Symbiotic Bradyrhizobium japonicum Reduces N ₂ O Surrounding the Soybean Root System via Nitrous Oxide Reductase

Abstract

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...

NAD-Malic Enzyme Affects Nitrogen Fixing Activity of Bradyrhizobium japonicum USDA 110 Bacteroids in Soybean Nodules

“…Mutagenesis targeting the dme gene was performed as reported by Sameshima-Saito et al 20,21) . The dme gene was excised as a 3.3-kb EcoRI fragment, and inserted into the EcoRI site of pK18mob 23) to generate the plasmid pKdme (7.0 kb).…”

Nitrate-Dependent N 2 O Emission from Intact Soybean Nodules via Denitrification by Bradyrhizobium japonicum Bacteroids

“…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.…”

“…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).…”

Effect of Rj Genotype and Cultivation Temperature on the Community Structure of Soybean-Nodulating Bradyrhizobia

“…This strain also has nitrous oxide (N 2 O) reductase activity (25,26,27). The N 2 O that is developed by defective denitrification is a strong greenhouse gas (2,11).…”