Isolation of a Mutant Bacteriophage T7 Deleted in Nonessential Genetic Elements, Gene19.5andm

Kim, Soo-Hyoung; Chung, Yeon-Bo

doi:10.1006/viro.1996.0030

Cited by 5 publications

(3 citation statements)

References 6 publications

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“…However, a potential role in lysis is not entirely unexpected. A phage with a deletion of gene 19.5 (along with the adjacent M-hairpin) lyses late and mutant phages make small plaques ( Kim and Chung 1996 ), although much of this effect may be caused by the deletion of the hairpin ( Kim et al 1997 ).…”

Section: Resultsmentioning

confidence: 99%

“…There is no function assigned to this gene, but it is predicted to encode a short, membrane-spanning protein (by TMHMM; Krogh et al 2001 ; data not shown). A deletion mutant lacking gene 19.5 lyses late ( Kim and Chung 1996 ), although the observed effect on lysis can perhaps be largely explained by the deletion also removing a nearby hairpin. However, Kim et al (1997) have suggested that gene 19.5 acts to generate the hairpin, and if the hairpin triggers holin activity, 19.…”

Section: Discussionmentioning

confidence: 99%

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Layers of Evolvability in a Bacteriophage Life History Trait

Heineman

Bull

Molineux

2009

Molecular Biology and Evolution

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Functional redundancy in genomes arises from genes with overlapping functions, allowing phenotypes to persist after gene knockouts. Evolutionary redundancy or evolvability of a genome is one step removed, in that functional redundancy is absent but the genome has the potential to evolve to restore a lost phenotype. Exploring the extent to which this recovery alters gene networks can illuminate how functional gene interactions change through time. Here, the evolvability of lysis was studied in bacteriophage T7, revealing hidden functional interactions. Lysis is the destruction of host cell wall and membranes that releases progeny and is therefore essential for phage propagation. In most phages, lysis is mediated by a two-component genetic module: a muralytic enzyme that degrades the bacterial cell wall (endolysin) and a holin that permeabilizes the inner membrane to allow the endolysin access to the cell wall. T7 carries one known holin, one endolysin, and a second muralytic enzyme that plays little role in lysis by wild-type phage. If the primary endolysin is deleted, the second muralytic enzyme evolves to restore normal lysis after selection for faster growth. Here, a second level of evolutionary redundancy was revealed. When the second muralytic enzyme was prevented from adapting in a genome lacking the primary endolysin, the phage reevolved lysis de novo in the absence of any known muralytic enzymes by changes in multiple genes outside the original lysis module. This second level of redundancy proved to be evolutionarily inferior to the first, and both result in a lower fitness and slower lysis than wild-type T7. Deletion of the holin gene delayed lysis time modestly; fitness was restored by compensatory substitutions in genes that lack known roles in lysis of the wild type.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Layers of Evolvability in a Bacteriophage Life History Trait

Heineman

Bull

Molineux

2009

Molecular Biology and Evolution

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“…It is well known that up to 50% of the genes of extensively studied bacteriophages such as T7 are non‐essential for plaque formation. These genes, although dispensable for phage growth under laboratory conditions, bear meaningful functions and some of them are known to interact with and inhibit host proteins (Kim and Chung, 1996).…”

Section: Discussionmentioning

confidence: 99%

Insights into the function of the WhiB‐like protein of mycobacteriophage TM4 – a transcriptional inhibitor of WhiB2

Rybniker¹,

Nowag²,

Gumpel³

et al. 2010

Molecular Microbiology

108

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SummaryWhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB-like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apoWhiB2 of Mycobacterium tuberculosis and apoWhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability.

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Defects in Concatemer Processing of Bacteriophage T7 DNA Deleted in the M-Hairpin Region

Kim

Chung

1997

Virology

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The intracellular replicating form of T7 DNA is a concatemer in which linear genomes are joined head to tail by sharing 160-bp terminally repeated sequences. A unique hairpin (M-hairpin) end generated on the left side of the TR was proposed to be responsible for the duplication of the concatemer junction for efficient packaging. We characterized the defects caused by loss of the M-hairpin by constructing a recombinant T7 (T7Deltam) deleted in the m region. Initially, the intracellular growth rate of progeny phage was normal in T7Deltam infection. However, the titer of progeny phage was eventually reduced by two- to threefold and lysis was significantly delayed. The restriction fragment, LEDelta160, generated simultaneously with the double-strand cleavage at the onset of packaging reaction was found more or less at the same intensity in both T7(+) and T7Deltam infection at the beginning but preferentially in T7Deltam infection during the later phase of infection. These observations suggest that the DNA packaging of T7 proceeds on the intact concatemer junctions during the early stage of infection while the duplication of the concatemer junction by the M-hairpin seemed to be important during the later phase, presumably due to reduced replication. While the generation of the M-hairpin involves DNA replication, the loss of m did not reduce DNA synthesis, suggesting that the role of the M-hairpin as an origin of replication is minimal.

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Isolation of a Mutant Bacteriophage T7 Deleted in Nonessential Genetic Elements, Gene19.5andm

Cited by 5 publications

References 6 publications

Layers of Evolvability in a Bacteriophage Life History Trait

Layers of Evolvability in a Bacteriophage Life History Trait

Insights into the function of the WhiB‐like protein of mycobacteriophage TM4 – a transcriptional inhibitor of WhiB2

Defects in Concatemer Processing of Bacteriophage T7 DNA Deleted in the M-Hairpin Region

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