Stable and efficient delivery of DNA to Bacillus subtilis (natto) using pLS20 conjugational transfer plasmids

Bacillus subtilis (natto) is generally regarded as a safe bacterium and used as a host for the production of several materials. However, genetic engineering of B. subtilis (natto) is not well established because of poor DNA delivery methods and the lack of a standard strain for the aim. Here, we developed a genetic delivery tool in B. subtilis (natto) using the pLS20 conjugational plasmid (65 kbp). Transmission of pLS20 from B. subtilis 168 to wild-type B. subtilis (natto) did not occur via established mating protocols. We isolated B. subtilis (natto) mutants showing dramatically increased recipient activity. Whole-genome sequence analyses revealed three common alterations: mutations in the restriction endonuclease gene and in the methyl-accepting chemotaxis protein gene, and a 43-kbp deletion at the genome replication termination locus. A representative strain named NEST116 was generated as the first B. subtilis (natto) strain suitable for exploring pLS20-based genetic engineering.