Identification and characterizatioon of mobile genetic elements carrying xenobiotics-degrading genes from soil bacteria

摘要土壤细菌的各种异生物质降解活性通常由质粒和转座子等移动的遗传元件编码，这种特性使得降解基因在环境细菌菌株中具有快速传播和动态特性。本研究的目的是鉴定和截留这些移动的元件和它们的详细特征。1.一个广宿主范围的质粒pUO 1携带两个卤乙酸脱卤酶基因dehH 1和dehH 2。后者基因位于IS 1071复合转座子TnHad 1中，而dehH 1和TnHad 1转座子位于Tn 21型转座子TnHad 2中。进一步明确了TnHad 2转座所必需的末端核苷酸序列。2.甲苯转座子Tn 4651的转座需要在同一分子上重复的Tn 4651拷贝之间进行位点特异性分辨。该反应在TnpS存在下在确定的res位点介导，TnpT大大增强了该反应的分辨率。 ...更多信息 动作频率TnpS在没有TnpT参与的情况下也表现出位点特异性整合活性。Tn 465 l型解析系统广泛分布于不同的土壤细菌菌株中。3.将质粒pWW 53上的甲苯降解基因簇定位于一个80-kb的转座子Tn 4657上。Tn 4657的一个41 kb的区域含有pWW 53特异性的复制基因，两侧是两个反向构型的res位点拷贝。Tn 4657通过TnpR介导的两个res位点之间的位点特异性倒位产生了另一个甲苯转座子Tn4656.4。一个萘转座子Tn 4655携带了几个不同转座子的残基片段，表明Tn 4655的现有结构是由几个转座子重复插入并随后重排形成的。Tn 4655 TnpR蛋白也具有位点特异性整合活性。5.构建了一株携带Tn 4655衍生物的土壤细菌菌株，该菌株在染色体上缺失一个萘降解基因。利用该菌株作为受体，在土壤样品的接合实验中产生了能够降解萘的接合子。接合子中含有携带萘降解基因的广宿主范围质粒。少

ABSTRACTVarious xenobiotics-degrading activities of soil bacteria are often encoded by mobile genetic elements such as plasmids and transposons, and this property allows the rapid dissemination and dynamic properties of the degradation genes in the environmental bacterial strains. The aim of this study is the identification and entrapment of such mobile elements and their detailed characterization.1.A broad-host-range plasmid, pUO1, carries two haloacetate dehalogenase genes, dehH1, and dehH2. The latter gene was located in an IS1071-composite transposon, TnHad1, and dehH1 and TnHad1 transposon were located in a Tn21-type transposon, TnHad2. The terminal nucleotide sequences essential for the TnHad2 transposition was further clarified.2.Transposition of toluene transposon Tn4651 requires the site-specific resolution between the duplicated copies of Tn4651 on the same molecule. This reaction was mediated at a defined res site in the presence of TnpS, and TnpT greatly enhanced the resolu … More tion frequency. TnpS also showed the site-specific integration activity without the involvement of TnpT. The Tn465l-type resolution systems were widely distributed among various soil bacterial strains.3.The toluene-degrading gene cluster on plasmid pWW53 was located on an 80-kb transposon, Tn4657. A 41-kb region of Tn4657 containing the pWW53-specified replication genes was flanked by two copies of the res sites in an inverted configuration. The Tn4657 TnpR-mediated site-specific inversion between the two res sites generated another toluene transposon, Tn4656.4.A naphthalene transposon Tn4655 carried several remnant segments of different transposons, suggesting that the present structure of Tn4655 was formed by repeated insertion of several transposons and their subsequent rearrangements. The Tn4655 TnpR protein also had the site-specific integration activity.5.A soil bacterial strain was constructed that carried a chromosomal copy of the Tn4655 derivative defective in one gene for the naphthalene degradation. Use of this strain as a recipient in the conjugation experiments with soil samples gave rise to the transconjugants able to degrade naphthalene. The transconjugants contained broad-host-range plasmids that carried the genes for the degradation of naphthalene. Less