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Bacillus subtilis DNA polymerase III is required for the replication of DNA of bacteriophages SPP-1 and phi 105
Abstract:
The replication of the Bacillus subtilis bacteriophages SPP-1 and phi 105 is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of replicative DNA synthesis of B. subtilis which acts specifically at the levels of a replication-specific polymerase, DNA polymerase III (pol III). The origin of the HPUra-sensitive polymerase required for phage replication was examined by comparison of the drug sensitivity of phage development in a normosensitive host with that in a host carrying azp-12, a polC mutation that specifies production of an HPUra-resistant pol III. azp-12 specified HPUra-resistant phage host pol III. The host polIII requirement for SPP-1 replication also was confirmed by the demonstration that phage development was temperature sensitive in a host mutant carrying the polC mutation mut-1 (ts). Examination of the pol III activity of crude and purified cell-free preparations derived from phage-infected cells did not indicate any detectable changes in the specific activity, purification behavior, or drug sensitivity of the enzyme.
Insights
Bacillus subtilis bacteriophage replication relies on host DNA polymerase III (pol III), which is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra). Mutations conferring HPUra resistance to host pol III also confer resistance to phage replication.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacteriophage SPP-1 and phi 105 replication in Bacillus subtilis are inhibited by 6-(p-hydroxyphenylazo)-uracil (HPUra).
- HPUra selectively targets the replicative DNA synthesis of B. subtilis by inhibiting DNA polymerase III (pol III).
Purpose of the Study:
- To investigate the origin of the HPUra-sensitive DNA polymerase required for SPP-1 and phi 105 bacteriophage replication.
- To determine if bacteriophage replication utilizes the host's DNA polymerase III (pol III).
Main Methods:
- Comparison of phage development sensitivity to HPUra in a normosensitive host versus a host with a polC mutation (azp-12) conferring HPUra-resistant pol III.
- Assessment of SPP-1 replication temperature sensitivity in a host carrying a temperature-sensitive polC mutation (mut-1).
- Analysis of pol III activity in cell-free extracts from phage-infected cells.
Main Results:
- The azp-12 mutation, conferring HPUra resistance to the host pol III, also resulted in HPUra-resistant bacteriophage replication.
- SPP-1 replication was temperature-sensitive in a host with the mut-1 polC mutation, indicating a dependence on the host pol III.
- No detectable changes in specific activity, purification, or drug sensitivity of pol III were observed in phage-infected cells.
Conclusions:
- Bacteriophage SPP-1 and phi 105 replication in Bacillus subtilis depend on the host's DNA polymerase III (pol III).
- The HPUra sensitivity observed during phage replication is attributed to the host pol III, not a phage-encoded polymerase.