A STREPTOMYCES PLASMID PHZ65 AND ITS DEVELOPMENT INTO CLONING VECTORS

FR-0001 and FR-005 are two fusants obtained through protoplast fusion between differently marked derivatives of Streptomyces hygroscopicus 5102, which produce three agriculturally important antibiotics. Although they show distinct differences in morphological and cultural characteristics, antimicrobial activity and metabolites, they all carry a 20.1 kb plasmid (pHZ65) with complete DNA homology. A detailed restriction map with 29 sites for restriction enzymes BamHI, BclI, EcoRV, KpnI, PstI, SphI, SstI and XhoI has been determined. Four Strepsomyces-E. coli bifunctional plasmids, pHZ200, pHZ201, pHZ206 and pHZ207, have been constructed by the in vitro insertion of an E. coli plasmid pIJ2703 which carries the tsr gene (expressible in Streptomyces) and amp gene (expressible in E. coli) into the BclI or BglII sites of pHZ65. The replicability of a series of plasmids obtained by the cloning of DNA fragments from pHZ65 into the BclI site of pIJ2703 in S. lividans shows that the essential replication region of pHZ65 is not more than 4.96 kb in size within two clustered BglII fragments. Bifunctional plasmids derived from pHZ65 can be transformed into S. hygroscopicus var. yingchengensis (the fusion parent of FR-001 and FR-005), whereas many other plasmid vectors developed so far are unable to do so.