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

摘要土壤细菌的各种外源降解活性通常由质粒和转座子等可移动遗传元件编码，这使得降解基因在环境菌株中具有快速传播和动态特性。本研究的目的是鉴别和圈闭这些流动元素，并详细描述它们的特征。广泛宿主范围的质粒pUO1携带两个卤乙酸脱卤酶基因，dehH1和dehH2。后者基因位于is1071型转座子TnHad1中，而deh1和TnHad1转座子位于tn21型转座子TnHad2中。进一步明确了TnHad2转位所必需的末端核苷酸序列。甲苯转座子Tn4651的转位需要在同一分子上的Tn4651的复制拷贝之间的位点特异性分辨率。在TnpS存在的情况下，该反应被介导在一个确定的位点上，而TnpT极大地提高了该反应的分辨率。TnpS在没有TnpT参与的情况下也表现出位点特异性整合活动。tn465l型分解体系广泛分布于各种土壤菌株中。质粒pWW53上的甲苯降解基因簇位于一个80 kb的转座子Tn4657上。Tn4657的41 kb区域包含pww53指定的复制基因，两侧有两个反向配置的res位点拷贝。Tn4657 tnpr介导的两个res位点之间的位点特异性倒置产生了另一个甲苯转座子Tn4656.4。萘转座子Tn4655携带不同转座子的几个残余片段，表明Tn4655目前的结构是由多个转座子的重复插入及其随后的重排形成的。Tn4655 TnpR蛋白也具有位点特异性整合活性。构建了一株土壤细菌，该细菌携带Tn4655衍生物的染色体拷贝，其中一个基因对萘的降解有缺陷。在与土壤样品的偶联实验中，使用该菌株作为受体，产生了能够降解萘的转偶联物。转偶联物含有广泛的宿主质粒，携带降解萘的基因。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