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The Bacteriophage Lambda CII Phenotypes For viruses Article The Bacteriophage Lambda CII Phenotypes for Complementation, Cellular Toxicity and Replication Inhibition Are Suppressed in cII-oop Constructs Expressing the Small RNA OOP Karthic Rajamanickam and Sidney Hayes * ID Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; [email protected] * Correspondence: [email protected] Received: 23 January 2018; Accepted: 5 March 2018; Published: 7 March 2018 Abstract: The temperate bacteriophage lambda (λ) CII protein is a positive regulator of transcription from promoter pE, a component of the lysogenic response. The expression of cII was examined in vectors devoid of phage transcription-modulating elements. Their removal enabled evaluating if the expression of the small RNA OOP, on its own, could suppress CII activities, including complementing for a lysogenic response, cell toxicity and causing rapid cellular loss of ColE1 plasmids. The results confirm that OOP RNA expression from the genetic element pO-oop-to can prevent the ability of plasmid-encoded CII to complement for a lysogenic response, suggesting that it serves as a powerful regulatory pivot in λ development. Plasmids with a pO promoter sequence of 45 nucleotides (pO45), containing the −10 and −35 regions for oop, were non-functional; whereas, plasmids with pO94 prevented CII complementation, CII-dependent plasmid loss and suppressed CII toxicity, suggesting the pO promoter has an extended DNA sequence. All three CII activities were eliminated by the deletion of the COOH-terminal 20 amino acids of CII. Host mutations in the hflA locus, in pcnB and in rpoB influenced CII activities. These studies suggest that the COOH-terminal end of CII likely interacts with the β-subunit of RNA polymerase. Keywords: bacteriophage lambda; CII regulation in lysogeny decision; OOP RNA 1. Introduction The regulatory circuits for control of the lysis-lysogeny decision made by bacteriophage λ upon infecting an E. coli host and the key involvement and modulation of CII activity have been extensively reviewed [1–9]. The elevated expression of gene cII, which is rightward and downstream from promoter pR, appears essential for lysogeny. CII serves as a positive transcriptional regulator to stimulate leftward transcription antisense to cro and sense for cI from promoter pE (Figure1A), which is partially embedded within the first eight codons of cII. The enhanced expression of cI, encoding the CI repressor, results in its binding to operator oL (preventing leftward transcription from pL) and to operator oR, thus shutting down sR (rightward mRNA start site)-cro-nutR-tR1-cII-O-P-ren ... tR2-Q transcription arising from pR. CII drives a lysogenic outcome by stimulating the transcription of int from promoter pI and antisense transcription of Q from promoter pAQ. The cII gene is regulated at multiple levels: by repressors CI and Cro binding at oR and limiting transcription from pR and by nutR and the tRI terminator site between cro and cII [1]. CII is degraded by the host ATP-dependent membrane protease FtsH (HflB), which is essential for cell growth [10–12]. This activity is modulated by the HflK-HflC proteins encoded by the hflA locus, which form a complex with FtsH [13], and by HflD [14]. The C-terminal 16 residues of CII are necessary and sufficient for rapid degradation by FtsH Viruses 2018, 10, 115; doi:10.3390/v10030115 www.mdpi.com/journal/viruses Viruses 2018, 10, 115 2 of 18 Viruses 2018, 10, x FOR PEER REVIEW 2 of 18 butFtsH are notbut sufficientare not sufficient for the activationfor the activation of transcription of transcription by CII by [15 CII]. Proteolytic[15]. Proteolytic degradation degradation of CII of by HflB(FtsH)-HflC-HflKCII by HflB(FtsH)-HflC-HflK is inhibited is inhibited by λ gpCIII by λ [ 16gpCIII–18]. [16–18]. At some At level, some the level, small the RNA small OOP RNA serves OOP to targetserves the tocII targetmRNA the for cII degradation mRNA for [degradation19,20], although, [19,20], the althou cleavagegh, the site cleavage for RNaseIII-dependent site for RNaseIII- (and dependent (and -independent) degradation is outside (12 nucleotides downstream) of the coding -independent) degradation is outside (12 nucleotides downstream) of the coding sequence for cII [20]. sequence for cII [20]. While suggesting a mechanism for OOP activity, these important studies While suggesting a mechanism for OOP activity, these important studies measured CII activity via its measured CII activity via its ability to stimulate galactokinase expression from plasmid encoded pE ability to stimulate galactokinase expression from plasmid encoded pE and did not directly measure and did not directly measure CII complementation reflected as limiting a lytic response from CII complementation reflected as limiting a lytic response from infecting phages. We show that OOP infecting phages. We show that OOP synthesis prevents CII-dependent stimulation of pE expression synthesisfrom infecting prevents heteroimmune CII-dependent imm stimulation434 phages, of pE limitingexpression them from to a infecting lytic, rather heteroimmune than lysogenic,imm434 phages,response. limiting them to a lytic, rather than lysogenic, response. FigureFigure 1. 