title: Lambda gpP-DnaB Helicase Sequestration and gpP-RpoB Associated Effects: On Screens for Auxotrophs, Selection for Rif(R), Toxicity, Mutagenicity, Plasmid Curing.
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.
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. sidney.hayes@usask.ca.
authors.forename: Sidney
authors.initials: S
authors.lastname: Hayes
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. wen.wang@me.com.
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. kar029@mail.usask.ca.
authors.forename: Karthic
authors.initials: K
authors.lastname: Rajamanickam
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. audreymchu@gmail.com.
authors.forename: Audrey
authors.initials: A
authors.lastname: Chu
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. abanerj7@uwo.ca.
authors.forename: Anirban
authors.initials: A
authors.lastname: Banerjee
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. clh127@outlook.com.
authors.forename: Connie
authors.initials: C
authors.lastname: Hayes
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D010582:Bacteriophage lambda / Q000254:growth & development*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D053488:DnaB Helicases / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000821:virology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006790:Host-Parasite Interactions; D009154:Mutation; D011485:Protein Binding; D012293:Rifampin / Q000494:pharmacology; D014764:Viral Proteins / Q000633:toxicity*
chemical_list: D000900:Anti-Bacterial Agents; C024013:DNA replication complex protein, Bacteriophage lambda; D029968:Escherichia coli Proteins; D014764:Viral Proteins; C540081:rpoB protein, E coli; D012321:DNA-Directed RNA Polymerases; C506730:dnaB protein, E coli; D053488:DnaB Helicases; D012293:Rifampin
keywords: ColE1 plasmid curing; E. coli DnaB replicative helicase; P-DnaB sequestration; Replicative Killing phenotype; bacteriophage lambda (λ) replication initiation protein P; cellular mutagenesis; rpoB encoding β-subunit of RNA polymerase (RNAP); rpoB mutations suppressing P-lethality; screening for auxotrophs; selecting for RifR mutants