The spectinomycin (Sp) resistance determinant from Streptomyces flavoperricus was cloned into Streptomyces lividans using the plasmid vector ~11699. A plasmid, pDGLl5, with a 3-65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from 5. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of Is1128 respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphorylates Sp and actinamine, the aminocyclitol moiety of Sp.

To clone bifunctional vectors in streptomycetes, it was necessary to define the restriction-modification system of Streptomyces flavopersicus. Plasmid DNA from bifunctional vectors pIJ699 and pXED3-13, isolated from E. coli strains with different methylation systems: E. coli DH5 alpha (dam+ dcm+), E. coli MB5386 (dam dcm), E. coli CB51 (dam dcm+), E. coli NM544 (dam+ dcm), was used for transformation of protoplasts from strain S. flavopersicus. Restriction of dcm-methylated DNA from S. flavopersicus was established. As a host in the intermediate cloning strain E. coli NM544 (dam+ dcm) should be used, as the dcm-transmethylase-dependent strain S. flavopersicus does not process DNA from this strain.

Protoplasts of Streptomyces flavopersicus with the highest regeneration frequency were isolated from late log phase mycelium grown in a two-stage culture system with 2% glycine in the medium. Of the 11 regeneration media tested, R9 was selected as the most efficient with 29 degrees C as the best temperature.

Stable mutants that are blocked in the production of the amino glycoside antibiotic spectinomycin were generated by treatment of Streptomyces flavopersicus NRRL 2820 with a combination of N-methyl-N-nitro-N-nitrosoguanidine (NTG) and ultraviolet light. On the basis of their co-synthetic properties in agar-strip and mixed culture methods, the mutants were grouped into five phenotypic groups, and arranged in the most probable linear sequence of metabolic blocks.