Structural basis for anti-CRISPR repression mediated by bacterial operon proteins Aca1 and Aca2.
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC8633003 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:CRISPR-Cas systems are bacterial defense systems for fighting against invaders such as bacteriophages and mobile genetic elements. To escape destruction by these bacterial immune systems, phages have co-evolved multiple anti-CRISPR (Acr) proteins, which inhibit CRISPR-Cas function. Many acr genes form an operon with genes encoding transcriptional regulators, called anti-CRISPR-associated (Aca) proteins. Aca10 is the most recently discovered Aca family that is encoded within an operon containing acrIC7 and acrIC6 in Pseudomonas citronellolis. Here, we report the high-resolution crystal structure of an Aca10 protein to unveil the molecular basis of transcriptional repressor role of Aca10 in the acrIC7-acrIC6-aca10 operon. We identified that Aca10 forms a dimer in solution, which is critical for binding specific DNA. We also showed that Aca10 directly recognizes a 21 bp palindromic sequence in the promoter of the acr operon. Finally, we revealed that R44 of Aca10 is a critical residue involved in the DNA binding, which likely results in a high degree of DNA bending.
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Project description:Many bacteria use CRISPR-Cas systems to defend against invasive mobile genetic elements (MGEs). In response, MGEs have developed strategies to resist CRISPR-Cas, including the use of anti-CRISPR (Acr) proteins. Known acr genes may be followed in an operon by a putative regulatory Acr-associated gene (aca), suggesting the importance of regulation. Although ten families of helix-turn-helix (HTH) motif containing Aca proteins have been identified (Aca1-10), only three have been tested and shown to be transcriptional repressors of acr-aca expression. The AcrIIA1 protein (a Cas9 inhibitor) also contains a functionally similar HTH containing repressor domain. Here, we identified and analysed Aca and AcrIIA1 homologs across all bacterial genomes. Using HMM models we found aca-like genes are widely distributed in bacteria, both with and without known acr genes. The putative promoter regions of acr-aca operons were analysed and members of each family of bacterial Aca tested for regulatory function. For each Aca family, we predicted a conserved inverted repeat binding site within a core promoter. Promoters containing these sites directed reporter expression in E. coli and were repressed by the cognate Aca protein. These data demonstrate that acr repression by Aca proteins is widely conserved in nature.
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Project description:CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by the Pectobacterium carotovorum temperate phage ZF40, is an autoregulator that represses the anti-CRISPR-aca2 operon. Aca2 is a helix-turn-helix domain protein that forms a homodimer and interacts with two inverted repeats in the anti-CRISPR promoter. The inverted repeats are similar in sequence but differ in their Aca2 affinity, and we propose that they have evolved to fine-tune, and downregulate, anti-CRISPR production at different stages of the phage life cycle. Specific, high-affinity binding of Aca2 to the first inverted repeat blocks the promoter and induces DNA bending. The second inverted repeat only contributes to repression at high Aca2 concentrations in vivo, and no DNA binding was detectable in vitro. Our investigation reveals the mechanism by which an Aca protein regulates expression of its associated anti-CRISPR.
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