To understand the mechanisms underlying the increased N 2 O reductase activity in the Bradyrhizobium japonicum 5M09 mutant from enrichment culture under N 2 O respiration, we analyzed the expression of genes encoding denitrification reductases and regulators. Our results suggest a common regulation of nap (encoding periplasmic nitrate reductase) and nos (encoding N 2 O reductase). N itrous oxide (N 2 O) is a key greenhouse gas that also damages the ozone layer. Soybean nodules emit N 2 O under field conditions in the late growth period (1-3). While N 2 O generation is due to a diversity of microbial enzymes, N 2 O consumption appears to be due exclusively to N 2 O reductase (Nos) (4-6). Nos catalyzes the two-electron reduction of N 2 O to N 2 . In strain USDA110 of the soybean symbiont Bradyrhizobium japonicum, the complete reduction of nitrate to N 2 depends on napEDABC (which encode periplasmic nitrate reductase [Nap]), nirK (copper-containing nitrite reductase [NirK]), norCBQD (c-type nitric oxide reductase [cNor]), and nosRZDYFLX (Nos) (7).A B. japonicum mutant, 5M09, with increased N 2 O reductase activity (Nos ϩϩ ) was isolated through the impairment of proofreading activity and the use of enrichment culture under selection pressure for N 2 O respiration (8). Recently, it was shown that postharvest N 2 O emissions from soybean fields can be mitigated by inoculation with 5M09 at the field scale (9). Because the mechanisms underlying the increased Nos activity in 5M09 remain unclear, the main objective of this work was to evaluate the expression of genes encoding denitrification reductases and regulators in 5M09. A B. japonicum mutant (PRNOS) that overexpresses the nos genes as controlled by the rRNA gene promoter (9) was used as a Nos ϩϩ reference. B. japonicum USDA110 (United States Department of Agriculture, Beltsville, MD) and the mutant derivative strains 5M09 (8) and PRNOS (9) were used in this study. The bacterial strains and plasmids used in this work are listed in Table S1 in the supplemental material. Cells were routinely cultured at 30°C in HM salt medium (10) supplemented with 0.1% arabinose, 0.025% (wt/vol) yeast extract, and trace metals (HMM medium) (11). For -galactosidase assays, methyl viologen (MV)-dependent nitrate reductase assays, and RNA isolation, aerobically grown cells were collected and washed twice. They were then adjusted to an optical density at 660 nm of about 0.1 by the addition of HMM medium. Cells (10 ml) were incubated for 24 h in 120-ml airtight vials with air (aerobic conditions) or N 2 (anaerobic conditions). Where appropriate, either N 2 O (1%, 5%, 20%, or 25% [vol/vol]) or KNO 3 (10 or 20 mM) was added.For -galactosidase assays, we used chromosomally integrated transcriptional lacZ fusions with the napE, nirK, norC, and nosZ promoters. The plasmids pBG0614 (12), pRJ2498 (13), pRJ2499 (13), and pNOSLZch were integrated by homologous recombination into the chromosome of each of USDA110 and 5M09. -Galactosidase activity was determined with permeabilized cells as descri...