Genomic ecology of bradyrhizobia for denitrification systems to repress N_2O evolution

反硝化作用是利用硝酸盐（NO_3^-）、亚硝酸盐（NO_2^-）、一氧化氮（NO）和氧化亚氮（N_2O）作为终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，通过辐射增温和平流层臭氧耗竭导致全球气候变化。农业用地是N_2O的主要来源之一，对大气N_2O的净增加有显著贡献。以天然丰度为^<15>N（0.366原子%）的n2为载气，用热导检测器气相色谱仪对^<15>N- n2进行了定量检测。在N-NO_3≤15>的培养基中厌氧培养日本慢生根瘤菌USDA110，化学计量学检测到n - no_2≤15>。63株缓生根瘤菌的反硝化能力因地而异。为了研究缓生日本根瘤菌的nos基因簇是否与共生过程中N_2O的减少有关，我们构建了缓生日本根瘤菌USDA110的nosZ缺失多离子突变体。N_2O还原酶的活性是通过N_2O的吸收和^<15>N_2O转化为^<15>N_2来测定的。USDA110的自由活细胞表现出N_2O还原酶活性，而nosZ突变体则没有。将含有日本芽孢杆菌USDA 110 nos基因簇的菌体与nosZ突变体互补，恢复了N_2O还原酶活性，表明该突变体的N_2O还原酶活性和有效性依赖于nosZ。当野生型USDA110形成的离体大豆根瘤向外注入^<15>的N_2O时，根瘤向外迅速排放^<15>的N_2O，其吸收量约为^<15>N-N_2O的98.5%。另一方面，接种了nosZ突变体的根瘤根本没有介导^<15>N_2O向^<15>N_2的转化。此外，接种野生型USDA110后，完整大豆植株根际N_2O浓度显著降低。这些结果表明，N_2O向N_2的转化完全取决于nos基因簇的功能，携带nos基因簇的结瘤大豆对根际环境中N_2O气体的去除能力较弱

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