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Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis

1991; Wiley; Volume: 5; Issue: 10 Linguagem: Inglês

10.1111/j.1365-2958.1991.tb02095.x

1365-2958

Carlos Martı́n, Philippe Mazodier, M. V. Mediola, Brigitte Gicquel, Tamara Smokvina, Charles J. Thompson, Julian Davies,

Microbial Natural Products and Biosynthesis

Summary A method which allowed the stable integration of DNA fragments at a single site ( attB ) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens , pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn 903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site ( attP ) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site ( attB ) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.