Basic techniques for DNA cloning and conditions required for streptomycetes as a host.

To develop a host-vector system in streptomycetes for DNA cloning, we examined the technical problems encountered and the conditions required for use of Streptomyces kasugaensis MB273 as the host. Basic techniques, such as plasmid DNA isolation, regeneration of mycelia from protoplasts and elimination of plasmids from cells were investigated. These techniques were found to be useful for many streptomycetes. Strain M518, a derivative of S. kasugaensis MB273, was found to have the following useful characteristics as a host. The plasmids of MB273 were easily cured by regeneration of mycelia from protoplasts. The protoplasts prepared from M518 regenerated mycelia at high frequency using an improved method and were efficiently transformed by plasmid DNA. The extra and intra cellular DNase activities were very weak, and no restriction endonuclease activity was detected. The sensitivity to various antibiotics was determined. This strain did not show any pathogenicity in mice nor suvival in the digestive organs of rats. MB273 and its derivatives died rather quickly in natural soil. M518 still forms aerial mycelial conidia. These results indicate that S. kasugaensis M518, derived from MB273, has useful characteristics as a host for DNA cloning. The techniques thus developed were found to be useful in other streptomycetes.