Mapping of the replication origin of the Bacillus subtilis chromosome by the two-dimensional gel method

Analysis of replication intermediates produced by in vitro replication of the Bacillus subtilis oriC plasmid revealed that replication initiated at an untranslatable DnaA box region downstream of the dnaA gene. In order to show that replication of the B. subtilis chromosome also starts at the same region in vivo, we have analyzed replication intermediates generated in vivo by the two-dimensional gel method. A bubble arc was detected when the downstream region was used as a probe. In contrast, only a simple Y arc was found when the upstream DnaA box region required for autonomous replication of the oriC plasmid was used as a probe. Furthermore, the bubble arc ranged from unit to almost double the size of a fragment in which the downstream region was located near the middle. These results indicate that replication of the B. subtilis chromosome initiates at the downstream DnaA box region of the dnaA gene and proceeds bidirectionally.