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Bacteriophage P4 Vis protein is needed for prophage excision
Simona Calì1, Elena Spoldi, Daniela Piazzolla
1Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, 20133 Milan, Italy.
Virology
|April 6, 2004
Summary
The P4 vis gene product is essential for satellite bacteriophage P4 prophage excision from Escherichia coli. Vis protein binds P4 DNA and regulates integrase expression, facilitating excision.
Area of Science:
- Molecular Biology
- Virology
- Bacteriology
Background:
- Satellite bacteriophage P4 integrates into Escherichia coli via site-specific recombination.
- Prophage excision is crucial for P4 replication and can occur spontaneously or be induced.
- The P4 vis gene regulates late promoters, but its role in excision was unknown.
Purpose of the Study:
- To investigate the role of the P4 vis gene product in prophage excision.
- To elucidate the mechanism by which Vis protein influences excision.
Main Methods:
- Site-directed mutagenesis to create vis mutants.
- Plasmid-based expression of Vis protein.
- Analysis of prophage excision efficiency.
- DNA binding assays (electrophoretic mobility shift assays) to detect Vis-DNA interactions.
- Primer extension to map the 5' end of the integrase transcript.
Main Results:
- P4 vis mutants were defective in both spontaneous and P2-induced prophage excision.
- Expression of Vis protein from a plasmid restored excision in vis mutants.
- Vis protein bound to specific DNA sites within the P4 attP region, causing DNA bending.
- Vis protein negatively regulated the integrase (int) gene promoter.
Conclusions:
- The P4 Vis protein is essential for prophage excision.
- Vis acts by facilitating the formation of the excision complex and negatively regulating integrase expression.
- Vis protein directly binds P4 DNA in the attP region, suggesting a role in organizing the recombination machinery.