Genetic Dissection of T4 Lysis

Moussa, Samir H.; Lawler, Jessica L.; Young, Ry

doi:10.1128/jb.01548-14

Cited by 17 publications

(17 citation statements)

References 30 publications

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“…More-recent studies on T and RI have confirmed aspects of this operational model for LIN and provided molecular details for the lysis pathway of T4 (14)(15)(16). Like other holins, including the wellstudied S105 holin of phage lambda, the T holin accumulates harmlessly in the host IM until it suddenly forms lethal, micrometer-scale membrane lesions at an allele-specific time.…”

mentioning

confidence: 80%

“…Since RIII has no membrane or export signals (Fig. 4A), the only possible target for RIII is the N-terminal cytoplasmic domain of T (nT), which has 34 aa and is required for holin function (15). To test this idea, we used the bacterial twohybrid system, based on intragenic complementation of CyaA function (34).…”

Section: Resultsmentioning

confidence: 99%

“…To address the role of RIII in LIN, we used a convenient system based on the inducible lambda prophage -t, in which the holin gene S is replaced by T4 t (14). Not only was this hybrid phage previously shown to recapitulate T4 lysis timing and LIN at physiological levels of expression; it allows the coexpression of selected T4 genes cloned in inducible plasmid vectors without the confounding ef- fects of T4-mediated host DNA degradation and translational repression (14,15,21,39). This system mimics the T-dependent lysis in the context, where effects of T4 genes other than t are excluded.…”

Section: Resultsmentioning

confidence: 99%

“…All plasmids used in this study are listed in Table 1. Isolation of plasmid DNA, DNA amplification by PCR, DNA transformation, and DNA sequencing were performed as described previously (15,22,29). The DNA sequences of oligonucleotides (primers) are listed in Table 2.…”

Section: Methodsmentioning

confidence: 99%

“…However, if the T4-infected cells are superinfected by other T4 (or T-even phages) after the first 5 min of the infection cycle, LIN is imposed (18). There has been progress on the molecular basis of LIN (15,(19)(20)(21). While most holins have two or more transmembrane domains (TMDs) and only short soluble loops connecting them (13), the T holin has a unique structure, with only a single TMD and significant N-terminal (34-amino-acid [34-aa]) and C-termi- …”

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confidence: 99%

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The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin

Chen

Young

2016

J Bacteriol

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The latent period of phage T4, normally ϳ25 min, can be extended indefinitely if the infected cell is superinfected after 5 min. This phenomenon, designated lysis inhibition (LIN), was first described in the 1940s and is genetically defined by mutations in diverse T4 r genes. RI, the main effector of LIN, has been shown to be secreted to the periplasm, where, upon activation by superinfection with a T-even virion, it binds to the C-terminal periplasmic domain of the T4 holin T and blocks its lethal permeabilization of the cytoplasmic membrane. Another r locus, rIII, has been the subject of conflicting reports. In this study, we show that RIII, an 82-amino-acid protein, is also required for LIN in both Escherichia coli B strains and E. coli K-12 strains. In T4⌬rIII infections, LIN was briefly established but was unstable. The overexpression of a cloned rIII gene alone impeded T-mediated lysis temporarily. However, coexpression of rIII and rI resulted in a stable LIN state. Bacterial two-hybrid assays and pulldown assays showed that RIII interacts with the cytoplasmic N terminus of T, which is a critical domain for holin function. We conclude that RIII is a T4 antiholin that blocks membrane hole formation by interacting directly with the holin. Accordingly, we propose an augmented model for T4 LIN that involves the stabilization of a complex of three proteins in two compartments of the cell: RI interacting with the C terminus of T in the periplasm and RIII interacting with the N terminus of T in the cytoplasm. IMPORTANCELysis inhibition is a unique feature of phage T4 in response to environmental conditions, effected by the antiholin RI, which binds to the periplasmic domain of the T holin and blocks its hole-forming function. Here we report that the T4 gene rIII encodes a cytoplasmic antiholin that, together with the main antiholin, RI, inhibits holin T by forming a complex of three proteins spanning two cell compartments. The r genes of the T-even phages, first identified by laboratories of the Phage Group in the 1940s (1, 2), have a special place in the history of molecular biology. Detailed studies of the first three loci discovered-rI, rIIAB, and rIII-were foundational in working out the fundamentals of inheritance, genetic code, mutation, recombination, DNA repair, and gene structure (3-7). These mutable loci were originally discovered by their distinctive plaque morphology: large, clear, sharply defined plaques, easily distinguished from the small, fuzzy-edged, turbid plaques of the parental phages (1). The "r" designation meant "rapid lysis," which refers to the observation that the mutant phages isolated from the r-type plaques caused rapid, culture-wide lysis at ϳ25 min after infection, whereas cultures infected with the parental phages continued to increase in mass and accumulate progeny virions intracellularly for hours, in a state called "lysis inhibition"(LIN) (8). In the ensuing decades, more loci were classified as r genes based on mutant plaque phenotypes; at one point, r genes numbering u...

