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, 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

Averaged EOP on host cells +/− plasmids with cloned <i>O</i> gene^a.

a<p>The average EOP per indicated phage was relative to that phage plating on strain 594 cells. The standard errors were all <0.05.</p>b<p>The precise <i>O</i> sequence (ATG = 38686–39582 plus TAA stop codon that replaces normal TGA stop at end of <i>O</i>) was cloned to make plasmids pcIpR-<i>O-</i>timm and p434′pR-<i>O</i>-timm. In each plasmid, gene <i>O</i> occupies the position corresponding to λ gene <i>cro</i> (in phage) and the consensus Shine Dalgarno sequence for <i>cro</i> was maintained ahead of <i>O</i> in pcIpR-<i>O-</i>timm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Espacenet1" target="_blank">[75]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Hayes14" target="_blank">[76]</a>. In p434′pR-<i>O</i>-timm, the SD differed by one bp compared to the SD in pcIpR-<i>O-</i>timm because of the slightly different sequence ahead of <i>cro</i> in <i>imm</i>434 DNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Grosschedl2" target="_blank">[77]</a>. The <i>O</i> gene within pcIpR-<i>O-</i>timm is transcribed from <i>p_R</i> and regulated by <i>cI</i>[Ts]857 repressor: at 30° <i>O</i> is repressed, at 39° and 42° <i>O</i> is expressed, or fully expressed. Gene <i>O</i> is constitutively expressed from <i>p_R</i> in p434′pR-<i>O</i>-timm.</p>c<p>The column for plating at 30°, 39° and 42° yielded equivalent phage titers on 594 and the EOP was set to 1. Plaques ranged between 0.5–2 mm in diameter.</p>d<p>Plaques formed were tiny.</p>e<p>Plaques ranged from 0.3–1 mm diameter.</p>f<p>nd is not done, since equivalent results were expected as seen for λ<i>cI</i>72.</p

Thermal Induction of <i>rep</i>λ or the <i>rep</i>P22 –hybrid λ<i>cI</i>857 prophages.

<p>Lysogenic cultures of strain 594 were grown at 30° and each prophage was thermally induced by shifting the culture from 30° to 42° at time 0. A. Thermally induced <i>rep</i>P22 prophage. B. Thermally induced <i>rep</i>λ prophage. The results represent the averages for 2 independent assays. Plasmids within lysogenic cells: square, <i>Po</i>⁺<i>oop</i>⁺<i>ori</i>⁺ (results shown for p27R, but identical results were observed for p27); triangle, <i>Po</i>− <i>oop</i>⁺<i>ori</i>⁺; inverted triangle, Δ (<i>to-oop-Po</i>) <i>ori</i>λ⁺ (ITN-AT⁺); diamond, <i>cII</i>-<i>oop-Po</i>⁺ Δ<i>ori</i>λ; circle, none (no plasmid). The standard deviation is shown for all of the data points, but is too small for visualization in some data intervals.</p

Replication-targeted inhibition of <i>rep</i>λ phage plating.

<p>A. Plasmid cloned λ DNA fragments used to map the sequence requirement(s) for an inhibition phenotype (IP). B. Genomic region spanning five contiguous and partially homologous genes of phages λ and P22 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498.s002" target="_blank">Fig. S2</a>). Phage λ is naturally missing the <i>orf48</i> gene between <i>oop</i> and <i>O</i> that is present between <i>oop</i> and <i>18</i> in P22 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Horbay1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Horbay2" target="_blank">[51]</a>. C. Assay for EOP, defined as phage titer on strain 594 (with one of the indicated plasmids) / titer on 594 cells, where plating on 594  =  EOP of 1.0. All of the plasmids shown were derived from pBR322. The <i>oop⁺ ori</i>λ⁺ plasmid used was p27. The DNA substitution of the “<i>ice</i>” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Lusky1" target="_blank">[16]</a> sequence of λ to make plasmid Δ<i>ice oop</i>⁺<i>ori</i>λ⁺ ( =  p50) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498.s003" target="_blank">Fig. S3A</a>. Numbers in brackets represent standard error values.</p

<i>E. coli</i> K12 and Bacteriophage λ Strains.

a<p>The λ prophage genes <i>int-xis-exo-bet-gam-kil</i> in strain Y836 were substituted with <i>bio</i>275 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Hayes3" target="_blank">[13]</a>. The strain carries the chromosomal deletion Δ431<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Hayes7" target="_blank">[33]</a> that removes genes rightward from ninB in prophage through <i>moaA</i> in host, including prophage genes <i>orf146</i> (<i>orf</i>) – <i>J</i>b2 (<i>i.e.</i>, all the late genes required for cell lysis and phage morphogenesis). A map of the cryptic lambda prophage in strain Y836 is drawn in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone-0036498-g004" target="_blank">Fig. 4A</a>.</p>b<p>Lysogenic strains show the prophage within the cell by “( )” bordering the prophage.</p>c<p>The NinR region deleted by Δnin5 removes λ bases 40,503–43,307, i.e., <i>ren-ninA – ninI</i> (including <i>orf -ninC</i> and <i>rap-ninH</i> ); the NinL region substituted by <i>bio</i>275 replaces genes <i>int-xis-hin-exo-bet-gam-kil</i>, representing λ bases 27,731–∼33,303 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Hayes5" target="_blank">[18]</a>.</p

<i>ori</i>λ-dependent DNA replication inhibition is bypassed in multiply infected cells.

a<p>Burst at 110 min after infecting cells. The results are expressed as phage burst (# phage particles released per infective center) +/− standard error. Each value represents the average of ≥2 separate trials.</p>b<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036498#pone.0036498-Taylor1" target="_blank">[78]</a> for host requirements for growth of λ-P22 hybrid.</p

Primers used for plasmid modification.

a<p>L and R primer sequences are from the lambda <i>l</i>-strand (coding strand for <i>cII-O</i>) and <i>r</i>-strand (coding strand for <i>cI</i> and <i>oop</i>) sequences, respectively.</p

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

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