In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii. The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus, and only when the distances between the two regions were relatively short (2–5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.

中文翻译：

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大异源DNA片段整合到热球菌kodakarensis的基因组中。

在这项研究中，为高嗜热古细菌Thermococcus kodakarensis开发了一种能够进行大规模基因重组的转化系统。使用尿嘧啶营养缺陷型T. kodakarensis KU216（Δ的pyrF）作为亲本株，构建多个宿主菌株从任一热古细菌的基因组中携带两个1 kbp的DNA区域激烈热球菌或Methanocaldococcus詹氏甲烷球菌。选择两个区域，使得在各自基因组上它们之间的区域将包括pyrF基因，其可以潜在地用于选择。使用这些宿主菌株和狂热假单胞菌或进行了M. jannaschii。仅当带有狂犬病假单胞菌区域的宿主菌株时，以及当狂犬病假单胞菌基因组的两个区域之间的距离相对较短（2–5 kbp）时，才能获得具有外源性pyrF的转化体。为了插入更长的DNA片段，我们研究了使用恶性疟原虫细胞提供完整基因组DNA的可能性。激烈疟原虫的细胞沉淀物与柯达卡尔斯氏菌的细胞沉淀物重叠，从而使细胞直接接触。结果，我们能够分离出带有P. furiosus DNA片段的T. kodakarensis菌株。 在单个转换步骤中的最大长度为75 kbp。