Recombinational transfer of 100-kilobase genomic DNA to plasmid in Bacillus subtilis 168

Transformation of Bacillus subtilis by a plasmid requires a circular multim eric form. In contrast, linearized plasmids can be circularized only when h omologous sequences are present in the host genome. A . recombinational tra nsfer system was constructed with this intrinsic B. subtilis recombinationa l repair pathway. The vector, pGETS103, a derivative of the theta -type rep licating plasmid pTB19 of thermophilic Bacillus, had the full length of Esc herichia coli plasmid pBR322. A multimeric form of pGETS103 yielded tetracy cline-resistant transformants of B. subtilis. In contrast, linearized pGETS 103 gave tetracycline-resistant transformants only when the recipient strai n had the pBR322 sequence in the genome. The efficiency and fidelity of the recombinational transfer of DNAs of up to 90 kb are demonstrated.