Molecular Biological Study on Nitrite-Oxidizing Enzyme of Heterotophic Bacteria

本研究的目的是从生化和分子生物学的角度阐明异养细菌亚硝酸盐氧化酶。首先，对各种异养细菌的亚硝酸盐氧化酶系统进行了广泛的研究。从活性污泥中分离出具有较高亚硝酸氧化活性的巴迪氏芽孢杆菌I-73，对该酶进行了纯化和性质研究。我们还分析了亚硝酸氧化酶的N-末端氨基酸序列，显示了它的23个残基的N-末端氨基酸序列，由此证明了地高辛标记的寡核苷酸混合物是化学合成的。这一证明是与巴迪芽孢杆菌I-73的约6.4kbp的Hind III-Digest染色体DNA片段特异性杂交的。用Hind III酶切、琼脂糖凝胶电泳法提取6.4kbp的DNA片段，连接Hind III和磷酸酶处理的pUC18，构建Badius I-73的染色体DNA文库，转化大肠杆菌JM 109。经菌落杂交筛选，共获得6个阳性转化子。另一方面，通过聚合酶链式反应扩增出约1.3kbp的片段，获得了巴氏芽孢杆菌I-73的16SrDNA基因，并将其克隆到大肠杆菌中。序列分析表明，该菌与巴迪氏芽孢杆菌ATCC 14574有较近的亲缘关系。这样就有可能设计一种特定的证明来追踪开放系统中的有机体，如活性污泥和土壤中的有机体。

The aim of this study was on elucidation of nitirite-oxidizing enzyme of heterotrophic bacteria from biochemical and molecular biological aspects.First, nitrite-oxidizing enzyme system of various heterotrophic bacteria was investigated widely. The enzyme was purified and characterized from Bacillus badius I-73, which has been isolated from activated-sludge and showed relatively higer nitrite-oxidizing activity in bacteria. We also analyzed the N-terminal amino acid sequence of the nitrite-oxidizing enzyme and showed its N-teminal amino acid sequence of 23 residues, from which information the mixture of digoxigenin-labeled oligonucleotide prove was cynthesized chemically. This prove was specifically hybridized with ca. 6.4 kbp fragments of Hind III-digest chromosomal DNA from Bacillus badius I-73. Then the chromosomal DNA library of Bacillus badius I-73 was constructed by ligasing Hind III-and phosphatase-treated pUC18 with 6.4 kbp fragments digested by Hind III and extracted from agarose gel electrophoresed, by which Escherichia coli JM 109 was transformed. As a result, 6 positive transformants were obtained after screening by colony hybridization test. The chimeraplasmids from these transformants are analyzing now.On the other hand, the 16SrDNA gene of Bacillus badius I-73 was obtained by amplifying ca.1.3kbp fragment by PCR reaction and cloned in Escherichia coli. The sequence analysis with RDP databases indicated that the microorganism was the close relative of B.badius ATCC 14574. So that it would be possible to design a specific prove to trace the organism in an open system such as in activated sludge and soil.