Identification of three cytotoxic early proteins of mycobacteriophage L5 leading to growth inhibition in Mycobacterium smegmatis

Rybniker, Jan; Plum, Georg; Robinson, Nirmal; Small, P. L. C.; Hartmann, Pia

doi:10.1099/mic.0.2008/017004-0

Cited by 30 publications

(25 citation statements)

References 26 publications

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“…We note that gp45 (encoding a single-stranded DNA [ssDNA] annealing protein, SSAP), gp60 (a recombination endonuclease VII), and gp72 (a RecB family exonuclease) are functional genes of homologous recombination. Genes 84, 85, and 86 are highly similar to genes encoding three cytotoxic early proteins of L5 (11). Phage-associated depolymerases have not been identified in the SWU1 genome.…”

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confidence: 83%

Biology of a Novel Mycobacteriophage, SWU1, Isolated from Chinese Soil as Revealed by Genomic Characteristics

Fan

Teng

Wang

et al. 2012

J Virol

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bMycobacteriophage SWU1 is a newly isolated phage from a soil sample collected at Gongping village, Pingchang County, Sichuan Province, China, using Mycobacterium smegmatis mc 2 155 as a host. Plaques of SWU1 appear as a unique bull's-eye on an M. smegmatis lawn. In this paper, we report the complete genome sequence of SWU1 and some major findings from the analysis result. Bacteriophages represent unprecedented bioresources and an indispensable quarry for novel genetic tools. This holds true for mycobacteriophages, a magic toolkit for Mycobacterium molecular manipulation and tuberculosis control (7-10, 13, 14). However, most mycobacteriophages are isolated from limited regions, and many prophages and the CRISPR spacers within mycobacterial genomes fail to find matching phages (4), suggesting that more work on the isolation and description of mycobacteriophages remains necessary.Using Mycobacterium smegmatis mc 2 155 as the host organism, a novel mycobacteriophage (SWU1) was isolated from a soil sample collected at Sichuan Province, China. Plaques of SWU1 appear a characteristic bull's-eye with a halo, which is thought an indicator for the appearance of phage-associated depolymerases. In order to understand the formation mechanism of halo, we sequenced and analyzed the genome of SWU1.We used phenol to break up SWU1 particles and isolate the DNA. Purified phage DNA was sheared with a HydroShear DNAfragmenting system (Gene Machines). The 1.5-to-3-kb fragmented DNAs were purified, repaired, and cloned into PUC118 to create a shotgun library. The resulting ligation mixture was used to transform Escherichia coli DH10B. Recovered plasmid was sequenced on an ABI 3730XL DNA Sequencer (Applied Biosystem) by GBI in China (approximately 4.8ϫ coverage). Predicted genes were subjected to database searches using Glimmer software (3). The putative functions of the open reading frames (ORFs) were determined by BLASTP searches (1). The use of a previously constructed L5 codon usage table assisted the identification of SWU1 predicted genes (6).The SWU1 genome was found to be 52,474 bp in length with 94 putative protein-coding genes and 3 tRNA genes with a GϩC content of 62.4%. SWU1 belongs to cluster A2 using the previously stated criteria (5). Among those genes, 91 have sequence similarity to other genes and 22 have been assigned functions, although 3 fail to match any genes. Many of the genes (genes 6, 13, 14, 16, 17, 23, 26, 27, and 28) are presumably involved in phage structure and assembly. Several genes (genes 44, 49, and 50) might encode proteins involved in phage DNA metabolism. Genes 10, 11, and 12 encode LysA, Holin, and LysB, which are components of the lytic cassette of SWU1. The phage integrase (gp33), the phage attachment site (bp 25,583 to 25,625), and the excisionase (gp36) form an integration cassette. We note that gp45 (encoding a single-stranded DNA [ssDNA] annealing protein, SSAP), gp60 (a recombination endonuclease VII), and gp72 (a RecB family exonuclease) are functional genes of homologous recombination. ...

