The autoregulator Aca2 mediates anti-CRISPR repression

Birkholz, Nils; Fagerlund, Robert D.; Smith, Lisa F.; Jackson, Simon A.; Fineran, Peter C.

doi:10.1093/nar/gkz721

Cited by 60 publications

(46 citation statements)

References 33 publications

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“…5 ). For example, we found a number of sequenced Pectobacterium genomes with integrated prophages that have similarities with Pectobacterium phage ZF40 (a phage carrying the acrIF8-aca2 locus) 18 , 20 , but in the remaining cases the prophage regions encoded different combinations of acr (s), together with aca5 (Fig. 3 ).…”

Section: Resultsmentioning

confidence: 99%

“…Motivated by this finding, we sought to investigate the potential Aca role of this gene further. A hallmark of all other previously identified Aca proteins is the presence of a helix-turn-helix (HTH) DNA-binding domain, necessary for the transcriptional repression of the acr operon 19 , 20 . Multiple sequence alignments of diverse representatives followed by functional domain prediction analyses revealed a conserved HTH motif in the hypothetical protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Apart from sharing a typically low molecular weight, Acrs lack conserved sequence and structural features, thus rendering de novo prediction largely impractical with current methods. However, acr genes tend to cluster within loci that encode more conserved Acr-associated (Aca) proteins 8 , 18 , which are transcriptional repressors of the acr locus 19 , 20 . The aca genes are often more broadly distributed than acr genes and have been used to uncover new acr loci 12 , 14 , 18 , 21 .…”

Section: Introductionmentioning

confidence: 99%

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Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements

et al. 2020

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Many prokaryotes employ CRISPR–Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have developed strategies to bypass immunity, including anti-CRISPR (Acr) proteins; yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we report the discovery of new Acrs by assaying candidate genes adjacent to a conserved Acr-associated (Aca) gene, aca5, against a panel of six type I systems: I–F (Pseudomonas, Pectobacterium, and Serratia), I–E (Pseudomonas and Serratia), and I–C (Pseudomonas). We uncover 11 type I–F and/or I–E anti-CRISPR genes encoded on chromosomal and extrachromosomal MGEs within Enterobacteriaceae and Pseudomonas, and an additional Aca (aca9). The acr genes not only associate with other acr genes, but also with genes encoding inhibitors of distinct bacterial defense systems. Thus, our findings highlight the potential exploitation of acr loci neighborhoods for the identification of previously undescribed anti-defense systems.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements

et al. 2020

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“…Furthermore, if the two systems are indeed related, the Aca proteins (equivalent of antitoxin), which are hypothesized to be regulator of Acrs, might play a role of negatively controlling the expression of Acr genes. Interestingly, a very recent paper experimentally proved that in an AcrIF8-Aca2 operon of the Pectobacterium carotovorum temperate phage ZF40, Aca2 served as a repressor of AcaIF8 (40). This might also explain why Acr genes found in lytic phages often do not have surrounding Aca genes, as they need to be immediately and highly expressed to turn off the host’s defense system to kill.…”

