Plasmid transformation of Streptomyces tendae after heat attenuation of restriction

Abstract

Streptomyces tendae ATCC 31160 produces nikkomycin, a fungicide and insecticide that inhibits chitin synthases. Exposure of S. tendae protoplasts to 50 degrees C for 30 min is required for transformation (10(2) thiostrepton-resistant transformants micrograms of DNA-1) with plasmid pIJ702 or pIJ680 from Streptomyces lividans. pIJ702 and pIJ680 DNA isolated from the S. tendae transformants is efficient (10(6) to 10(7) transformants micrograms of DNA-1) in subsequent transformations of S. tendae protoplasts generated at 30 degrees C. PstI fails to cut the single PstI site in pIJ702 and cuts only one of the two PstI sites in pIJ680 DNA isolated from S. tendae transformants. Digests of plasmid DNA mixtures showed that plasmid DNA from S. tendae does not inhibit PstI activity. pIJ702 and pIJ680 DNA from S. tendae transformants was used to transform S. lividans to show that plasmid DNA remains unchanged, except for modification at some PstI sites in S. tendae, as a consequence of passage through S. tendae. The DNA modification is lost when S. lividans is transformed with plasmid DNA from S. tendae transformants. Since S. tendae modifies only some PstI sites, it appears the modification (presumably restriction activity also) activity in S. tendae recognizes a sequence that includes or overlaps the PstI hexanucleotide recognition sequence.