Construction of plasmid vectors from Streptomyces kasugaensis plasmids, pSK1 and pSK2

Streptomyces kasugaensis G3 was transformed by pIJ702 DNA carrying the thiostrepton-resistance gene at a frequency of 2 X 10(5) transformants/micrograms DNA, but it was found that the introduced pIJ702 was very unstable in this strain. This result led us to make useful vectors using the stable plasmids resident in S. kasugaensis. The Bcl I-fragment, containing the thiostrepton-resistance gene obtained from pIJ702, was inserted into the pSK1 and pSK2 plasmids isolated from S. kasugaensis. Two composite plasmids, pSK11-1 (8.0 Md) and pSK21-1 (4.8 Md), were isolated from the thiostrepton-resistant transformants of strain Ge. The constructed pSK11-1 consisted of the entire pSK1 molecule and the thiostrepton-resistance gene fragment. pSK21-1 consisted of the large Bcl I-fragment of pSK2 (4.1 Md) and the same thiostrepton-resistance gene. These plasmids were stably maintained in S. kasugaensis G3. Small derivatives of these composite plasmids were prepared by restriction enzyme cleavage and self-ligation, and several unique insertion sites were also constructed in these small plasmids. By analysis of the physical maps of these plasmids, the essential regions of pSK1 and pSK2 were determined from their DNA segments to be 2.5 Md in pSK11-1 and 1.9 Md in pSK21-1. pSK21-B5, one of these plasmid vectors, showed a wide host range in the genus Streptomyces and was stably maintained in all streptomycete species tested, except S. kasugaensis M338.