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Published on: May 15, 2018
Lambda gpP-DnaB Helicase Sequestration and gpP-RpoB Associated Effects: On Screens for Auxotrophs, Selection for
Sidney Hayes1, Wen Wang2, Karthic Rajamanickam3
1Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. sidney.hayes@usask.ca.
Abstract:
The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (Rif(R)) cells acquiring mutations within the rpoB gene encoding the β-subunit of RNA polymerase (RNAP), nine of which proved unique. Using fluctuation assays, we show that the intracellular expression of P evokes a mutator effect. Most of the Rif(R) mutants remained P(S) and localized to the Rif binding pocket in RNAP, but a subset acquired a P(R) phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One P(R) mutation was identical to rpo*Q148P, which alleviates the UV-sensitivity of ruv strains defective in the migration and resolution of Holliday junctions and destabilizes stalled RNAP elongation complexes. The results suggest that P-DnaB sequestration is mutagenic and supports an earlier observation that P can interact with RNAP.
Insights
Bacteriophage P protein causes mutations in E. coli by sequestering DnaB helicase. This interaction with RNA polymerase leads to a mutator effect, impacting DNA replication fidelity.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacteriophage lambda protein P is toxic to Escherichia coli (E. coli) by forming a complex with DnaB helicase.
- Protein P expression causes cellular filamentation and plasmid curing, independent of the SOS response.
Purpose of the Study:
- To investigate if bacteriophage lambda protein P influences E. coli replication fidelity.
- To determine the mutagenic potential of protein P buildup within E. coli cells.
Main Methods:
- Screening for auxotrophic mutants and selecting for rifampicin-resistant (Rif(R)) E. coli.
- Analyzing mutations in the rpoB gene encoding the RNA polymerase (RNAP) β-subunit.
- Utilizing fluctuation assays to assess the mutator effect of protein P.
Main Results:
- Intracellular expression of protein P induces a mutator effect in E. coli.
- Rifampicin-resistant mutants showed altered phenotypes, with some P(R) mutations affecting RNAP interaction.
- One P(R) mutation (rpo*Q148P) destabilizes stalled RNAP elongation complexes.
Conclusions:
- P-DnaB sequestration by bacteriophage lambda protein P is mutagenic in E. coli.
- Protein P's interaction with RNAP contributes to its mutagenic effect and cellular toxicity.
- The study supports previous findings on protein P's interaction with RNAP.
