Development of a small mobilizable plasmid system: A foundation for genetic engineering of Bacillus subtilis strains

Although Bacillus subtilis natto is a highly beneficial probiotic bacterium, its genetic engineering is heavily constrained by low natural transformability. To provide a competence-independent route for DNA delivery, we developed a minimized mobilizable plasmid system. We demonstrated that the pCJ plasmid was successfully transferred into B. subtilis natto via conjugation, achieving knockout of the BSNT10618 gene, selected for its predicted role in restricting incoming DNA. Furthermore, systematic replicon evaluation revealed that a pE194-derived rolling-circle replicon maintained high transfer efficiency, enabling successful marker-free genome editing at the malS locus in the laboratory strain B. subtilis 168 via both transformation and conjugation. While demonstrated primarily in strain 168, this system provides a reliable foundation to expand future genome editing and genetic engineering applications in the industrial natto strain.