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Abstract：
Integrative and conjugative elements (ICEs) are chromosomally-integrated mobile genetic elements that excise from their host chromosome and transfer to other bacteria via conjugation. ICEMlSymR7A is the prototypical member of a large family of \"symbiosis ICEs\" which confer upon their hosts the ability to form a nitrogen-fixing symbiosis with a variety of legume species. Mesorhizobial symbiosis ICEs carry a common core of mobilisation genes required for integration, excision and conjugative transfer. IntS of ICEMlSymR7A enables recombination between the ICEMlSymR7A attachment site attP and the 3\' end of the phe-tRNA gene. Here we identified putative IntS attP arm (P) sites within the attP region and demonstrated that the outermost P1 and P5 sites demarcated the minimal region for efficient IntS-mediated integration. We also identified the ICEMlSymR7A origin-of-transfer (oriT) site directly upstream of the relaxase-gene rlxS. The ICEMlSymR7A conjugation system mobilised a plasmid carrying the cloned oriT to Escherichia coli in an rlxS-dependent manner. Surprisingly, an in-frame, markerless deletion mutation in the ICEMlSymR7A recombination directionality factor (excisionase) gene rdfS, but not a mutation in intS, abolished mobilisation, suggesting the rdfS deletion tentatively has downstream effects on conjugation or its regulation. In summary, this work defines two critical cis-acting regions required for excision and transfer of ICEMlSymR7A and related ICEs.
摘要：
整合和接合元件(ICE)是染色体整合的可移动遗传元件,其从其宿主染色体切除并通过接合转移到其他细菌。ICEMlSymR7A是“共生ICE”大家族的原型成员，该家族赋予宿主与各种豆科植物形成固氮共生的能力。中根瘤菌共生ICEs携带整合所需的动员基因的共同核心，切除和共轭转移。ICEMlSymR7A的IntS能够在ICEMlSymR7A附着位点attP和phe-tRNA基因的3'端之间进行重组。在这里，我们确定了attP区域内推定的IntSattP臂（P）位点，并证明了最外面的P1和P5位点划定了有效IntS介导整合的最小区域。我们还鉴定了位于松弛酶基因rlxS上游的ICEMlSymR7A转移起点（oriT）位点。ICEMlSymR7A接合系统以rlxS依赖性方式将携带克隆的oriT的质粒动员到大肠杆菌中。令人惊讶的是,一个内框，ICEMlSymR7A重组定向因子（切除酶）基因rdfS中的无标记缺失突变，但不是intS的突变，废除了动员，这表明rdfS缺失对共轭或其调控有初步的下游影响。总之,这项工作定义了ICEMlSymR7A和相关ICE切除和转移所需的两个关键顺式作用区.
