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DNA packaging by the Bacillus subtilis defective bacteriophage PBSX
Abstract:
Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144.
Insights
Defective bacteriophage PBSX in Bacillus subtilis packages 13-kilobase DNA fragments from host chromosomes and plasmids. This process, triggered by mitomycin C, involves a head-filling mechanism and alters plasmid replication.
Area of Science:
- Molecular Biology
- Microbiology
- Virology
Background:
- Bacteriophage PBSX is a defective phage integrated into Bacillus subtilis 168 chromosomes.
- Mitomycin C induces phage activity and DNA packaging in Bacillus subtilis.
Purpose of the Study:
- To investigate the physical process of DNA packaging by bacteriophage PBSX.
- To determine if PBSX exhibits selectivity in packaging chromosomal and plasmid DNA.
- To understand the role of the prophage region in mitomycin C-induced plasmid alterations.
Main Methods:
- Utilized mitomycin C induction in Bacillus subtilis strains.
- Employed genetically engineered strains with chromosomal genetic flags.
- Analyzed DNA packaging using DNase I resistance assays.
- Examined plasmid DNA structure and replication dynamics.
Main Results:
- Wild-type PBSX packages discrete 13-kilobase DNA fragments via a DNase I-resistant, head-filling mechanism.
- No significant selectivity was observed for packaging specific chromosomal regions.
- Mitomycin C induced increased size and packaging of plasmid DNA in wild-type strains.
- Plasmid replication alterations and subsequent packaging occurred in a strain lacking parts of the PBSX prophage.
Conclusions:
- Bacteriophage PBSX packaging is a size-limited process involving host DNA.
- Mitomycin C induction can lead to the 'cannibalization' of plasmid replication for phage packaging.
- The PBSX prophage region plays a role in mediating mitomycin C-induced plasmid alterations.