to a combined OD₆₀₀ of 2.7 (two times, 50 μl). Arrowheads in f to j mark the addition of membrane proteins: wild type (0.17 mg in 20 μl), NorB deficient (0.59 mg in 50 μl), NorQ deficient (0.28 mg, 75 μl), NorB deficient complemented (0.20 mg, 20 μl) (twice), and Fnr deficient (0.28 mg in 75 μl).

FIG. 3.

(a) Growth curves of cells of wild-type and NorB-deficient strains of N. europaea during cultivation in O₂-limited batch cultures. Squares, wild-type cells; circles, NorB-deficient cells. Error bars indicate the 95% confidence interval of replicate cultures (n = 3). (b and c) Growth curves of wild-type (b) and NorB-deficient (c) cells of N. europaea in aerobic batch cultures to which 0 (squares), 50 (circles), 100 (triangles), and 200 (diamonds) μM SNP was added at a t of 20 h. Arrowheads indicate the addition of SNP. Error bars indicate the 95% confidence interval of replicate cultures (n = 3).