Tsuge K, Itaya M
Mitsubishi Kasei Institute of Life Sciences, Machida-Shi, Tokyo 194-8511, Japan.
J Bacteriol. 2001 Sep;183(18):5453-8. doi: 10.1128/JB.183.18.5453-5458.2001.
Transformation of Bacillus subtilis by a plasmid requires a circular multimeric form. In contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. A recombinational transfer system was constructed with this intrinsic B. subtilis recombinational repair pathway. The vector, pGETS103, a derivative of the theta-type replicating plasmid pTB19 of thermophilic Bacillus, had the full length of Escherichia coli plasmid pBR322. A multimeric form of pGETS103 yielded tetracycline-resistant transformants of B. subtilis. In contrast, linearized pGETS103 gave tetracycline-resistant transformants only when the recipient strain had the pBR322 sequence in the genome. The efficiency and fidelity of the recombinational transfer of DNAs of up to 90 kb are demonstrated.
质粒对枯草芽孢杆菌的转化需要环状多聚体形式。相比之下，线性化质粒只有在宿主基因组中存在同源序列时才能环化。利用枯草芽孢杆菌固有的重组修复途径构建了一个重组转移系统。载体pGETS103是嗜热芽孢杆菌的θ型复制质粒pTB19的衍生物，含有大肠杆菌质粒pBR322的全长序列。pGETS103的多聚体形式产生了对四环素耐药的枯草芽孢杆菌转化体。相比之下，只有当受体菌株基因组中含有pBR322序列时，线性化的pGETS103才能产生对四环素耐药的转化体。研究证明了长达90 kb的DNA重组转移的效率和保真度。
