Autoregulation of the Plasmid Addiction Operon of Bacteriophage P1

Magnuson, Roy David; Lehnherr, Hansjörg; Mukhopadhyay, Gauranga; Yarmolinsky, Michael B.

doi:10.1074/jbc.271.31.18705

Cited by 76 publications

(91 citation statements)

References 39 publications

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“…The DNA site bound by Phd is 23 base pairs in length and includes two 10-base pair subsites that are roughly palindromic and separated by 3 base pairs (12) (see Sequence 1). In footprinting experiments, Phd occupies the left operator subsite at roughly 10-fold lower concentrations than the right subsite (12). Studies in vivo show that Phd alone is sufficient to repress transcription from the plasmid addiction operon (12).…”

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confidence: 99%

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Stability and DNA Binding of the Phd Protein of the Phage P1 Plasmid Addiction System

Gazit

Sauer

1999

Journal of Biological Chemistry

123

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The plasmid addiction module of bacteriophage P1 encodes two proteins, Doc, a toxin that is stable to proteolytic degradation, and Phd, the toxin's antidote that is proteolytically unstable. Phd has been shown to autoregulate its expression by specific DNA binding. Here, we investigate the secondary structure and thermal stability of Phd, the effect of operator DNA binding on the structure and stability of Phd, and the stoichiometry, affinity, and cooperativity of Phd binding to operator subsites and intact operator DNA. Phd folds as a monomer at low temperatures or in the presence of osmolytes but exists predominantly in an unfolded conformation at 37°C. The native state of Phd is stabilized by operator binding. Two Phd monomers bind to each operator subsite, and four monomers bind to the intact operator. The subsite binding reaction shows a second-order dependence on protein concentration and monomer-bound DNA species are unpopulated, suggesting that two Phd molecules bind cooperatively to each operator subsite. In intact operator binding experiments, both dimerbound and tetramer-bound DNA species are populated, and binding occurs at protein concentrations similar to those required for subsite binding, suggesting that there is no significant dimer-dimer cooperativity.The stable and efficient maintenance of low-copy plasmids within bacterial cells is ensured, in part, by addiction mechanisms mediated by specific proteins (1, 2). For example, when bacteriophage P1 lysogenizes Escherichia coli as a low copy plasmid, the rate of spontaneous plasmid loss is only about one per 10 5 generations (3). Two proteins comprise the plasmid addiction system of bacteriophage P1: Doc (death on cure), a 126-residue toxic protein, and Phd (prevent host death), a 73-residue antidote (4). This system functions to kill cells that have been cured of the plasmid. The addiction mechanism depends on significant differences in the proteolytic stability of Doc, which is resistant to proteolysis, and Phd, which is degraded in a manner dependent on the host-encoded ClpXP protease complex (5). In P1 lysogens, a concentration of Phd sufficient to suppress Doc toxicity is maintained by a continuous synthesis of Phd molecules de novo. In a bacterial cell that has lost the P1 genome, existing Phd is degraded and because there is no further synthesis of new Phd, levels fall, and the cytoplasmically inherited Doc kills the P1-free daughter cells.Post-segregational killing of plasmid-cured cells is also used by other low-copy number plasmids, including the F (6), RK2 (7), and R1 (8) plasmids of E. coli, and similar systems in Streptomyces lividans and Klebsiella oxytoca (for review, see Refs. 1 and 9). In each case studied, a long-lived toxin and short-lived antidote are part of the addiction mechanism. Interestingly, a functionally similar two-protein module is encoded by the mazE and mazF genes of E. coli (10), which are regulated by the cellular level of ppGpp, an indicator of amino acid starvation. Functional homologues of the Phd and Doc prot...

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confidence: 99%

“…In footprinting experiments, Phd occupies the left operator subsite at roughly 10-fold lower concentrations than the right subsite (12). Studies in vivo show that Phd alone is sufficient to repress transcription from the plasmid addiction operon (12).…”

mentioning

confidence: 99%

Stability and DNA Binding of the Phd Protein of the Phage P1 Plasmid Addiction System

Gazit

Sauer

1999

Journal of Biological Chemistry

123

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“…Phd-Doc and their corresponding genes exhibit characteristic properties of TA systems. The genes encoding Phd and Doc reside in an operon (16), and the antitoxin (Phd, 73 aa/8.1 kDa) and toxin (Doc, 126 aa/13.6 kDa) are relatively small proteins. Two Phd antitoxin polypeptides form a 2:1 heterotrimer with one Doc toxin polypeptide (17) to both inhibit the toxin activity of Doc and autoregulate antitoxin-toxin module transcription (18,19).…”

