Development of improved cloning vectors for Bacillus and Staphylococcus species

Abstract

A shuttle vector was constructed which was stably maintained in Escherichia coli, Bacillus subtilis and Staphylococcus aureus. It was made by ligating E. coli positive selection plasmid pEcoR251 and S. aureus resistance plasmid pC194 via their respective BamHI and XhoII sites. Designated pNDW1, it was shown to be effective in genomic library construction. The number of restriction sites in the EcoRI (“suicide”) gene was increased by successive addition of XbaI and XhoI using site-directed mutagenesis. SpeI, NheI and AvrII generate DNA fragments with compatible cohesive ends to XbaI while SalI digestion gives rise to ends compatible with XhoI. Therefore the number of different genomic libraries which can be made using this system is augmented by six. A principal impediment to full exploitation of these shuttle vectors is apparently the severe restriction by B. subtilis and S. aureus of DNA coming from E. coli.