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Effect of elevated temperature on deoxyribonucleic acid synthesis in bacteriophage phi-29-infected Bacillus
Abstract:
Deoxyribonucleic acid (DNA) synthesis in bacteriophage phi29-infected Bacillus amyloliquefaciens was studied at 37 and 45 C. Infectious intracellular particles appear at the same time at both temperatures, but the average burst size is reduced 45 to 50% at 45 C. There is a transient inhibition of cellular mass increase at 45 C which is not observed at the lower temperature. In addition, the rate of host DNA synthesis is reduced and the onset of viral-specific DNA replication is delayed for 6 to 9 min at 45 C. These findings allowed us to screen phage phi29 mutants which are sensitive to growth at 45 C for their ability to synthesize phi29 DNA in the absence of host DNA replication. We obtained mutants which make no viral DNA, reduced levels of DNA, or normal quantities of DNA under nonpermissive conditions. Pulse-labeled viral DNA which sediments more rapidly than mature phi29 DNA molecules was observed after gentle cell lysis and zone sedimentation. This DNA is not a precursor of normally sedimenting phi29 DNA and apparently consists of mature phi29 DNA molecules aggregated with large pieces of bacterial DNA.
Insights
Bacteriophage phi29 DNA synthesis is impaired at 45°C, affecting host cell growth and viral replication. Mutants were screened for their ability to synthesize viral DNA under these conditions.
Area of Science:
- Molecular Biology
- Virology
- Microbiology
Background:
- Bacteriophage phi29 infects Bacillus amyloliquefaciens, a model system for studying DNA replication.
- Viral DNA synthesis is crucial for phage propagation and understanding host-pathogen interactions.
Purpose of the Study:
- To investigate the effects of elevated temperature (45°C) on bacteriophage phi29 DNA synthesis and replication in Bacillus amyloliquefaciens.
- To identify and characterize phi29 mutants with altered DNA synthesis capabilities at non-permissive temperatures.
Main Methods:
- Comparative analysis of viral DNA synthesis at 37°C and 45°C.
- Screening of phi29 mutants for temperature sensitivity in DNA replication.
- Zone sedimentation analysis of pulse-labeled viral DNA.
Main Results:
- Elevated temperature (45°C) reduced burst size by 45-50% and transiently inhibited cellular mass increase.
- Host DNA synthesis was reduced, and viral DNA replication onset was delayed by 6-9 minutes at 45°C.
- Mutants were isolated exhibiting no, reduced, or normal viral DNA synthesis at 45°C; rapidly sedimenting viral DNA aggregated with bacterial DNA was observed.
Conclusions:
- Temperature significantly impacts phi29 DNA synthesis and replication, affecting both viral yield and host cell physiology.
- The study successfully identified phi29 mutants with distinct DNA synthesis phenotypes under temperature stress.
- Aggregated viral and bacterial DNA complexes were observed, suggesting potential interactions during replication or lysis at elevated temperatures.