LETTERS PUBLISHED: 13 JUNE 2016 | ARTICLE NUMBER: 16085 | DOI: 10.1038/NMICROBIOL.2016.85 Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species April Pawluk1, Raymond H.J. Staals2, Corinda Taylor2, Bridget N.J. Watson2, Senjuti Saha3, Peter C. Fineran2, Karen L. Maxwell4* and Alan R. Davidson1,3* CRISPR-Cas systems provide sequence-specific adaptive immu- MGE-encoded mechanisms that inhibit CRISPR-Cas systems. In nity against foreign nucleic acids1,2. They are present in approxi- support of this hypothesis, phages infecting Pseudomonas aeruginosa mately half of all sequenced prokaryotes3 and are expected to were found to encode diverse families of proteins that inhibit constitute a major barrier to horizontal gene transfer. We pre- the CRISPR-Cas systems of their host through several distinct viously described nine distinct families of proteins encoded in mechanisms4,5,17,18. However, homologues of these anti-CRISPR Pseudomonas phage genomes that inhibit CRISPR-Cas function4,5. proteins were found only within the Pseudomonas genus. Here, We have developed a bioinformatic approach that enabled us to we describe a bioinformatic approach that allowed us to identify discover additional anti-CRISPR proteins encoded in phages five novel families of functional anti-CRISPR proteins encoded in and other mobile genetic elements of diverse bacterial phages and other putative MGEs in species spanning the diversity species. We show that five previously undiscovered families of Proteobacteria. of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of The nine previously characterized anti-CRISPR protein families both Pseudomonas aeruginosa and Pectobacterium atrosepticum, possess no common sequence motifs, so we used genomic context to and a dual specificity anti-CRISPR inactivates both type I-F search for novel anti-CRISPR genes. A highly conserved gene and I-E CRISPR-Cas systems. Mirroring the distribution of the encoding a putative transcriptional regulator (Pfam family CRISPR-Cas systems they inactivate, these anti-CRISPRs were HTH_24) was found downstream of all known phage anti- found in species distributed broadly across the phylum CRISPR gene loci4,5 and was absent in related phages lacking Proteobacteria. Importantly, anti-CRISPRs originating from anti-CRISPRs. We conducted a BLAST search with this protein, species with divergent type I-F CRISPR-Cas systems were which we refer to as anti-CRISPR-associated protein 1 (Aca1), able to inhibit the two systems we tested, highlighting their and analysed the proteins encoded by genes immediately upstream broad specificity. These results suggest that all type I-F of putative Aca1 homologues. Proteins were identified as candidate CRISPR-Cas systems are vulnerable to inhibition by anti- anti-CRISPRs if they were encoded on the same strand as the Aca1 CRISPRs. Given the widespread occurrence and promiscuous homologue and were less than 200 amino acids long, because pre- activity of the anti-CRISPRs described here, we propose that viously described anti-CRISPRs range from 50 to 139 residues in anti-CRISPRs play an influential role in facilitating the move- length. A predicted promoter was identified upstream of each puta- ment of DNA between prokaryotes by breaching the barrier tive anti-CRISPR-aca1 gene pair, similar to the arrangement of imposed by CRISPR-Cas systems. the known anti-CRISPR operons. In this way, we identified four dis- Horizontal gene transfer (HGT) is a major driving force of bac- tinct putative anti-CRISPR gene families present in a variety of terial evolution6,7. However, many HGT events are maladaptive. Pseudomonas prophages unrelated to previously described phages Bacteria have therefore evolved numerous mechanisms to destroy bearing anti-CRISPRs (Fig. 1a). To facilitate the description of or silence foreign DNA. One of the most widespread of these anti-CRISPR genes, we have established a naming convention defences is the CRISPR-Cas system, which confers adaptive, whereby anti-CRISPRs targeting a type I-F CRISPR-Cas system sequence-specific immunity against mobile genetic elements are referred to as AcrF, and different numbers are given to each (MGEs) including bacteriophages (phages) and plasmids1,2,8,9. family. The species from which a particular gene originated may This adaptive immunity is powerful, as it allows organisms to be designated by a subscript. The numbering of new functional build a memory of earlier invasions and deploy targeted defence AcrF families discovered here begins at 6, because five families strategies when related invaders return. CRISPR-Cas systems were discovered in our previous work4. employ small CRISPR RNAs (crRNAs), often matching foreign All previously identified anti-CRISPR proteins inhibited either genetic sequences, that guide CRISPR-associated (Cas) proteins to the type I-E or type I-F CRISPR-Cas systems of P. aeruginosa4,5, recognize and destroy DNA, or in some cases, RNA1,10–13. These so we tested one representative from each new family for activity adaptive defence systems seem likely to pose a major barrier to in strains of P. aeruginosa possessing active type I-E or I-F HGT, but evolutionary studies have implied that the global impact systems. Anti-CRISPR genes were expressed in these strains on of CRISPR-Cas on HGT is minimal14. Despite the prevalence of their own from a plasmid using an inducible promoter, and their CRISPR-Cas systems, HGT occurs frequently and is ongoing ability to support replication of a CRISPR-targeted phage was 15,16 4,5 within diverse bacterial niches . Why is HGT still pervasive assessed . AcrF6Pae, a putative anti-CRISPR from a prophage among organisms possessing these sophisticated adaptive defence region in P. aeruginosa (Fig. 1a), completely inhibited the type I-F systems? One possible answer is the widespread existence of CRISPR-Cas system, as demonstrated by a 106-fold increase in 1Department of Biochemistry, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada. 2Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand. 3Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada. 4Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada. *e-mail: [email protected]; [email protected] NATURE MICROBIOLOGY | VOL 1 | AUGUST 2016 | www.nature.com/naturemicrobiology 1 © 2016 Macmillan Publishers Limited. All rights reserved LETTERS NATURE MICROBIOLOGY DOI: 10.1038/NMICROBIOL.2016.85 a Known anti-CRISPR phages Capsid Tail acr genes aca1 Capsid Capsid P. aeruginosa ATCC 33349 acrF6 aca1 Tail Pseudomonas sp. BAY1663 cand1 aca1 Lysis Tail Tail P. fluorescens AU14440 Capsid cand2 aca1 Capsid P. aeruginosa PACS458 acrF7 aca1 Tail b Type I-F Type I-E Vector ΔCRISPR-Cas acrF6 cand1 cand2 acrF7 Tenfold phage dilutions Tenfold phage dilutions Figure 1 | Discovery and characterization of aca1-associated anti-CRISPR genes. a, Representative genome regions from Aca1 BLAST results with candidate anti-CRISPR genes (coloured) identified upstream. Arrows representing genes are not drawn to scale. Genes coloured in light grey are associated with mobile DNA, and gene functions are annotated (when known) to illustrate the unique genomic contexts of anti-CRISPRs. The putative functions of the proteins encoded by some open reading frames are shown to highlight the distinct genomic contexts of these anti-CRISPR genes. These functions are defined as follows: ‘Capsid’ is involved phage capsid morphogenesis; ‘Tail’ is involved in phage tail morphogenesis; ‘Lysis’ is required for cell lysis; ‘Ter’ denotes a phage terminase; ‘Int’ denotes an integrase; ‘Transp’ denotes a transposase; ‘Mobil’ is involved in mobilization of plasmids and conjugative elements; ‘cand’ denotes candidate anti-CRISPR gene. b, Putative anti-CRISPRs shown in a were assayed for biological activity. Tenfold dilutions of lysates of CRISPR-sensitive phages were applied to bacterial lawns of P. aeruginosa strain UCBPP-PA14 (possessing a type I-F CRISPR-Cas system) or P. aeruginosa strain SMC4386 (possessing a type I-E CRISPR-Cas system). As indicated, some strains carried plasmids expressing the anti-CRISPR genes or candidate anti-CRISPR genes. Clearing of the bacterial lawn indicates phage replication. Strains UCBPP-PA14 ΔCRISPR-Cas and SMC4386 ΔCRISPR-Cas are included as positive controls for loss of I-F and I-E CRISPR-Cas activity, respectively. The replication efficiency of a CRISPR-insensitive phage was unchanged following expression of each candidate gene (Supplementary Fig. 1a). Photographs are representative images from three biological replicates. a * Vibrio nigripulchritudo (Vni) Pasteurella multocida Acinetobacter radioresistens Plesiomonas sp. * Pseudomonas aeruginosa (Pae) * Oceanimonas smirnovii (Osm) Methylophaga nitratireducenticrescens * Methylophaga aminisulfidivorans (Mam) * Methylophaga frappieri (Mfr) * Acinetobacter baumanii (Aba) bcType I-F Type I-E Type I-F Type I-E Pae WT Osm K3stop Mfr Y38A Aba Δ99-100 Vni Tenfold phage dilutions Tenfold phage dilutions Mam Tenfold phage dilutions Tenfold phage dilutions Figure 2 | Investigation of AcrF6Pae dual anti-CRISPR activity. a, Sequence alignment of AcrF6 homologues coloured with the ClustalX scheme. The homologues tested are indicated with an asterisk. The conserved tyrosine residue (Y38 of AcrF6Pae) is indicated by an arrow. b, Phage spotting experiments