1.λ λimm imm-region-region map map and and synthetic synthetic cII cIIplasmidplasmid expression expression vector. vector. (A) Map (A of) the Map imm of-O the regionimm -O regionof λof showingλ showing immunity immunity region region genes genes rexB—crorexB—cro, several, several promoters promoters (those (those relevant relevant are areshown shown in in orange)orange) and and terminators, terminators,tImm tImm, ,tR1 tR1and andtO tO.. TheThe cI maintenance promoter, promoter, pMpM enablesenables the the expression expression of gene cI, encoding a repressor that binds to operators oL and oR and blocks transcription initiation of gene cI, encoding a repressor that binds to operators oL and oR and blocks transcription initiation from the major leftward and rightward promoters pL and pR. pO is required for transcription of the from the major leftward and rightward promoters pL and pR. pO is required for transcription of the short OOP RNA [21]. The product of gene cII, transcribed from pR, stimulates repressor establishment short OOP RNA [21]. The product of gene cII, transcribed from pR, stimulates repressor establishment transcription from promoter pE. The level of pE transcription can be 30- to 100-fold greater than the transcription from promoter pE. The level of pE transcription can be 30- to 100-fold greater than the level level of cI transcription from promoter pM upon thermal induction of a cro defective prophage [22– of cI transcription from promoter pM upon thermal induction of a cro defective prophage [22–24] or 10- 24] or 10- to 20-fold after λ infection [25]. tImm, terminates repressor establishment transcription from λ tImm to 20-foldpromoters after pM infectionand pE, [preventing25]. , terminatesread-through repressor into the establishment oL/pR site. (B transcription) Plasmid pcIpR-cII-timm from promoters pM(abbreviatedand pE, preventing herein as read-through[cII] in tables) intois a thesyntheticoL/pR cIIsite. expression (B) Plasmid system pcIpR-cII-timm (see Table S1 for (abbreviated related hereinconstructs) as [cII] that in tables) eliminates is a syntheticgene cro, ninRcII expression and the tR1system terminator, (see as Table shown S1 forin the related plasmid constructs) insert (not that eliminatesdrawn to gene scale).cro ,GeneninR expressionand the tR1 fromterminator, the pR promoter as shown is in regulated the plasmid by the insert encoded (not drawntemperature to scale). Genesensitive expression CI [Ts857] from therepressor,pR promoter via binding is regulated to the byoR theoperator encoded sequences. temperature Gene sensitivecII is positioned CI [Ts857] repressor,immediately via binding downstream to the ofoR theoperator WT ribosomal sequences. binding Gene site cII(RBS)is positioned for cro, which immediately perfectly matches/can downstream 0 of thebase WT pair ribosomal with the binding3′ terminal site AUUCCUCCA (RBS) for cro, whichsequence perfectly of 16Smatches/can rRNA [26]. Potential base pair stem-loop with the 3 terminalconfigurations AUUCCUCCA in pR mRNA sequence ahead of of 16S cII rRNAfor the [various26]. Potential plasmid stem-loop constructs configurations used are shown in inpR FiguremRNA ahead2. Sequence of cII for variations the various (1–3) plasmid of pcIpR-cII-timm constructs used enabling are shown cII expression in Figure2 arrangements. Sequence variations are shown (1–3) in of pcIpR-cII-timmFigure S1. The enabling overall organizationcII expression of arrangements genetic elements are on shown plasmid in Figure pcIpR-[…]-timm S1. The overall was organizationdescribed of genetic[27–29],elements where a synthetic on plasmid version pcIpR-[ of tImm... is]-timm inserted was between described the ClaI [27– and29], EcoRI where sites a synthetic to prevent version read- of tImmthroughis inserted of transcription between the arisingClaI andfromEcoRI promotersites pR to. prevent read-through of transcription arising from promoter pR. Viruses 2018, 10, 115 3 of 18 Other aspects of CII function required further study. CII, as expressed from a multicopy plasmid, is highly toxic/lethal to E. coli [30] and this activity is ascribed to its inhibition of host cell DNA replication [31–33]. Localization of the toxic effect to sites within the CII protein have not been determined, nor has localization of the
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