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Characterization of PBSX, a defective prophage of Bacillus subtilis
H E Wood1, M T Dawson, K M Devine
1Department of Genetics, Trinity College, Dublin, Ireland.
Abstract:
PBSX, a defective Bacillus subtilis prophage, maps to the metA-metC region of the chromosome. DNA (33 kilobases) from this region of the chromosome was cloned and analyzed by insertional mutagenesis with the integrating plasmid pWD3. This plasmid had a promoterless alpha-amylase gene (amyL) that provided information on the direction and level of transcription at the site of integration. Transcription under the control of the PBSX repressor proceeded in the direction metA to metC over a distance of at least 18 kilobases. Electrophoretic analysis of proteins produced by different integrant strains upon PBSX induction and by fragments subcloned in Escherichia coli allowed the identification of early and late regions of the prophage. A set of contiguous fragments directing mutagenic integration suggested that the minimum size of an operon that encodes phage structural proteins is 19 kilobases. The adaptation of PBSX transcriptional and replicational functions to a chromosomally based, thermoinducible expression system is discussed.
Insights
Researchers mapped the defective Bacillus subtilis prophage PBSX using DNA cloning and mutagenesis. They identified transcriptional regions and determined the minimum operon size for phage structural proteins.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The defective Bacillus subtilis prophage PBSX is located in the metA-metC chromosomal region.
- Understanding prophage organization and gene expression is crucial for microbial genetics.
Purpose of the Study:
- To clone and analyze the DNA region of the Bacillus subtilis chromosome containing the PBSX prophage.
- To identify transcriptional units and gene regions within the PBSX prophage.
- To determine the minimum size of the operon encoding PBSX structural proteins.
Main Methods:
- Cloning of 33 kilobases of Bacillus subtilis chromosomal DNA containing the PBSX prophage.
- Insertional mutagenesis using the integrating plasmid pWD3, which contains a promoterless alpha-amylase gene (amyL).
- Analysis of transcriptional direction and level, protein expression via electrophoresis, and subcloning in Escherichia coli.
Main Results:
- Transcription analysis revealed expression from the PBSX repressor in the metA to metC direction over at least 18 kilobases.
- Identification of early and late prophage regions through protein analysis and fragment subcloning.
- A minimum operon size of 19 kilobases was suggested for phage structural protein encoding.
Conclusions:
- The study successfully mapped and characterized transcriptional elements of the PBSX prophage.
- The findings provide insights into the genetic organization and expression of defective prophages.
- The research discusses the adaptation of PBSX functions for a thermoinducible expression system.