Construction of improved vectors for gene cloning in Micromonospora melanosporea

Two vectors for Micromonospora melanosporea have been constructed with the Streptomyces plasmid pIJ702 along with the sgm gene (sisomicin-gentamicin resistance gene from M. zionensis) as the second antibiotic resistance marker. These plasmids, containing sgm gene as the second selectable marker, may be an attractive alternative to pIJ702, which is incapable of conferring melanin production in M. melanosporea and consequently is not useful for insertional inactivation in this bacterium. The constructions remove restriction site for M. melanosporea restriction endonuclease and provide additional unique sites for the insertional inactivation of selectable markers, which enhance the use of these plasmids as general cloning vectors in both M. melanosporea and S. lividans. On the other side, in S. lividans, plasmid pMK33-1 facilitates isolation and studies of promoters based on detection of extremely convenient phenotype of melanin production. This has been proved by shotgun cloning of chromosomal DNA fragments of M. melanosporea and chromogenic identification of S. lividans transformants which were capable of producing a melanin