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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.
Insights
DNA replication initiation is crucial for bacterial chromosome and plasmid membrane association. Inhibiting initiation disrupts this link, highlighting DNA-membrane association
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- DNA replication initiation is a tightly regulated process essential for cell division.
- The association of DNA replication origins with the cell membrane is a known phenomenon in bacteria.
- The precise role of membrane association in replication initiation remains incompletely understood.
Purpose of the Study:
- To investigate the relationship between DNA replication initiation and membrane association in Bacillus subtilis.
- To determine if plasmid DNA replication initiation is also dependent on membrane association.
- To elucidate the role of specific initiation mutants in affecting DNA-membrane interactions.
Main Methods:
- Utilized temperature-sensitive initiation mutants (dna-1 and dnaB19) of Bacillus subtilis.
- Assessed membrane association of chromosomal origin markers (purA16) and a chimeric plasmid (pSL103) under restrictive and permissive conditions.
- Performed in vitro experiments involving purified DNA-membrane complexes to analyze DNA release.
Main Results:
- Inhibition of initiation in dna-1 mutants decreased membrane association of both chromosomal DNA and pSL103.
- Resuming initiation at permissive temperatures restored membrane association for both chromosomal DNA and pSL103.
- The dnaB19 mutant showed impaired chromosomal DNA initiation and membrane association, but pSL103 remained unaffected.
- In vitro studies confirmed selective release of DNA from membrane complexes upon inhibition of initiation.
Conclusions:
- DNA-membrane association is critically coupled to the initiation of DNA replication for both the Bacillus subtilis chromosome and the pSL103 plasmid.
- Specific initiation proteins or pathways may differentially regulate the membrane association of chromosomal versus plasmid DNA.
- These findings suggest that the cell membrane plays an integral role in the fundamental process of DNA replication initiation in bacteria.