A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

Abstract

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, pO 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.

Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

“…594[pcIpR- P Δ76 -timm] transformants were unable to complement for P , suggesting that the region of P removed by the in-frame deletion of 76 codons, localized to the N-teminal end of the 233 amino acid P protein (fusing part of codon 9 with 86) is essential for the role of P in λ replication. The immune state, i.e., imm λ[Ts] phenotype, of the pcIpR-[ ]-timm plasmids was examined (Table 5, and [36]) by plating λ cI 72 on hosts with plasmids encoding intact P or O genes.…”

“…Plasmid p434’pR- O -timm has a hybrid cI gene fusion made from λ and 434 cI genes and the resulting repressor does not repress transcription from pR , allowing constitutive O expression at all temperatures [36]. …”

“…Plasmid pcIpR- O -timm confers high imm λ immunity in cells grown between 25 and 39 ˚C, and loses it at 42 ˚C where the CI[Ts]857 repressor is fully inactive. Additional immune characterization of these plasmids was reported (Table 4 in [36]). Although pcIpR- O -timm only carries oR operators for repressor binding, CI immune repression is maintained through 39 ˚C, but lost at 42 ˚C where the CI repressor is denatured.…”

“…Table 7 shows the influence of expression of lambda gene O from plasmids pcIpR- O -timm and p434’pR- O -timm on plasmid loss at 30, 35, 37, 39 and 42 ˚C. The immunity properties of these two plasmids was shown in Table 4 of [36]. The expression of gene O is constitutive at all temperatures from plasmid p434’pR- O -timm.…”

Bacteriophage lambda: Early pioneer and still relevant

“…Thus, the early study of lambda replication contributed greatly to an understanding of the detailed steps required to accomplish controlled initiation. In addition, the origin is activated by rightward transcription from P R , but this phenomenon remains poorly understood (Leng and McMacken, 2002; Hayes et al , 2012; Olszewski et al , 2014). In some other lambdoid phages like P22, the P gene is replaced by a gene that encodes an actual DnaB type helicase (Wickner, 1984a and 1984b), and phages APSE-1 and N15 encode a putative DNA polymerase (van der Wilk et al , 1999) or a bifunctional helicase-primase (Ravin et al , 2000; Mardanov and Ravin, 2006), respectively.…”