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Defective bacteriophage PBSH in Bacillus subtilis. II. Intracellular development of the induced prophage
Abstract:
Treatment of Bacillus subtilis strain 168 with mitomycin C caused induction of a defective prophage, PBSH. During induction, extensive deoxyribonucleic acid (DNA) synthesis took place. Concurrently, a change in marker frequency of the bacterial DNA was noticed. The frequency of only one marker, ade-16, the marker closest to the origin of the bacterial chromosome, was enhanced manyfold. DNA from whole phage particles transformed all bacterial markers at a frequency equal to that of DNA in the noninduced culture, except ade-16, the frequency of which was enhanced 30 to 100 times. Analysis of a double isotope experiment demonstrated that 14% of the phage DNA was derived from preinduction bacterial DNA. The other 86% of DNA in phage particles was DNA replicated after induction. Density label experiments with 5-bromodeoxyuridine showed that postinduction DNA synthesis took place preferentially at the origin region of the bacterial chromosome. Measurement of the molecular weight of DNA replicated after induction clearly showed that postinduction DNA replication is chromosomal. No evidence for prophage detachment and autonomous phage DNA replication was found. The data indicated that, after mitomycin C action, the bacterial chromosome under-went multiple reinitiation at the origin, while normal sequential DNA replication was stopped. The pool of replicated bacterial DNA was fragmented randomly. This DNA was packaged into PBSH particles which were released after cell lysis.
Insights
Mitomycin C treatment induced Bacillus subtilis prophage PBSH, causing extensive DNA replication. This replication preferentially occurred at the bacterial chromosome origin, leading to marker ade-16 enrichment in phage particles.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacillus subtilis strain 168 harbors a defective prophage, PBSH.
- Mitomycin C is known to induce prophages in bacteria.
- Understanding prophage induction mechanisms is crucial for bacterial genetics.
Purpose of the Study:
- To investigate the mechanism of DNA replication during PBSH prophage induction in Bacillus subtilis.
- To characterize the origin and nature of DNA synthesis following mitomycin C treatment.
- To determine the fate of bacterial DNA during phage production.
Main Methods:
- Prophage induction using mitomycin C in Bacillus subtilis strain 168.
- Analysis of bacterial DNA synthesis and marker frequency changes.
- Transformation assays using DNA from phage particles.
- Isotope labeling (double isotope and density labeling with 5-bromodeoxyuridine) experiments.
- Molecular weight determination of replicated DNA.
Main Results:
- Mitomycin C induced PBSH prophage, triggering extensive deoxyribonucleic acid (DNA) synthesis.
- A significant enrichment of the bacterial marker ade-16, located near the chromosome origin, was observed in phage DNA.
- Density labeling revealed preferential DNA synthesis at the bacterial chromosome origin after induction.
- Molecular weight analysis confirmed that post-induction DNA replication was chromosomal, not autonomous phage replication.
- 14% of phage DNA originated from pre-induction bacterial DNA, while 86% was newly replicated.
Conclusions:
- Mitomycin C treatment causes multiple reinitiation events at the bacterial chromosome origin, halting normal replication.
- Replicated bacterial DNA is randomly fragmented and packaged into PBSH phage particles.
- Prophage detachment and autonomous phage DNA replication were not observed, indicating a unique replication strategy.