Lambda gpP-DnaB Helicase Sequestration and gpP-RpoB Associated Effects: On Screens for Auxotrophs, Selection for RifR, 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 (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.

The Bacteriophage Lambda CII Phenotypes for Complementation, Cellular Toxicity and Replication Inhibition Are Suppressed in cII-oop Constructs Expressing the Small RNA OOP

“…Several of the rpoB mutants, B1 (Q148P), B8 (L571Q), C4 (ΔGEV440-442V), D2 (R451S) and D6 (G537C) had been shown to confer cellular resistance to the highly toxic λ gpP, while C1 (P564L) and C10 (R529H) remained P-sensitive [ 37 ]. Except for B1, both P R and P S mutants, suppressed CII toxicity at 39 °C (cell viabilities: 594[cII], 0.03; rpoB [cII]: B1, 0.003; B8, 0.65; C1, 0.96; C4, 0.15; C10, 0.14; D2, 0.95; D6, 0.11) and 42 °C.…”

“…What is responsible for CII toxicity? It was shown that the single base substitution G537C responsible for rpoB D2 [ 37 ] can completely suppress CII toxicity and plasmid loss at the highest level of cII expression at 42 °C (with cell viability of 0.95 at 39 °C and no plasmid loss), while reducing but not eliminating CII complementation at 39 °C. This suggests that both CII toxicity and plasmid loss depend upon a binding interaction between CII and E. coli RNA polymerase holoenzyme, or a direct interaction with the β-subunit encoded by rpoB .…”

“…Cellular growth medium, buffers, PCR amplification, DNA sequencing, transformation and P1 transduction methods were previously described [ 37 ], as were techniques for the construction of plasmid expression vectors for λ genes, gel analysis of plasmids, insertion localization and assays for plasmid loss and for cell viability resulting from the toxicity of plasmid-expressed λ genes [ 38 ]. E. coli strain employed: 594 [ 38 ], the rpoB mutants employed were isolated, mapped and sequenced in 594 [ 37 ]; pcnB ::kan and hflA ::kan alleles were moved by P1 from strains MG1655 pcnB ::kan and X9368 hflA ::kan (kindly provided by Drs. Grzegorz Węgrzyn and Susan Gottessman, respectively).…”

“…This was done as previously described [ 37 ]. In brief, a single colony of E. coli cells with plasmid was inoculated in 20 mL of LB + Amp50 broth and incubated ON at 30 °C.…”

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes

“…Each of these alleles was examined for their ability to complement the growth of a λ O amber mutant(s) in trans . We have explored the influence of O:P interactions on cell growth and toxicity, since the expression of P by itself, using the same system, is highly toxic [ 25 , 36 ]. These studies reveal that some alleles of O with internal deletions can complement as well or better than O + and that the co-expression of O-P , or of O with portions of the N-terminal end of P , prevents an ability of O to complement in trans .…”