Genomic ecology of bradyrhizobia for denitrification systems to repress N_2O evolution

反硝化作用是以硝酸盐（NO_3^-）、亚硝酸盐（NO_2^-）、一氧化氮（NO）和一氧化二氮（N_2O）为末端电子受体的厌氧呼吸作用。一氧化二氮（N_2O）是一种重要的大气温室气体，它通过辐射增暖和平流层臭氧的消耗，对全球气候变化起着重要作用。农田是大气N_2O的主要来源之一，对大气N_2O的净增加贡献显著。我们用天然丰度为^<15>N（0.366atom%）的N_2作载气，在带热导检测器的气相色谱仪上定量检测^<15>N-N_2。当大豆慢生根瘤菌（Bradyrhizobium japonicum）USDA 110在含N-NO_3的培养基中厌氧培养时<15>，<15>可检测到化学计量的N-N_2。对63株慢生型根瘤菌的分析表明，不同田间条件下，其反硝化能力不同。为了研究日本慢生根瘤菌nos基因簇是否与共生过程中N_2O的还原有关，我们构建了一个nosZ缺失的 关于我们 日本B.japonicum USDA 110离子突变体。N_2O还原酶的活性是通过N_2O的吸收和^ N_2O转化<15>为^<15>N_2来确定的。USDA 110的游离细胞具有N_2O还原酶活性，而nosZ突变体则没有。将含有nos基因簇的B.japonicum USDA 110互补到nosZ突变体上，恢复了N_2O还原酶活性，表明nosZ依赖的N_2O还原酶活性和突变体的有效性。野生型USDA 110形成的大豆离体结瘤，在喂入^<15>N_2O后，结瘤迅速向外排放^<15>N_2，其吸收率约为^<15>N-N_2 O的98.5%。另一方面，接种nosZ突变体的根瘤不介导^<15>N_2O向^<15>N_2的转化。接种USDA 110的大豆根际N_2O浓度显著降低。这些结果表明，N_2O向N_2的转化完全依赖于nos基因簇的功能，携带nos基因簇的B.japonicum对大豆根际环境中N_2O气体的去除作用较小

Denitrification is anaerobic respiration using nitrate (NO_3^-), nitrite (NO_2^-), nitric oxide (NO), and nitrous oxide (N_2O) as terminal electron acceptors. Nitrous oxide (N_2O) is a key atmospheric greenhouse gas that contributes to global climatic change through radiative warming and depletion of stratospheric ozone. Agricultural lands are one of major sources, and significantly contribute the net increase in atmospheric N_2O. We used N_2, which has a natural abundance of ^<15>N (0.366 atom%), as a carrier gas in a gas chromatograph with a thermal conductivity detector to detect ^<15>N-N_2 quantitatively. When Bradyrhizobium japonicum USDA110 was cultured anaerobically in medium containing ^<15>N-NO_3, ^<15>N-N_2 was detected stoichiometrically. Analysis of 63 bradyrhizobial isolates revealed that denitrification ability varied with field site. To examine whether nos gene cluster of Bradyrhizobium japonicum is responsible for N_2O reduction in symbiosis, we constructed a nosZ delet … More ion mutant of B.japonicum USDA110. The activity of N_2O reductase was determined by N_2O uptake and conversion of ^<15>N_2O into ^<15>N_2. Free-living cells of USDA110 showed N_2O reductase activity, while nosZ mutant did not. Complementation of cosmids containing nos gene cluster of B.japonicum USDA 110 to the nosZ mutant restored the N_2O rductase activity, indicating nosZ-dependent N_2O reductase activity and validity of the mutant. When the detached soybean nodules formed with wild-type USDA110 was fed with ^<15>N_2O, the nodules rapidly emitted ^<15>N_2 outside at a ratio of approximately 98.5% of ^<15>N-N_2O uptake. On the other hand, nosZ mutant-inoculated nodules did not mediate the conversion of ^<15>N_2O to ^<15>N_2 at all. Moreover, substantial decrease of N_2O concentration was detected in the rhizosphere of intact soybean plant inoculated with wild-type USDA110. These results demonstrate that the conversion of N_2O to N_2 depends exclusively to the function of nos gene cluster, and soybeans nodulated with B.japonicum carrying nos cluster are able to remove N_2O gas in rhizosphere environments Less