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

Abstract

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.

Fig. 1. Bioinformatic search of aca5-associated acrs.

a Phylogenetic distribution of Aca5 homologs across bacterial taxa. Tree branches are color-coded according to the genus from which the Aca5 orthologs originate. Black circles in the outer ring depict instances where Aca5 is associated with AcrIF11, marking the starting points for the guilt-by-association search; black lines represent the genomes from which acr candidates were selected for functional testing. b Genomic organization of the acrs selected for testing. Genes are colored by Acr family and the surrounding prophage genomic contexts are depicted in gray. Specific orthologs selected for testing are highlighted in bold. The presence/absence of I–F and I–E CRISPR–Cas systems and self-targeting spacers in the host genomes are summarized on the right (for details, see Supplementary Data 1 and 2). Self-targeting analysis was not possible in genomes where CRISPR–Cas systems were not detected, marked here as “NA”. c Graphic representation of the self-targeting instances observed in two Pectobacterium parmentieri genomes from which acrIF16 and acrIF20 genes were selected for testing. Colored arrowheads indicate the positions within the prophages that are targeted by a spacer in one of the host’s I–F CRISPR arrays. Genes are colored according to the legend in (b). When possible, the name of the targeted gene is provided. All protospacers were flanked by the I–F 5’-GG-3’ PAM (Supplementary Data 2). Asterisks next to arrowheads indicate the number of spacer-protospacer mismatches; Two out of the seven protospacers were found to have a mismatch within the seed region (Supplementary Fig. 1).

Fig. 2. Newly identified acr genes inhibit diverse type I CRISPR–Cas systems.

a Schematic of the experimental setup employed for acr validation. Bacteria carrying their native type I–F, I–E, or I–C CRISPR–Cas systems and a plasmid expressing each acr candidate were challenged with a CRISPR-targeted phage and infectivity was assessed. b Percent sequence identity comparison between the model CRISPR–Cas systems challenged (type I–F: Pectobacterium, Serratia, and Pseudomonas; type I–E: Serratia and Pseudomonas; type I–C: Pseudomonas) and the type I–F/I–E systems found in the endogenous hosts of the acr candidates, when available. Average values for the individual percentage identity comparisons between Cas orthologs forming the type I–E/I–F interference complexes are shown, excluding the adaptation modules (see top gene map) (Supplementary Data 3). The specific Acr orthologs found in the genomes which were selected for testing are noted below; asterisks indicate dual anti-I–F/I–E function. c Efficiency of plaquing (EOP) of CRISPR-targeted phages in bacterial lawns expressing the different acr candidates, or the empty vector control (ev +CRISPR), compared to EOP of the same phage in non-targeting bacterial lawns carrying the empty vector (ev −CRISPR). Positive controls for Acr inhibition include AcrIF9 (Pectobacterium and Serratia I–F), AcrIE5 (Serratia I–E), AcrIE4-F7 (Pseudomonas I–F and I–E), and AcrIC1 (Pseudomonas I–C). Data are presented as mean ± SEM (n = 3 biologically independent samples). In Serratia, CRISPR immunity provided an EOP of ~1 × 10⁻⁶ and the absence of EOP data indicates that no single plaques were detected with ~1 × 10⁹ pfu mL⁻¹ of phage JS26. d Percentage distribution of the MGE origin (phage, plasmid/ICE, or unclear) for the collection of orthologs of each of the validated acrs. The isoelectric point (pI) and molecular weights (MW) of the validated acrs are shown. Source data are available in the Source data file.

Fig. 3. Genomic comparison between Pectobacterium phage ZF40 and different Pectobacterium genomes with related prophage regions.

The alignment of the acr loci in related prophages/MGEs shows variability in nearby genes. Genes are color-coded according to their functional annotations. Gene annotations are based on protein sequence searches against pfam and pVOG (Supplementary Data 5). The black dot denotes a pseudogene (early stop codon); domain of unknown function (DUF).

Fig. 4. AcrIF18* and AcrIF15 prevent type I–F CRISPRi.

a Schematic of the CRISPRi assay. In the absence of a crRNA, phzM is transcribed (green rhomb) and pyocyanin is produced at normal levels (green medium). In the presence of a crRNA and an Acr acting upstream of the target DNA-binding stage (purple circle), transcription of phzM by Cascade is de-repressed (no color change; green). If the Acr’s activity manifests downstream of the target DNA-binding stage (orange circle), phzM expression is repressed (color change; yellow). b Average pyocyanin production levels for four CRISPRi lysogens expressing a prophage-encoded Acr (AcrIF15–18*) in the presence and absence of a crRNA (indicated by the ± sign in the black circles on the x-axis). “Bg” represents the background pyocyanin detection levels for the assay. Error bars indicate the standard deviation of the mean for three biological replicates (unpaired two-sided t-test; *p < 0.05, **p < 0.005, ns = not significant; p_AcrIF18* = 0.1, p_AcrIF15 = 0.7, p_AcrIF16 = 0.0003, p_AcrIF17 = 0.006). Representative colors displayed in the graph have been derived from pictures of the results, see Supplementary Fig. 7. Source data are available in the Source data file.

Fig. 5. Discovery of Aca9 uncovers two potent I–F Acrs.

a Identification of an AcrIF22* homolog reveals a previously undescribed aca marker gene (aca9). b Alignment of diverse Aca9 representatives carried by Proteobacterial MGEs. Predicted secondary structure alpha-helix regions are illustrated as ribbons for the K. pneumoniae homolog (top). The HTH DNA-binding domain is highlighted in gray. c Phylogenetic diversity of Aca9 across publicly available sequences. Tree branches are colored by the bacterial host from where they originate (Order level). The MGE origin is shown with an empty circle (plasmid-like elements), a black diamond (phage), and left blank when the genomic context was unclear. The relative percentage distribution of aca9 orthologs by MGE origin is depicted as the area of the gray shaded circles in the legend. The positions in the tree where acrIF15 and acrIF22* are found associated with aca9 are marked with yellow and red circles, respectively. d Guilt-by-association searches following aca9 led to the discovery of two candidate acr genes in P. aeruginosa. e Functional testing for inhibition of the PA14 I–F CRISPR–Cas system was performed by phage plaque assays in strains carrying plasmids expressing the indicated acr candidates. Ten-fold serial dilutions of CRISPR-targeted DMS3m phage were titered on lawns of P. aeruginosa naturally expressing its I–F CRISPR–Cas system. The ΔCRISPR strain shows phage replication in the absence of CRISPR–Cas targeting. AcrIF11 was employed as a positive control for strong I–F inhibition. Source data are available in the Source data file.

Fig. 6. Acrs cluster with other antagonists of host defense functions.

a Genome organization of the Klebsiella pneumoniae ABFQB plasmid unitig_1 (CP036439.1), highlighting the anti-defense cluster region. Genes are colored according to their predicted functions as shown in the key. Coding regions encoded on the plus and minus strands are shown on the outer and inner lanes of the gene map, respectively. b Examples of genomic regions within diverse MGEs where acrs were found in the vicinity of known anti-RM genes and other putative anti-defense determinants. Genes are colored according to the gene key in (a).