Mechanism of Anucleate Cell Production in the oriC-Deleted Mutants of Bacillus subtilis

We constructed oriC-deleted mutants of Bacillus subtilis byintegrating the minimal replication region of plasmid pLS32into the proA (115°), spoIIIJ (360°) and thrS (256°)loci of the chromosome, respectively. All three mutants producedanucleate cells and the DNA/protein ratio was lower than thatof the wild-type strain when grown in nutrient broth. However,when grown in minimal-glucose medium, the frequency of anucleatecells was reduced in all of them and the DNA/protein ratio wasrestored to normal. Especially, the oriC-deleted mutant in whichthe plasmid was integrated near oriC produced almost no anucleatecell. These results indicate that initiation frequency of chromosomereplication from the integrated plasmid origin were reduceddisproportionately to cell mass increase in rich medium, whichin turn disrupted coordination between DNA replication cycleand cell division cycle. The locations of the plasmid originrelative to the natural oriC locus affected the production ofanucleate cell remarkably, suggesting that partition mechanismof chromosome was also impaired by the translocation of itsreplication origin.