Vectors for Cloning Recombinant DNA in Escherichia coli and Streptomyces griseofuscus C 581

The streptomycete plasmid SCP2 and its mutant SCP2* (3, 4, 15) are 31-kilobase (kb) (10) sex factors (3) with one to five copies per cell (16). The SCP2 sequence can be stably cloned in Escherichia coli (12). SCP2* is suitable as a cloning vector in streptomycetes (2). Further developments of SCP2* into useful cloning vectors are described in this report. (Portions of this work were presented at the annual meeting of the Indiana branch of the American Society of Microbiologists, 23 April 1983, Crawfordsville, Ind., and the Engineering Foundation Conference, 19 to 24 September 1982, Santa Barbara, Calif. [Ann. N.Y. Acad. Sci., in press].) E. coli K-12 C600 hsdR hsdM (11) (designated E. coli) was used throughout. Streptomyces coelicolor Mi110 (5) was used to isolate plasmid SCP2* (3, 4). Methods for growth, isolation of DNA, analysis of DNA, construction of recombinant plasmids, and transformation have been described previously (8; P. R. Rosteck, Jr., and C. L. Hershberger, Gene [Amsterdam], in press). Plasmids were initially desginated pJL; however, pHJL was approved as the official designation by the Plasmid Reference Center. Streptomyces griseofuscus C581 is the preferred host for transformation and propagation of the streptomycete plasmids. It was selected because it grows rapidly, sporulates well, and is easily protoplasted and regenerated (1). Additionally, it appears to be nonrestricting (K. Cox and R. Baltz, manuscript in preparation). A collection of chimeric plasmids was constructed with deletions in the SCP2* sequence so that the segment responsible for self-propagation in strain C581 could be identified. Two chimeric plasmids, pHJL120 and pHJL121, contained the entire sequence of SCP2*. They were either completely or paitially digested with restriction enzyme BglII, PstI, Sall, SstI, or XorlI, ligated under conditions that favored circularization rather than formation of recombinants (7), and transformed into E. coli. Eighteen isolates were retained for further investigations. Additional deleted derivatives of SCP2* were generated by shotgun cloning of PstI fragments and BamHI fragments into the PstI site or the BamHI site of pBR322 or pBR325. Restriction maps for the plasmid derivatives are summarized in Fig. 1.