Chan Y K, Wheatcroft R
Plant Research Centre, Agriculture Canada, Ottawa, Ontario.
J Bacteriol. 1993 Jan;175(1):19-26. doi: 10.1128/jb.175.1.19-26.1993.
The gene encoding a denitrification enzyme, nitrous oxide reductase (EC 1.7.99.6), in Rhizobium meliloti and other gram-negative bacteria was detected by hybridization to an internal 1.2-kb PstI fragment of the structural gene (nosZ) cloned from Pseudomonas stutzeri Zobell (W.G. Zumft, A. Viebrock-Sambale, and C. Braun, Eur. J. Biochem. 192:591-599, 1990). Homology to the probe was detected in the DNAs of two N2-fixing strains of P. stutzeri, two denitrifying Pseudomonas species, one Alcaligenes eutrophus strain, and 36 of 56 R. meliloti isolates tested. Except for two isolates of R. meliloti, all showed nitrous oxide reduction activity (Nos+). Therefore, at least part of the nosZ sequence appears to be conserved and widely distributed among denitrifiers, which include free-living and symbiotic diazotrophs. By using Agrobacterium tumefaciens transconjugants harboring different megaplasmids of R. meliloti JJ1c10 and SU47, sequence homology with the nosZ probe was unequivocally located on the nod megaplasmid. A cosmid clone of JJ1c10 in which nosZ homology was mapped on a 4.2-kb BamHI fragment was selected. This cosmid, which conferred Nos+ activity to the R. meliloti wild-type strains ATCC 9930 and Balsac (Nos- and nondenitrifying, respectively) also restored Nos+ activity in the mutants of JJ1c10 and SU47 in which the 4.2-kb BamHI segment was deleted. Therefore, this segment contains sequences essential for nos gene expression in JJ1c10 and SU47 and thus confirms that the nod megaplasmid in JJ1c10 and SU47 which carries genes essential for symbiotic dinitrogen fixation also carries genes involved in the antagonistic process of denitrification.
通过与从施氏假单胞菌Zobell克隆的结构基因（nosZ）的1.2 kb PstI内部片段杂交，检测了苜蓿根瘤菌和其他革兰氏阴性细菌中编码反硝化酶一氧化二氮还原酶（EC 1.7.99.6）的基因（W.G. Zumft、A. Viebrock-Sambale和C. Braun，《欧洲生物化学杂志》192:591 - 599，1990年）。在两株固氮施氏假单胞菌、两种反硝化假单胞菌、一株嗜糖产碱杆菌菌株以及所检测的56株苜蓿根瘤菌分离株中的36株的DNA中检测到与探针的同源性。除了两株苜蓿根瘤菌分离株外，所有菌株均表现出一氧化二氮还原活性（Nos +）。因此，nosZ序列的至少一部分似乎是保守的，并且在反硝化细菌中广泛分布，这些反硝化细菌包括自由生活和共生的固氮菌。通过使用携带苜蓿根瘤菌JJ1c10和SU47不同大质粒的根癌农杆菌转接合子，与nosZ探针的序列同源性明确位于结瘤大质粒上。选择了一个JJ1c10的黏粒克隆，其中nosZ同源性定位在一个4.2 kb的BamHI片段上。这个黏粒赋予苜蓿根瘤菌野生型菌株ATCC 9930和Balsac（分别为Nos - 和非反硝化型）Nos + 活性，也恢复了JJ1c10和SU47中4.2 kb BamHI片段被缺失的突变体的Nos + 活性。因此，该片段包含JJ1c10和SU47中nos基因表达所必需的序列，从而证实了JJ1c10和SU47中携带共生固氮所需基因的结瘤大质粒也携带参与反硝化拮抗过程的基因。
