Construction and characterization of new cloning vectors derived from Streptomyces griseobrunneus plasmid pBT1 and containing amikacin and sulfomycin resistance genes.

Three cryptic plasmids, designated pBT1 (5.6 kb), pBT2 (9.7 kb), and pBT3 (16.6 kb), were isolated from Streptomyces griseobrunneus ISP5066 and physically characterized. pBT1 and pBT2, which differ by a 4.1-kb segment, are high copy-number plasmids (40-100 copies per chromosome) that coexist with each other. pBT3 is a low copy-number plasmid. Vectors containing amikacin (or kanamycin) and sulfomycin (or thiostrepton) resistance genes from Streptomyces litmocidini ISP5164 and Streptomyces viridochromogenes subsp. sulfomycini ATCC 29776, respectively, were constructed from pBT1. One such vector, pBT37, has unique restriction sites for cloning, including BglII, XhoI, PvuII, ClaI, and SacI, with the PvuII and ClaI sites allowing clone recognition by insertional inactivation of sulfomycin resistance. Since many Streptomyces species were very sensitive to amikacin and sulfomycin, these resistance genes serve as useful selective markers. pBT37 could transform several Streptomyces strains that produce antibiotics such as tetracyclines, macrolides, beta-lactams, and aminoglycosides. This plasmid is a potentially useful vector for cloning antibiotic biosynthetic genes.