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Bacillus subtilis DNA polymerase III is required for the replication of the virulent bacteriophage phi e
Abstract:
The virulent phage phie of Bacillus subtilis which contains hydroxymethyluracil in its DNA requires host DNA polymerase III for its DNA replication. DNA polymerase III(ts) mutant cells infected with phie at restrictive temperatures do not support phage DNA synthesis. However, phie grows normally both at low and high temperatures in the mutant's parent strain and in spontaneous DNA polymerase III(+) revertants isolated from the mutant strain. Temperature-shift-down experiments with phie-infected cells having thermosensitive DNA polymerase III (pol III(ts)) indicate that at 48 C the thermolabile DNA polymerase III is irreversibly inactivated and has to be synthesized de novo after the shift to 37 C, before phage DNA synthesis can begin. Temperature-shift-up experiments with phie-infected mutant cells show that phage replication is arrested immediately after the temperature shift and indicate that phie requires DNA polymerase III throughout its replication stage.
Insights
Bacillus subtilis phage phiE requires host DNA polymerase III for replication. Its DNA synthesis is blocked in temperature-sensitive DNA polymerase III mutants but restored in parent strains or revertants.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacillus subtilis phage phiE possesses hydroxymethyluracil in its DNA.
- Bacterial DNA replication relies on complex enzymatic machinery, including DNA polymerases.
Purpose of the Study:
- To investigate the role of host DNA polymerase III in the replication of Bacillus subtilis phage phiE.
- To determine the essentiality of DNA polymerase III activity throughout the phage replication cycle.
Main Methods:
- Utilizing temperature-sensitive (ts) DNA polymerase III mutant strains of Bacillus subtilis.
- Infecting mutant and wild-type strains with phage phiE under various temperature conditions.
- Performing temperature-shift-up and temperature-shift-down experiments.
Main Results:
- Phage phiE DNA replication was inhibited in DNA polymerase III (ts) mutants at restrictive temperatures.
- Phage phiE replicated normally in the parent strain and in spontaneous DNA polymerase III-positive revertants.
- Temperature-shift-down experiments revealed that inactivated DNA polymerase III must be resynthesized for phage DNA synthesis to resume.
- Temperature-shift-up experiments demonstrated immediate arrest of phage replication, indicating continuous requirement for DNA polymerase III.
Conclusions:
- Host DNA polymerase III is essential for the replication of Bacillus subtilis phage phiE.
- Phage phiE requires functional DNA polymerase III activity throughout its entire replication process.