Identification of the ssDNA-binding protein of bacteriophage T5

Werten, Sebastiaan

doi:10.4161/bact.27304

Cited by 9 publications

(7 citation statements)

References 27 publications

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“…Several ArV2 gene products are potentially involved in nucleic acid replication or recombination processes. ORF54 was predicted to encode a ssDNA binding protein, which builds the scaffold of DNA replication and is reported to be essential both for DNA replication and for genetic recombination processes [48] . The protein encoded by ArV2 ORF61 shared 42% amino acid identity with the DNA helicase from Streptomyces sp.…”

Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of vB_ArS-ArV2 – First Arthrobacter sp. Infecting Bacteriophage with Completely Sequenced Genome

et al. 2014

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This is the first report on a complete genome sequence and biological characterization of the phage that infects Arthrobacter. A novel virus vB_ArS-ArV2 (ArV2) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. Based on transmission electron microscopy, ArV2 belongs to the family Siphoviridae and has an isometric head (∼63 nm in diameter) with a non-contractile flexible tail (∼194×10 nm) and six short tail fibers. ArV2 possesses a linear, double-stranded DNA genome (37,372 bp) with a G+C content of 62.73%. The genome contains 68 ORFs yet encodes no tRNA genes. A total of 28 ArV2 ORFs have no known functions and lack any reliable database matches. Proteomic analysis led to the experimental identification of 14 virion proteins, including 9 that were predicted by bioinformatics approaches. Comparative phylogenetic analysis, based on the amino acid sequence alignment of conserved proteins, set ArV2 apart from other siphoviruses. The data presented here will help to advance our understanding of Arthrobacter phage population and will extend our knowledge about the interaction between this particular host and its phages.

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Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of vB_ArS-ArV2 – First Arthrobacter sp. Infecting Bacteriophage with Completely Sequenced Genome

et al. 2014

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“…Among these is AcrIIA22’s closest structural relative in the PC4-like family: a predicted single-stranded binding (SSB) protein from phage T5 ( Fig 4E ) [ 28 ]. This putative SSB protein has been predicted to directly stimulate recombination during the recombination-dependent replication of phage T5’s genome [ 29 ]. This prediction, together with our inference from genomic analyses ( Fig 2 and S4 Fig ), led us to hypothesize that AcrIIA22 may have similar recombination-stimulating activity.…”

Section: Resultsmentioning

confidence: 99%

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

et al. 2021

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To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements (MGEs) encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic acid–binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and MGEs.

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“…Among these is AcrIIA22’s closest structural relative in the PC4 family: a predicted single-stranded binding (SSB) protein from phage T5 (Figure 4B) 26 . This putative SSB protein has been predicted to directly stimulate recombination during the recombination-dependent replication of phage T5’s genome 27 . This prediction, together with our inference from genomic analyses (Figure 2), led us to hypothesize that AcrIIA22 may have similar recombination-stimulating activity.…”

Section: Resultsmentioning

confidence: 99%

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

Forsberg

Schmidtke

Werther

et al. 2020

Preprint

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To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all mechanistically characterized Acrs directly bind their cognate Cas protein to inactivate CRISPR immunity. Here, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of Clostridial bacteria and their prophages from the human gut microbiome. Uncovered in a functional metagenomic selection, AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic-acid binding proteins. This homology helped us deduce that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids, rendering them less susceptible to SpyCas9, which is highly dependent on negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and mobile genetic elements.

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Identification of the ssDNA-binding protein of bacteriophage T5

Cited by 9 publications

References 27 publications

Isolation and Characterization of vB_ArS-ArV2 – First Arthrobacter sp. Infecting Bacteriophage with Completely Sequenced Genome

Isolation and Characterization of vB_ArS-ArV2 – First Arthrobacter sp. Infecting Bacteriophage with Completely Sequenced Genome

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

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