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-ge ntamicin resistance gene from M zionensis) as the second antibiotic re sistance marker. These plasmids, containing sgm gene as the second sel ectable marker, may be an attractive alternative to pIJ702, which is i ncapable of conferring melanin production in M melanosporea and conseq uently is not useful for insertional inactivation in this bacterium. T he constructions remove restriction site for M melanosporea restrictio n endonuclease and provide additional unique sites for the insertional inactivation of selectable markers, which enhance the use of these pl asmids as general cloning vectors in both M melanosporea and S. livida ns. On the other side, in S. lividans, plasmid pMK33-1 facilitates iso lation and studies of promoters based on detection of extremely conven ient phenotype of melanin production. This has been proved by shotgun cloning of chromosomal DNA fragments of M. melanosporea and chromogeni c identification of S. lividans transformants which were capable of pr oducing a melanin.