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Involvement of Escherichia coli K-12 DNA polymerase I in the growth of bacteriophage Mu
Abstract:
We examined several aspects of bacteriophage Mu development in Escherichia coli strains that carry mutations in the polA structural gene for DNA polymerase I (PolI). We found that polA mutants were markedly less efficient than PolI wild-type (PolI+) strains in their capacity to form stable Mu lysogens and to support normal lytic growth of phage Mu. The frequency of lysogenization was determined for polA mutants and their isogenic PolI+ derivatives, with the result that mutants were lysogenized 3 to 8 times less frequently than were PolI+ cells. In one-step growth experiments, we found that phage Mu grew less efficiently in polA cells than in PolI+ cells, as evidenced by a 50 to 100% increase in the latent period and a 20 to 40% decrease in mean burst size in mutant cells. A further difference noted in infected polA strains was a 10-fold reduction in the frequency of Mu-mediated transposition of chromosomal genes to an F plasmid. Pulse labeling and DNA-DNA hybridization assays to measure the rate of phage Mu DNA synthesis after the induction of thermosensitive prophages indicated that phage Mu replication began at about the same time in both polA and PolI+ strains, but proceeded at a slower rate in polA cells. We conclude that PolI is normally involved in the replication and integration of phage Mu. However, since phage Mu does not exhibit an absolute requirement for normal levels of PolI, it appears that residual PolI activity in the mutant strains, other cellular enzymes, or both can partially compensate for the absence of normal PolI activity.
Insights
Mutations in the polA gene, which codes for DNA polymerase I (PolI), significantly impair bacteriophage Mu development in Escherichia coli. PolI is crucial for Mu lysogenization, lytic growth, and transposition.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacteriophage Mu is a versatile genetic element used extensively in molecular biology.
- DNA polymerase I (PolI) is a key enzyme in DNA replication and repair in Escherichia coli.
Purpose of the Study:
- To investigate the role of DNA polymerase I (PolI) in the development of bacteriophage Mu in Escherichia coli.
- To determine the impact of polA gene mutations on Mu lysogenization, lytic growth, and transposition.
Main Methods:
- Comparative analysis of bacteriophage Mu development in polA mutant and wild-type Escherichia coli strains.
- Lysogenization frequency assays.
- One-step growth experiments.
- Mu-mediated transposition assays.
- Pulse labeling and DNA-DNA hybridization for Mu DNA synthesis analysis.
Main Results:
- polA mutants showed a 3-to-8-fold lower frequency of Mu lysogenization compared to wild-type strains.
- Phage Mu exhibited a longer latent period (50-100% increase) and reduced burst size (20-40% decrease) in polA mutants.
- A 10-fold reduction in Mu-mediated transposition of chromosomal genes to an F plasmid was observed in polA strains.
- Mu DNA replication initiated similarly but proceeded at a slower rate in polA cells.
Conclusions:
- DNA polymerase I (PolI) plays a significant role in the replication and integration of bacteriophage Mu.
- Phage Mu development is impaired but not completely abolished in the absence of normal PolI activity, suggesting partial compensation by residual activity or other enzymes.