A high-transformation-efficiency cloning vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal re plicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gen e cassette encoding a thermostable resistance to kanamycin, and the replica tive origin and multiple cloning site of pUC18. Plasmid pMK18 showed transf ormation efficiencies from 10(8) to 10(9) per microgram of plasmid in Therm us thermophilus HB8 and HB27, both by natural competence and by electropora tion. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DN A. To demonstrate its usefulness as a cloning vector, a gene encoding the b eta-subunit of a thermostable nitrate reductase was directly cloned in T. t hermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector. (C) 1999 Academic Press.