Construction of DNA transfer system of Streptomyces tenebrarius

To establish a gene transfer ssystem in Streptomyces tenebrarius, several methods including PEG-mediated transformation of protoplasts, conjugal transfer were investigated. Many attempts were made to introduce plasmid pIJ702 into Sreptomyces tenebrarius. It was found that plasmid pIJ702 isolated from S. lividans TK24 failed to transform the protoplasts of Sreptomyces tenebrarius. No transformant was achieved even if the protoplast was inactivated by heat treatment or dsDNA was converted ssDNA before transformation. All the results suggested that Sreptomyces tenebrarius exists a strong restriction and modification system for the transformation of foreign DNA. A recombinant E. coli ET12567 (pUZ8002, pHZ132) was obtained by transforming E. coli ET12567(pUZ8002) with oriT-containing E. coli-Sreptomyces shuttle plasmid pHZ132. In mating experiments, E. coli ET12567 (pUZ8002, pHZ132) was the donor, and the recipient was Sreptomyces tenebrarius 9904 spores after pregerminating by heat shock. Matings between dornor and recipient were conducted. Plasmid pHZ132 was introduced into Sreptomyces tenebrarius 9904 by conjugation from E. coli ET12567. The transfer system of Sreptomyces tenebrariu was established by conjugation. S. tenebrarius 9904 protoplasts were transformed by plasmid DNA modified by the host itself, and the transformation frequency was about 10(3)/microgram DNA (pHZ132).