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

We constructed oriC-deleted mutants of Bacillus subtilis by integrating the minimal replication region of plasmid pLS32 into the proA (115 degrees), spoIIIJ (360 degrees) and thrS (256 degrees) loci of the chromosome, respectively. All three mutants produced anucleate cells and the DNA/protein ratio was lower than that of the wild-type strain when grown in nutrient broth. However, when grown in minimal-glucose medium, the frequency of anucleate cells was reduced in all of them and the DNA/protein ratio was restored to normal. Especially, the oriC-deleted mutant in which the plasmid was integrated near oriC produced almost no anucleate cell. These results indicate that initiation frequency of chromosome replication from the integrated plasmid origin were reduced disproportionately to cell mass increase in rich medium, which in turn disrupted coordination between DNA replication cycle and cell division cycle. The locations of the plasmid origin relative to the natural oriC locus affected the production of anucleate cell remarkably, suggesting that partition mechanism of chromosome was also impaired by the translocation of its replication origin.