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

λ genes O and P are required for replication initiation from the bacteriophage λ origin site, oriλ, located within gene O. Questions have persisted for years about whether O-defects can indeed be complemented in trans. We show the effect of original null mutations in O and the influence of four origin mutations (three are in-frame deletions and one is a point mutation) on complementation. This is the first demonstration that O proteins with internal deletions can complement for O activity, and that expression of the N-terminal portion of gene P can completely prevent O complementation. We show that O-P co-expression can limit the lethal effect of P on cell growth. We explore the influence of the contiguous small RNA OOP on O complementation and P-lethality.

Lambda display phage as a mucosal vaccine delivery vehicle for peptide antigens.

Bacteriophage are structurally stable in the gastro-intestinal tract and have favorable traits of safety, stability, ease of production, and immunogenicity. These attributes make them potential candidates as oral vaccine delivery vehicles but little is known about their capacity to induce mucosal immune responses in the small intestine. Whole body imaging of mice confirmed lambda bacteriophage (LP) were distributed throughout the gastro-intestinal tract 24 h after oral delivery. In newborn calves, targeted delivery of LP within the small intestine confirmed LP were immunogenic in a dose-dependent manner and were taken up by Peyer's patches. LP-specific IgA responses were induced within both Peyer's patches and draining mesenteric lymph nodes. A lambda display phage (LDP) was constructed to present three immunogenic disease specific epitopes (DSE) from cervid prion protein (amino acids 130-140 [YML]; 163-170 [YRR]; and 171-178[YRR]) fused to phage capsid head protein D (LDP-DSE). Targeted delivery of purified LDP-DSE to intestinal segments induced IgA responses to all three peptide epitopes. Further, delivery of bacteria expressing soluble D-DSE also induced epitope-specific IgA responses in the targeted Peyer's patches. These are the first studies to report use of LDP to induce epitope-specific IgA responses in the small intestine andconfirm Peyer's patchesfunction as a site for LP uptake. Furthermore, IgA responses to peptide epitopes on LDP were observed in the absence of a mucosal adjuvant. These observations confirm LDP have the capacity to function as a mucosal delivery vehicle with protein D as an effective carrier for peptide epitopes.

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

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 ß-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.

Phage Lambda P protein: trans-activation, inhibition phenotypes and their suppression.

The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step.

A CI-independent form of replicative inhibition: turn off of early replication of bacteriophage lambda.

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, p(O) is required for IP, as are iterons ITN3-4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop(+)oriλ(+) sequence.

Dual expression system for assembling phage lambda display particle (LDP) vaccine to porcine Circovirus 2 (PCV2).

The bacteriophage lambda small capsid protein D forms trimers on the phage head. D-fusion polypeptides can be expressed from plasmids in E. coli and remain soluble without aggregation. We report a dual expression system for the display of four immunodominant regions of porcine Circovirus 2 (PCV2) capsid protein (CAP) as D-CAP fusions on lambda display particles (LDP). The LDP-D-CAP preparation proved an effective vaccine in pigs, eliciting both cellular and humoral immune responses and PCV2 neutralizing antibodies. In our dual system wild type D expression was encoded by a heteroimmune infecting phage. The D-fusion protein expression in the infected cells was from an inducible plasmid, enabling the deferral of D-fusion expression until needed. The effective vaccine preparation depended upon the gradient purification of very high concentration, essentially tail-less display particles, not previously described.

NinR- and red-mediated phage-prophage marker rescue recombination in Escherichia coli: recovery of a nonhomologous immlambda DNA segment by infecting lambdaimm434 phages.

We examined the requirement of lambda recombination functions for marker rescue of cryptic prophage genes within the Escherichia coli chromosome. We infected lysogenic host cells with lambdaimm434 phages and selected for recombinant immlambda phages that had exchanged the imm434 region of the infecting phage for the heterologous 2.6-kb immlambda region from the prophage. Phage-encoded activity, provided by either Red or NinR functions, was required for the substitution. Red(-) phages with DeltaNinR, internal NinR deletions of rap-ninH, or orf-ninC were 117-, 12-, and 5-fold reduced for immlambda rescue in a Rec(+) host, suggesting the participation of several NinR activities. RecA was essential for NinR-dependent immlambda rescue, but had slight influence on Red-dependent rescue. The host recombination activities RecBCD, RecJ, and RecQ participated in NinR-dependent recombination while they served to inhibit Red-mediated immlambda rescue. The opposite effects of several host functions toward NinR- and Red-dependent immlambda rescue explains why the independent pathways were not additive in a Rec(+) host and why the NinR-dependent pathway appeared dominant. We measured the influence of the host recombination functions and DnaB on the appearance of orilambda-dependent replication initiation and whether orilambda replication initiation was required for immlambda marker rescue.