Isolation and characterization of plasmids from Streptomyces.

In view of the importance of actinomycetes in the production of antibiotics, it would be useful to find plasmids in these organisms in order to construct molecular breeding systems. Since the first discovery of plasmid involvement in antibiotic production", several plasmids associated with the production of antibiotics have been found in Streptomyces (references are listed in AKAGAWA et al.-'). For the study of gene cloning, plasmids with smaller molecular weights are preferable for use as cloning vehicle. In order to find candidates for vector plasmids and to extend the study of plasmid involvement in antibiotic production, we examined thirty-four strains of actinomycetes for plasmids (Table 1). Among them, we found a linear plasmid-like DNA from a lankacidin producer, Streptomyces sp. 7434-AN,3'. In this communication, we report the isolation and characterization of covalently closed circular (CCC) DNA molecules from four strains among the strains we tested. Cells of Streptomyces puniceus KCC-S-0406, Streptomyces sp. 2217-G,, Streptotni'ces sp. 7068-CC, and Streptomyces hygroscopicus 434 were grown at 27°C in a medium which contained (in grams per liter): glucose, 3; peptone, 3; yeast extract, 4; pH 7.0. The methods for isolation of CCC DNA and the preparation of ['H]thymidine-labeled DNA were as described previously for the isolation of plasmids from Bacillus subtilis4). ['H]Labeled CCC DNAs obtained from CsCI-ethidium bromide (EtBr) density gradients were subjected to neutral 5 to 20% (w/v) linear sucrose gradient centrifugation with [14C]Iabeled l DNA added as an internal standard (33.6S) for determination of molecular weights.