Departments of Biochemistry and Agronomy, The University of Georgia, Athens, Georgia 30602.
Appl Environ Microbiol. 1993 Jun;59(6):1883-6. doi: 10.1128/aem.59.6.1883-1886.1993.
There have been no previous studies on the genetics of Azotobacter paspali, an aerobic bacterium which forms a highly specific diazotrophic association with Bahia grass (Paspalum notatum). We constructed A. paspali strains defective in the molybdenum nitrogenase so that alternative N(2)ases could be studied. The cosmid vector pTBE and genomic DNA fragments ( approximately 50 kb) of A. paspali ATCC 23367 were used to construct a gene library in Escherichia coli. Recombinant cosmids containing sequences homologous to molybdenum nitrogenase nifDK structural genes were identified by hybridization. A 2.9-kb fragment bearing the putative nifDK genes of A. paspali was subcloned and mutagenized in vitro by the insertion of a kanamycin resistance gene cassette. The mutation was recombined into the chromosome of A. paspali with the suicide vector pCU101. One resultant mutant strain, AP2, was incapable of diazotrophic growth in a molybdenum-containing medium (Nif) without vanadium but grew well in a molybdenum-deficient medium with vanadium. The nitrogenase system in AP2 reduced acetylene to ethylene and produced ethane as 2.4% of the total products. Molybdenum levels as low as 10 nM prevented the diazotrophic growth of AP2, even in the presence of vanadium at levels up to 10 muM. These results are consistent with the existence of a vanadium nitrogenase system in A. paspali.
先前尚未有研究关注固氮菌（Azotobacter paspali）的遗传学特性，这种需氧细菌与百喜草（Paspalum notatum）形成高度专一的共生固氮关系。我们构建了固氮菌缺陷型菌株以研究其它固氮酶，这些菌株的钼氮酶失活。我们使用 cosmid 载体 pTBE 和固氮菌 ATCC 23367 的基因组 DNA 片段（约 50kb），在大肠杆菌中构建基因文库。通过杂交鉴定到与钼氮酶 nifDK 结构基因同源的重组 cosmid。含固氮菌 nifDK 基因的 2.9kb 片段被亚克隆并通过插入卡那霉素抗性基因盒进行体外诱变。突变通过自杀载体 pCU101 重组入固氮菌染色体。一个突变株 AP2 在含钼但不含钒的培养基（Nif）中不能进行固氮生长，但在含钼且含钒的培养基中生长良好。AP2 的固氮酶系统将乙炔还原为乙烯，并产生 2.4%的乙烷作为总产物。即使在 10μM 钒存在的情况下，钼水平低至 10nM 也会阻止 AP2 的固氮生长。这些结果与固氮菌存在钒氮酶系统一致。