Section: Resultsmentioning

confidence: 99%

Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches

2019

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Anti-CRISPR (Acr) loci/operons encode Acr proteins and Acr-associated (Aca) proteins. Forty-five Acr families have been experimentally characterized inhibiting seven subtypes of CRISPR-Cas systems. We have developed a bioinformatics pipeline to identify genomic loci containing Acr homologs and/or Aca homologs by combining three computational approaches: homology, guilt-by-association, and self-targeting spacers. Homology search found thousands of Acr homologs in bacterial and viral genomes, but most are homologous to AcrIIA7 and AcrIIA9. Investigating the gene neighborhood of these Acr homologs revealed that only a small percentage (23.0% in bacteria and 8.2% in viruses) of them have neighboring Aca homologs and thus form Acr-Aca operons. Surprisingly, although a self-targeting spacer is a strong indicator of the presence of Acr genes in a genome, a large percentage of Acr-Aca loci are found in bacterial genomes without self-targeting spacers or even without complete CRISPR-Cas systems. Additionally, for Acr homologs from genomes with self-targeting spacers, homology-based Acr family assignments do not always agree with the self-targeting CRISPR-Cas subtypes. Last, by investigating Acr genomic loci coexisting with self-targeting spacers in the same genomes, five known subtypes (I-C, I-E, I-F, II-A, and II-C) and five new subtypes (I-B, III-A, III-B, IV-A, and V-U4) of Acrs were inferred. Based on these findings, we conclude that the discovery of new anti-CRISPRs should not be restricted to genomes with self-targeting spacers and loci with Acr homologs. The evolutionary arms race of CRISPR-Cas systems and anti-CRISPR systems may have driven the adaptive and rapid gain and loss of these elements in closely related genomes. IMPORTANCE As a naturally occurring adaptive immune system, CRISPR-Cas (clustered regularly interspersed short palindromic repeats–CRISPR-associated genes) systems are widely found in bacteria and archaea to defend against viruses. Since 2013, the application of various bacterial CRISPR-Cas systems has become very popular due to their development into targeted and programmable genome engineering tools with the ability to edit almost any genome. As the natural off-switch of CRISPR-Cas systems, anti-CRISPRs have a great potential to serve as regulators of CRISPR-Cas tools and enable safer and more controllable genome editing. This study will help understand the relative usefulness of the three bioinformatics approaches for new Acr discovery, as well as guide the future development of new bioinformatics tools to facilitate anti-CRISPR research. The thousands of Acr homologs and hundreds of new anti-CRISPR loci identified in this study will be a valuable data resource for genome engineers to search for new CRISPR-Cas regulators.

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“…We observed related phage genomes harboring completely distinct acr loci, in some cases carrying a different aca gene (Figure 3, Supplementary Figure S5), and sometimes lacking a known aca (Supplementary Figure S6). For example, we found a number of sequenced Pectobacterium genomes with integrated prophages that have similarities with Pectobacterium phage ZF40 (a phage carrying the acrIF8-aca2 locus) (Pawluk, Staals, et al, 2016;Birkholz et al, 2019), but in the remaining cases the prophage regions encoded different combinations of acr(s), together with aca5 ( Figure 3). This observation, together with the common detection of closely related aca and acr homologs within taxonomically mixed bacterial clades (Figure 1a, Supplementary Figure S4, Supplementary Datasheet S4), indicates that these genes are prone to frequent horizontal transfer between diverse MGEs.…”

Section: Identified Acrs Are Spread Across Diverse Proteobacteria Andmentioning

confidence: 99%

Discovery of multiple anti-CRISPRs uncovers anti-defense gene clustering in mobile genetic elements

Pinilla‐Redondo

Shehreen

Marino

et al. 2020

Preprint

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Many prokaryotes employ CRISPR-Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have evolved Anti-CRISPR (Acr) proteins to bypass this immunity, yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we uncover 11 new type I anti-CRISPR genes encoded on numerous chromosomal and extrachromosomal mobile genetic elements within Enterobacteriaceae and Pseudomonas. Candidate genes were identified adjacent to anti-CRISPR associated gene 5 (aca5) and assayed against a panel of six type I systems: I-F (Pseudomonas, Pectobacterium, and Serratia), I-E (Pseudomonas and Serratia), and I-C (Pseudomonas), revealing the type I-F and/or I-E acr genes and a new aca (aca9). We find that acr genes not only associate with other acr genes, but also with inhibitors of distinct bacterial defense systems. These genomic regions appear to be "anti-defense islands", reminiscent of the clustered arrangement of "defense islands" in prokaryotic genomes. Our findings expand on the diversity of CRISPR-Cas inhibitors and reveal the potential exploitation of acr loci neighborhoods for identifying new anti-defense systems.

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The autoregulator Aca2 mediates anti-CRISPR repression

Cited by 60 publications

References 33 publications

Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements

Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements

Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches

Discovery of multiple anti-CRISPRs uncovers anti-defense gene clustering in mobile genetic elements

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