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confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin

Chen

Young

2016

J Bacteriol

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Bacteriophage T4

Mathews

2015

Encyclopedia of Life Sciences

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Bacteriophage T4 and its host bacterium, Escherichia coli , can be considered among the earliest model organisms – biological systems that attract large numbers of investigators who, because of technical or conceptual advantages of the system, use the system to investigate processes and mechanisms of general significance in biology. Beginning in the mid‐twentieth century, studies focused on bacteriophage T4 revealed essential features of the molecular nature of genes and genomes, mechanism and fidelity of DNA replication, genetic recombination, DNA repair, control of gene expression, genome organisation, assembly of complex macromolecular structures and pre‐emption of cell metabolism by virus infection. Although much of the molecular biology research community has moved on to eukaryotic model organisms, such as yeast, nematode worms, fruitflies, the plant Arabidopsis , zebrafish and mice, bacteriophage T4 still presents the best opportunities for understanding at the molecular level DNA replication and recombination, and macromolecular assembly. In addition, T4 and related phages are being used in investigations of bacteriophage therapy. Finally, recent analyses of DNA packaging and morphogenesis have suggested applications of T4 as a gene delivery vehicle. Key Concepts Bacteriophage T4 presents numerous technical advantages as a model for studying virus reproduction or gene organisation and expression. Bacteriophage T4 is representative of a large group of closely related phages found in varied environments worldwide. Bacteriophage T4 is a virulent phage, which always lyses and kills its host bacterium. The T4 virion has a complex multiprotein structure with a contractile tail that serves both for adsorption to host cells and for intracellular delivery of the viral genome. Bacteriophage T4 contains a large, linear double‐stranded DNA genome, with chemical modifications of its cytosine residues. The T4 genome is circularly permuted and terminally redundant with respect to base sequence; these features protect against information loss during replication of a linear DNA. The T4 genome encodes numerous enzymes, used to support replication of the viral genome and to synthesise deoxyribonucleotides to support the enormous rate of DNA accumulation in infected cells. T4 uses host cell RNA polymerase for transcription of its own genes, but it modifies the bacterial enzyme, both to prevent its transcription of bacterial genes after infection and to contribute towards a timed viral gene transcription program. T4 morphogenesis involves separate subassembly pathways for viral heads, tails, tail baseplates and fibres, with the substructures assembling spontaneously.

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First complete genome sequence of a virulent bacteriophage infecting the opportunistic pathogen Serratia rubidaea

Xing

Zhang

et al. 2017

Arch Virol

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A Serratia rubidaea phage, vB_Sru IME250, was isolated from hospital sewage. The morphology suggested that phage vB_Sru IME250 should be classified as a member of the family Myoviridae. High-throughput sequencing revealed that the phage genome has 154,938 nucleotides and consists of 193 coding DNA sequences, 90 of which have putative functions. The genome of vB_Sru IME250 is a linear, double-stranded DNA with an average GC content of 40.04%. The phage has a relatively high similarity to Klebsiella phage 0507-KN2-1, with a genome coverage of 84%.

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Genetic Dissection of T4 Lysis

Cited by 17 publications

References 30 publications

The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin

The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin

Bacteriophage T4

First complete genome sequence of a virulent bacteriophage infecting the opportunistic pathogen Serratia rubidaea

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