Lambda gpP-DnaB Helicase Sequestration and gpP-RpoB Associated Effects: On Screens for Auxotrophs, Selection for Rif(R), Toxicity, Mutagenicity, Plasmid Curing.

Sidney Hayes,Karthic Rajamanickam,Audrey Chu,Wen Wang,Anirban Banerjee,Connie Hayes

doi:10.3390/v8060172

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.

Highlights

A lambda (λ) prophage is maintained within the chromosome of Escherichia coli (E. coli) by an action of the CI repressor encoded by gene cI
We showed that: (a) very limited oriλ initiation required for λ replication [35]; and (b) that λ replication resulting in high phage low levels of P are necessary for the limited oriλ initiation required for λ replication [35]; and (b) that burst from an induced λcI[temperature sensitive (Ts)]857 Sam7 prophage blocked for cell lysis occurred in cells that were λ replication resulting in high phage burst from an induced λcI[Ts]857 Sam7 prophage blocked for simultaneously fully derepressed for P expression from pcIpR-P-timm—conditions where DnaB would cell lysis occurred in cells that were simultaneously fully derepressed for P expression from pcIpR‐
We found that the RK+ phenotype was uniquely dependent upon multiple, non-repairable λ replication forks arising from oriλ within a trapped prophage fragment, resulting in very rapid, nonreversible cell death, whereas there are likely multiple possibilities for P-lethality, which can be reversed even after hours of

Summary

Introduction

A lambda (λ) prophage is maintained within the chromosome of Escherichia coli (E. coli) by an action of the CI repressor encoded by gene cI. DnaB functions as a hexamer, with about up to six ATP [20,21] It forms a complex with the host replication initiation protein DnaC to which the 20 hexamers per cell [18,19], with each binding up to six ATP [20,21]. It forms a complex with the host majority of DnaB in a cell is bound [22] This interferes with the intrinsic single-stranded (ss) DNA replication initiation protein DnaC to which the majority of DnaB in a cell is bound [22]. This binding activity of DnaB [20,22,23,24,25,26]. The specific to the bacterial origin of replication, oriC, where it begins unwinding dsDNA after a number

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Conclusion