Nitrous oxide (N 2 O) is a key atmospheric greenhouse gas that contributes to global climate change through radiative warming and depletion of stratospheric ozone (1, 24). Agricultural land is a major source through denitrification and nitrification (14, 35) and contributes significantly to the net increase in atmospheric N 2 O (1, 34). Several attempts have been made to reduce the emission of N 2 O from agricultural systems (34,35).The complete denitrification of nitrate by bacteria to dinitrogen (N 2 ) is generally an anaerobic respiratory process, where the last step is mediated by N 2 O reductase (54). The corresponding structural gene is nosZ and is assembled in the nosRZDFYL gene operon (54). Several species capable of denitrification are also nitrogen-fixing bacteria, including rhizobia such as Bradyrhizobium japonicum (4,6,44,49) and Sinorhizobium meliloti (7,20). Indeed, genes responsible for denitrification have been found in rhizobial genomes (15,23).Earlier studies (19,37) reported the evolution of 15 N-N 2 from 15 N-N 2 O from sliced or detached soybean nodules. Recently, Velasco et al. (50) reported that nosZ and nosR insertion mutants of B. japonicum USDA110 accumulate N 2 O when cultured microaerobically in the presence of nitrate. The nosZ gene was also expressed in soybean nodules (29). However, it has not yet been fully proved that N 2 evolution from N 2 O by soybean nodules is mediated by N 2 O reductase encoded by the nosZ gene in B. japonicum (4,9,38). The aims of this work were to confirm whether the nos gene cluster of B. japonicum is responsible for respiratory N 2 O reduction to N 2 in nodules and to evaluate the capability of the nodulated roots to transform N 2 O into N 2 . MATERIALS AND METHODSBacterial strains, plasmids, and media. The bacterial strains and plasmids used are listed in Table 1. Bradyrhizobium cells were grown at 30°C in HM salt medium (8) supplemented with 0.1% arabinose and 0.025% (wt/vol) yeast extract (Difco, Detroit, MI), which is termed HM medium here. HM medium was further supplemented with 0.55 M Na 2 MoO 4 · 2H 2 O, 1 M FeCl 3 , and 1 M CuSO 4 · 5H 2 O (HMM medium) for the denitrification assay (44). Escherichia coli cells were grown at 37°C in Luria-Bertani medium (42). Antibiotics were added to the media at the following concentrations: for B. japonicum, 100 g of tetracycline (Tc)/ml, 100 g of spectinomycin (Sp)/ml, 100 g of streptomycin (Sm)/ml, 100 g of kanamycin (Km)/ml, and 50 g of polymyxin B/ml; for E. coli, 15 g of Tc/ml, 50 g of Sp/ml, 50 g of Sm/ml, 50 g of Km/ml, and 100 g of ampicillin/ml. DNA manipulations. Isolation of plasmids, DNA ligation, and transformation of E. coli were performed as described by Sambrook et al. (42). DNA preparation and Southern hybridization were carried out as described previously (22,30,43).Construction of a B. japonicum USDA110 nosZ mutant. A 4-kb BamHI DNA fragment identified from the genome sequence of B. japonicum USDA110 (23) was excised from BamHI-digested total DNA and inserted into the BamHI site of pTZ18R (Fi...

In the presence of nitrate, N 2 O emission increased markedly from soybean roots inoculated with nosZ mutant of Bradyrhizobium japonicum, but not from soybean roots inoculated with a napA nosZ double mutant, indicating that B. japonicum bacteroids in soybean nodules are able to convert the exogenously supplied nitrate into N 2 O via a denitrification pathway.Nitrous oxide (N 2 O) is a key atmospheric greenhouse effect gas that not only affects global warming but also leads to destruction of the stratospheric ozone layer (15). Agricultural land is one of the major sources of N 2 O (8, 12, 23). In particular, more N 2 O is emitted from agricultural fields with legume crops than from fields with nonlegume crops (5, 11). Kim et al. (10) found that N 2 O emission from fields with nodulating soybean was several times higher than that from fields with nonnodulating soybean at a flowering stage of soybean growth in the field, suggesting that nodulation enhanced N 2 O emission. On the other hand, Sameshima-Saito et al. (17) reported that soybean nodules can take up a low concentration of N 2 O from outside the nodules, equivalent to the natural concentration of N 2 O in the air (approximately 0.31 ppm) and that this uptake depends on the nosZ gene, which encodes N 2 O reductase in Bradyrhizobium japonicum. Therefore, the N 2 O metabolism of nodulated soybean roots is complex, and the mechanism underlying N 2 O emission from soybean fields has not yet been identified.Under field conditions, N fertilization generally increases the nitrate concentration in the soil solution (7). Thus, we hypothesized that the increased nitrate supply may lead to an increase in N 2 O emission from intact soybean root systems via a denitrification pathway in Bradyrhizobium japonicum. To test this hypothesis, we constructed a napA nosZ double mutant of B. japonicum USDA110; the wild-type (WT) versions of these genes encode dissimilatory nitrate reductase (3) and N 2 O reductase (17), respectively.The bacterial strains and plasmids we used are listed in Table 1. Bradyrhizobium cells were grown at 30°C in HM salt medium supplemented with 0.1% arabinose and 0.025% (wt/ vol) yeast extract (Difco, Detroit, MI) (18). HM medium was further supplemented with trace metals (HMM medium) for the denitrification assay (17). Escherichia coli cells were grown at 37°C in Luria-Bertani medium (16). Antibiotics were added to the media: tetracycline (Tc) at 100 g/ml, spectinomycin (Sp) at 100 g/ml, streptomycin (Sm) at 100 g/ml, kanamycin (Km) at 100 g/ml, and polymyxin B at 50 g/ml for B. japonicum and Tc at 15 g/ml, Sp at 50 g/ml, Sm at 50 g/ml, Km at 50 g/ml, and ampicillin (Ap) at 100 g/ml for E. coli.