pFJ269, a new plasmid isolated from a beta-lactam antibiotic producing streptomycete.

Streptomyces are Gram-positive, filamentous bacteria which have a complex life cycle that includes formation of substrate and aerial mycelia and spores. Streptomyces produce approximately 70% of all naturally occurring antibiotics, 2) and it is in the commercial production of antibiotics that this genus has been exploited. While the genetics and molecular biology of many Streptomyces are poorly understood, recent improvements in protoplast preparation, regeneration, fusion, and transformation3~6) have facilitated the study of genetic organization, gene expression, and gene regulation in Streptomyces. Development of broad host range plasmid mediated gene cloning systems has also aided molecular studies in Streptomyces. Although plasmid vectors currently available appear to have utility in many Streptomyces species7~9), restriction barriers between species and the possible instability of some plamids in a certain species could result in the need for different plasmid replicons. We report here the isolation and partial characterization of a new 8.8 kilobase plasmid from a 8lactam antibiotic producing streptomycete and its initial development into a gene cloning vehicle. Total cellular DNA was isolated from Streptomyces strain A57284, an uncharacterized /9lactam producer, by procedures cited in RICHARDSON et al.8). Horizontal agarose gel electrophoresis revealed the presence of covalently closed circular DNA (cccDNA). The cccDNA was designated pFJ276. Although the strain from which pFJ276 was isolated produces a Q-lactam antibiotic, no /9-lactamase activity was detected in crude cell extracts. A restriction map of pFJ276 (Fig. 1) was constructed following digestion with several restriction endonucleases in various combinations. A 1.7 kilobase BamH I fragment containing the Streptomyces azureus gene conferring resistance to thiostrepton, described by THOMPSON et al.6), subcloned in our laboratory, was ligated to BamH I digested pFJ276. The