PMID:24736222

A new group of phage anti-CRISPR genes inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa.

Plasmid-borne expression of DMS3 gene 30 (ACR3-30) in Pseudomonas aeruginosa SMC4386, a strain with a natively active type I-E CRISPR-Cas system, allowed the CRISPR-sensitive phage JBD8 to form plaques three to five orders of magnitude more efficiently than empty vector. Three other unrelated genes, from phages JBD88a, D3112 and JBD5, had the same effect, defining four families of type I-E anti-CRISPRs.

"As shown in Fig. 3C, expression of putative anti-CRISPR genes from phages JBD88a, DMS3, D3112, and JBD5 allowed CRISPR-sensitive phage JBD8 to form plaques with 103- to 105-fold-higher efficiency than on cells containing empty vector."
The protein encoded by DMS3 gene 30 is named ACR3-30 in this paper. The other three are ACR88a-32 (phage JBD88a), ACR3112-31 (phage D3112, UniProtKB:Q6TM71) and ACR5-34 (phage JBD5).

"We designate the proteins encoded by these anti-CRISPR genes as ACR88a-32, ACR3-30, ACR3112-31, and ACR5-34"
The effect is specific to the four new families. Five type I-F anti-CRISPR genes and the conserved JBD30-36 gene, assayed in the same figure, did not rescue JBD8 plaquing.

"In contrast, plasmids expressing any one of five different type I-F anti-CRISPR genes, or gene JBD30-36 (homologs of which are found at the 3′ ends of all anti-CRISPR regions) had no effect on JBD8 plaquing."
Gene 30 is also required in the phage's own genome. A CRISPR-targeted derivative of DMS3 plaqued robustly on SMC4386, whereas isogenic phages in which gene 30 had been replaced by a type I-F anti-CRISPR gene, or by a frameshifted version of it, could not plaque at all, and all three plaqued equally on a CRISPR-Cas-negative strain.

"In contrast, a mutant phage in which the DMS3m type I-E anti-CRISPR gene was replaced with a type I-F anti-CRISPR gene (DMS3m30-35 [9]) was not able to form plaques on SMC4386."
The authors state the conclusion for gene 30 explicitly, as an activity exercised during lytic infection.

"These results demonstrate that gene 30, the type I-E anti-CRISPR gene of phage DMS3m, functions during lytic infection to inhibit the type I-E CRISPR-Cas system of SMC4386 and, thus, allow robust phage replication within this strain."
Activity is host-specific. None of the four type I-E anti-CRISPRs, including ACR3-30, inhibited the type I-E CRISPR-Cas system of Escherichia coli K-12, although all four were shown to be robustly expressed there.

"These data show that the anti-CRISPR proteins encoded by P. aeruginosa phages are not active against the type I-E system in E. coli."
A DMS3 prophage inactivates the host type I-E system from the lysogenic state as well, restoring protospacer-plasmid transformation to roughly empty-vector efficiency.

"the lysogens of phages JBD5, JBD24, DMS3, and JBD26 could be transformed with ~100% efficiency compared to the empty vector"

PMID:39541974

Structural and biochemical insights into the mechanism of the anti-CRISPR protein AcrIE3.

AcrIE3 acts by binding one subunit of the host type I-E surveillance complex, Cas8e, rather than the complex as a whole or the Cas3 nuclease.

"Our results reveal that AcrIE3 selectively binds to the Cas8e subunit of the Cascade complex."
Mutational analysis localises the inhibitory surface to acidic residues that contact the protospacer-adjacent-motif recognition site of Cas8e, the site Cascade uses to license target DNA, making this a competitive block on target recognition.

"Through extensive mutational analyses, we show that AcrIE3 interacts with the protospacer adjacent motif (PAM) recognition site in Cas8e through its negatively charged surface residues."
The crystal structure is an all-helical fold presenting a negatively charged surface.

"The crystal structure of AcrIE3 exhibits an all-helical fold with a negatively charged surface."

PMID:38797150

Novel structure of the anti-CRISPR protein AcrIE3 and its implication on the CRISPR-Cas inhibition.

An independently determined high-resolution crystal structure of AcrIE3, described as a four-helix bundle with an extended N-terminal loop.

"Our structural examination showed that AcrIE3 adopts a helical bundle fold comprising four α-helices, with a notably extended loop at the N-terminus."
Three acidic surface patches are identified and proposed to carry the inhibitory function, agreeing with the mutagenesis in PMID:39541974.

"surface analysis of AcrIE3 revealed the presence of three acidic regions, which potentially play a crucial role in the inhibitory function of this protein"