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confidence: 99%

Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit

Liu¹,

Zhang²,

Inouye³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

119

103

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Bacterial toxin-antitoxin (TA) systems (or ''addiction modules'') typically facilitate cell survival during intervals of stress by inducing a state of reversible growth arrest. However, upon prolonged stress, TA toxin action leads to cell death. TA systems have also been implicated in several clinically important phenomena: biofilm formation, bacterial persistence during antibiotic treatment, and bacterial pathogenesis. TA systems harbored by pathogens also serve as attractive antibiotic targets. To date, the mechanism of action of the majority of known TA toxins has not yet been elucidated. We determined the mode of action of the Doc toxin of the Phd-Doc TA system. Doc expression resulted in rapid cell growth arrest and marked inhibition of translation without significant perturbation of transcription or replication. However, Doc did not cleave mRNA as do other addiction-module toxins whose activities result in translation inhibition. Instead, Doc induction mimicked the effects of treatment with the aminoglycoside antibiotic hygromycin B (HygB): Both Doc and HygB interacted with 30S ribosomal subunits, stabilized polysomes, and resulted in a significant increase in mRNA half-life. HygB also competed with ribosome-bound Doc, whereas HygB-resistant mutants suppressed Doc toxicity, suggesting that the Doc-binding site includes that of HygB (i.e., helix 44 region of 16S rRNA containing the A, P, and E sites). Overall, our results illuminate an intracellular target and mechanism of TA toxin action drawn from aminoglycoside antibiotics: Doc toxicity is the result of inhibition of translation elongation, possibly at the translocation step, through its interaction with the 30S ribosomal subunit.antitoxin ͉ hygromycin B ͉ bacteriophage P1 ͉ 16S rRNA ͉ postsegregational killing

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“…In E. coli, some extrachromosomal elements are known to contain addiction modules causing the bacterial programmed cell death by the so-called postsegregational killing effect. The most studied extrachromosomal addiction modules are the phd-doc system on bacteriophage P1 (2)(3)(4)(5), the ccdA-ccdB system on factor F (6 -9), the kis-kid system on plasmid R1 (10 -13), and the pemIpemK system on plasmid R100 (14 -17). Interestingly, the E. coli chromosome also contains several addiction module systems, such as the relBE system (18 -21), the mazEF system (22)(23)(24)(25), and the chpB system (26 -28).…”

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Interference of mRNA Function by Sequence-specific Endoribonuclease PemK

Zhang

Zhu

et al. 2004

Journal of Biological Chemistry

122

149

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In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin. The pemI-pemK system is an addiction module present on plasmid R100. It helps to maintain the plasmid by post-segregational killing in E. coli population. Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E. coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis. Further studies reveal that PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the endoribonuclease activity of PemK. PemK cleaves only single-stranded RNA preferentially at the 5 or 3 side of the A residue in the "UAH" sequences (where H is C, A, or U). Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E. coli. The pemK homologue genes have been identified on the genomes of a wide range of bacteria. We propose that PemK and its homologues form a novel endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.In Escherichia coli, programmed cell death is proposed to be mediated through the system called "addiction module," which consists of a pair of genes encoding a toxin and an antitoxin (1). The addiction module has the following properties. (a) The toxic protein is stable, whereas the antitoxin is a labile protein. (b) The toxin and the antitoxin are coexpressed from an operon and interact with each other to form a stable complex. (c) Their expression is auto-regulated either by the toxin-antitoxin complex or by the antitoxin alone. When the co-expression is inhibited under stress conditions, the antitoxin is degraded by proteases, enabling the toxin to act on its target. In E. coli, some extrachromosomal elements are known to contain addiction modules causing the bacterial programmed cell death by the so-called postsegregational killing effect. The most studied extrachromosomal addiction modules are the phd-doc system on bacteriophage P1 (2-5), the ccdA-ccdB system on factor F (6 -9), the kis-kid system on plasmid R1 (10 -13), and the pemIpemK system on plasmid R100 (14 -17). Interestingly, the E. coli chromosome also contains several addiction module systems, such as the relBE system (18 -21), the mazEF system (22-25), and the chpB system (26 -28).The cellular effects of the toxins in the addiction modules have been studied quite extensively. CcdB, the toxin in the ccdA-ccdB system, interacts with DNA gyrase to block DNA replication (7,29), and RelE, the toxin in the relBE system, is not able to degrade free RNA but cleaves mRNA in the ribosome A site with high codon specificity (21). Recently, it was demonstrated that the A-site mRNA cleavage can occur in the absence of RelE (30). The exact mechanism of the A-site mRNA cleavage is still unknown. It has been proposed that MazF (ChpAK)...

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Autoregulation of the Plasmid Addiction Operon of Bacteriophage P1

Cited by 76 publications

References 39 publications

Stability and DNA Binding of the Phd Protein of the Phage P1 Plasmid Addiction System

Stability and DNA Binding of the Phd Protein of the Phage P1 Plasmid Addiction System

Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit

Interference of mRNA Function by Sequence-specific Endoribonuclease PemK

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