Experimental surgery to create subgenomes of Bacillus subtilis 168

The 4,188-kb circular genome of Bacillus subtilis 168 was artificially dissected into two stable circular chromosomes in vivo, one being the 3,878-kb main genome and the other the 310-kb subgenome that was recovered as covalently closed circular DNA in CsCl-ethidium bromide ultracentrifugation. The minimal requirements to physically separate the 310-kb DNA segment out of the genome were two interrepeat homologous sequences and an origin of DNA replication between them. The subgenome originated from the 1,255–1,551-kb region of the B. subtilis genome was essential for the cell to survive because the subgenome was not lost from the cell. The finding that the B. subtilis genome has a potential to be divided and the resulting two replicons stably maintained may shed light on origins and formation mechanisms of giant plasmids or second chromosomes present in many bacteria. Similar excision or its reversal process, i.e., integration of large sized covalently closed circular DNA pieces into the main genome, implies significant roles of subgenomes in the exchange of genetic information and size variation of bacterial genomes in bacterial evolution.