A novel, highly efficient gene-cloning system in Micromonospora applied to the genetic analysis of fortimicin biosynthesis.
We have developed a gene-cloning system in Micromonospora olivasterospora, a fortimicin A (astromicin) producer. Plasmids of Micromonospora from two strains of M. olivasterospora were used for construction of the vectors. Two antibiotic-resistance genes, nmrA and nmrB, cloned from a neomycin-producing Micromonospora, were introduced into these plasmids for the selection of transformants. In a new protoplasting protocol for lysozyme-resistant bacteria, protoplasts of M. olivasterospora were found in short-time incubation with lysozyme and transformed efficiently, indicating that the method was suitable to shotgun cloning. Using this system, seven biosynthetic genes for fortimicin A were cloned. Their physical maps revealed that at least four of these genes were clustered. Analysis of a cosmid library of M. olivasterospora showed that eleven biosynthetic genes and a self-defense gene existed in a region of approx. 25 kb of DNA.
Hasegawa
M
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Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008847:Micromonospora / Q000235:genetics*; D005810:Multigene Family; D010957:Plasmids; D011523:Protoplasts / Q000378:metabolism / Q000382:microbiology / Q000502:physiology; D014169:Transformation, Bacterial
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; C023521:fortimicins