A SPECTINOMYCIN RESISTANCE DETERMINANT FROM THE SPECTINOMYCIN PRODUCER STREPTOMYCES-FLAVOPERSICUS

The spectinomycin (Sp) resistance determinant from Streptomyces flavop ersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicu s conferring resistance to Sp was isolated. DNA sequence analysis of t he 3651 bp DNA insert revealed four open reading frames (ORFs). The am ino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a s econd one (SpcR) to a regulatory protein (StrR) of the streptomycin bi osynthesis gene cluster from S. griseus. The two other ORFs were incom plete and the deduced amino acid sequences showed similarities to an a midinotransferase encoded in the streptomycin biosynthesis gene cluste r of S. griseus and to the transposase of IS112, respectively. Express ion of the spcN gene in E. coli under the control of tac promoter conf erred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransfe rase which phosphorylates Sp and actinamine, the aminocyclitol moiety of Sp.