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Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX
Abstract:
Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since ø105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome.
Insights
Researchers identified Bacillus subtilis mutants affecting PBSX prophage. Mutations impact phage capsid formation and regulation, revealing insights into prophage replication and chromosome integration.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacillus subtilis harbors the cryptic prophage PBSX.
- Prophage induction is typically mediated by DNA-damaging agents like mitomycin C.
- Understanding PBSX gene regulation and function is crucial for bacterial genetics.
Purpose of the Study:
- To isolate and characterize Bacillus subtilis mutants with defects in PBSX prophage genes.
- To investigate the roles of specific PBSX genes in phage assembly and regulation.
- To elucidate the mechanism of PBSX prophage replication and its interaction with the host chromosome.
Main Methods:
- Isolation and characterization of Bacillus subtilis mutants with lesions in PBSX prophage genes.
- Analysis of mitomycin C-induced PBSX prophage derepression.
- Immunoprecipitation assays to detect PBSX structural proteins.
- Serological reactivity testing with anti-PBSX antiserum.
- Genetic mapping of PBSX-specific mutations on the host chromosome.
Main Results:
- Isolated mutants defective in PBSX regulation and capsid formation.
- Identified specific head (X4, X7) and tail (X5, X6) protein assembly defects.
- Observed abnormal serological reactivity of synthesized PBSX proteins.
- Demonstrated that mutations are specific to PBSX and clustered on the host chromosome.
- Evidence suggests prophage replication occurs while integrated, extending into adjacent chromosomal regions.
Conclusions:
- Specific PBSX genes are essential for proper phage capsid assembly.
- A regulatory mutation affects mitomycin C-induced PBSX derepression.
- PBSX prophage replication initiates from an integrated state, involving host chromosome expansion.
- The metC marker's amplification indicates replication fork progression into adjacent DNA.