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confidence: 83%

Biology of a Novel Mycobacteriophage, SWU1, Isolated from Chinese Soil as Revealed by Genomic Characteristics

Fan

Teng

Wang

et al. 2012

J Virol

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“…The protein displays significant similarity to the phage protein gp79 having inhibitory effect on host transcription [23] or having cytotoxic effect on bacterial host [24]. These characters might be useful for development of phage therapy or study of potential drug targets against bacterial pathogens.…”

Section: Figure1 Bacteriophages Isolated From Cultures Of Facultativmentioning

confidence: 99%

Proteomic and Electron Microscopy Study of Bacteriophages From Bartonella Henselae And Bartonella Grahamii

Nahálková¹,

Ml²

2014

IJVSR

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“…Cloning of pQE80::77 and pQE80::79 was performed as described recently [7]. Primers for amplifying gene 77 for subsequent cloning in pET29b were 5′-GGAGATATACATATGATCGACGGCAAGACG-3′ and 5′-GTGCGGCCGCAAGCTTCAGCCCGCCAGCGC-3′.…”

Section: Dna Manipulation and Cloningmentioning

confidence: 99%

“…Recently, we have focussed on a set of early proteins of mycobacteriophage L5 (gp77, 78 and 79) which are cytotoxic for the host Mycobacterium smegmatis upon expression from an inducible mycobacterial shuttle vector and from a newly identified L5 promoter. It is most likely that these proteins function as shut-off proteins during the early stages of phage propagation [7]. Mycobacteriophage L5 is a temperate phage with a broad host range including pathogens of clinical relevance such as Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium ulcerans [8].…”

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The cytotoxic early protein 77 of mycobacteriophage L5 interacts with MSMEG_3532, an L‐serine dehydratase of Mycobacterium smegmatis

Rybniker

Krumbach

Gumpel

et al. 2011

J. Basic Microbiol.

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Mycobacteriophage L5 is a temperate phage infecting a broad range of mycobacterial species. Upon induction of lytic growth, L5 rapidly switches off host protein synthesis. We have recently identified the mycobacteriophage L5 early protein gp77 as a host shut-off protein that acts growth inhibitory in the mycobacterial host when expressed through the corresponding phage promoter. Here we present data showing that this purified phage protein of unknown function specifically binds to protein MSMEG_3532 when incubated with cell lysates of Mycobacterium smegmatis. This interaction was confirmed by pull-down assays using purified MSMEG_3532 as bait which co-purified with gp77. The amino acid sequence of MSMEG_3532 is nearly identical to that of threonine dehydratases, serine dehydratases and an L-threo-3-hydroxyaspartate dehydratase. An enzymatic assay identified this host protein as a pyridoxal-5'-phosphate-dependent L-serine dehydratase (SdhA) which converts L-serine to pyruvate. This is the first biochemical characterization of a SdhA derived from mycobacteria. Though the addition of purified gp77 to the established in vitro assay had no influence on SdhA activity at a saturating L-serine concentration, the specific interaction of phage protein and dehydratase in vivo may well have a role in altering the amino acid pool or the products of amino acid metabolism in favour of phage maturation.

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Identification of three cytotoxic early proteins of mycobacteriophage L5 leading to growth inhibition in Mycobacterium smegmatis

Cited by 30 publications

References 26 publications

Biology of a Novel Mycobacteriophage, SWU1, Isolated from Chinese Soil as Revealed by Genomic Characteristics

Biology of a Novel Mycobacteriophage, SWU1, Isolated from Chinese Soil as Revealed by Genomic Characteristics

Proteomic and Electron Microscopy Study of Bacteriophages From Bartonella Henselae And Bartonella Grahamii

The cytotoxic early protein 77 of mycobacteriophage L5 interacts with MSMEG_3532, an L‐serine dehydratase of Mycobacterium smegmatis

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