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Viruses 2016, 8(6), 172; doi:10.3390/v8060172

Lambda gpP-DnaB Helicase Sequestration and gpP-RpoB Associated Effects: On Screens for Auxotrophs, Selection for RifR, Toxicity, Mutagenicity, Plasmid Curing

Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
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Author to whom correspondence should be addressed.
Academic Editor: Rob Lavigne
Received: 18 March 2016 / Revised: 25 May 2016 / Accepted: 9 June 2016 / Published: 22 June 2016
(This article belongs to the Section Bacterial Viruses)
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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 (RifR) 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 RifR mutants remained PS and localized to the Rif binding pocket in RNAP, but a subset acquired a PR phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One PR 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. View Full-Text
Keywords: bacteriophage lambda (λ) replication initiation protein P; E. coli DnaB replicative helicase; Replicative Killing phenotype; rpoB encoding β-subunit of RNA polymerase (RNAP); rpoB mutations suppressing P-lethality; ColE1 plasmid curing; screening for auxotrophs; selecting for RifR mutants; P-DnaB sequestration; cellular mutagenesis bacteriophage lambda (λ) replication initiation protein P; E. coli DnaB replicative helicase; Replicative Killing phenotype; rpoB encoding β-subunit of RNA polymerase (RNAP); rpoB mutations suppressing P-lethality; ColE1 plasmid curing; screening for auxotrophs; selecting for RifR mutants; P-DnaB sequestration; cellular mutagenesis
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MDPI and ACS Style

Hayes, S.; Wang, W.; Rajamanickam, K.; Chu, A.; Banerjee, A.; Hayes, C. Lambda gpP-DnaB Helicase Sequestration and gpP-RpoB Associated Effects: On Screens for Auxotrophs, Selection for RifR, Toxicity, Mutagenicity, Plasmid Curing. Viruses 2016, 8, 172.

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