DNA-membrane association is necessary for initiation of chromosomal and plasmid replication in Bacillus subtilis

Abstract

We examined the effect of the inhibition of initiation of DNA replication on the membrane association of the chromosomal origin of replication of Bacillus subtilis and the Staphylococcus aureus-Bacillus pumilus chimeric plasmid pSL103, using temperature-sensitive mutants of B. subtilis that have specifically affected initiation. Inhibition of initiation of the chromosome and pSL103 in the initiation mutant dna-1 results in a decrease in the membrane association of both a marker near the chromosomal origin, purA16, and the plasmid pSL103. The membrane association of both purA16 and pSL103 can be recovered by allowing initiation to resume at the permissive temperature. In another initiation mutant, dnaB19, only the initiation and membrane association of the host chromosome are affected at the nonpermissive temperature, whereas both initiation and membrane association are not affected in the plasmid pSL103. In experiments in vitro, DNA containing the purA16 marker and pSL103 DNA molecules are both selectively released during incubation of purified DNA-membrane complexes prepared from dna-1 cells at the nonpermissive temperature. On the other hand, only purA16 DNA is released in vitro from the DNA-membrane complex prepared from dnaB19 cells. This consistent coupling between initiation and membrane association indicates that DNA-membrane association is critical for the initiation of the B. subtilis chromosome and the plasmid pSL103.