Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110

Abstract

Plasmid pUB110 (approximately 2.8 x 10(6) daltons), originally detected in Staphylococcus aureus, specifies resistance to neomycin and has been transformed into Bacillus subtilis 168, In B. subtilis, pUB110 is stably maintained at about 50 copies per chromosome and renders the host resistant to neomycin sulfate at 5 microgram/ml. pUB110 isolated from B. subtilis transforms Rec+ and recE4-containing strains of B. subtilis at frequencies greater than or equal to 10(3) transformants per microgram of plasmid. pUB110 was transferred by PBS1 or SP10 transduction from B. subtilis to strains of B. pumilus and B. licheniformis. pUB110 is compatible with each of four previously described Bacillus plasmids, including pPL576, pPL10, pPL7065, and pPL2. pUB110 contains a single EcoR1-sensitive site and was used as vector to clone DNA fragments that complement the trpC2 mutation in B. subtilis 168 from EcoR1 digests of the chromosome DNA isolated from B. pumilus strains NRRL B-3275 and NRS576, B. licheniformis strains 9945A and 749C, and B. subtilis 168. Genetic and physical properties of each of the constructed Trp derivatives of pUB110 are described.