Stable Mutants of Restriction-deficient/modification-proficient Bacillus Subtilis 168: Hub Strains for Giant DNA Engineering

Bacillus subtilis 168 has been explored as a platform for the synthesis and transmission of large DNA. Two inherent DNA incorporation systems, natural transformation and pLS20-based conjugation transfer, enable rapid handling of target DNA. Both systems are affected by the Bsu restriction-modification system that recognizes and cleaves unmethylated XhoI sites, limiting the choice of target DNA. We constructed B. subtilis 168 with stable mutation for restriction-deficient and modification-proficient (r-m+). It was demonstrated that the r-m+ strains can incorporate and transfer synthesized DNA with multiple XhoI sites. These should be of value as hub strains to integrate and disseminate giant DNA between B. subtilis 168 derivatives.