ID Name PDB Structure Type of the 3D structure MoA category MoA Defence subtype Defences Defence links Protein source name Protein source link DOI Multicomponent system Existing Pfam domain Homologs from eukaryotic viruses Evidence Protein sequence pnk Polynucleotide_kinase 1LY1 pnk.pdb PDB entry synthesises and restores essential molecules depleted by bacterial defence Adds a 5'-phosphate group to the 3'-OH end of the tRNA fragment cleaved by PrrC, making it ready for ligation. Escherichia coli PrrC PrrC https://defensefinder.mdmlab.fr/wiki/defense-systems/prrc Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1002/j.1460-2075.1987.tb02532.x rna_ligase PF13238;PF13671;PF24694 pnk_model.html Polynucleotide kinase (pnk) was shown to be essential for the repair of tRNAs cleaved by the T4-encoded anticodon nuclease. In T4-infected E. coli prr strains, specific tRNA fragments accumulated in infections with pnk mutants, while these fragments were transient during wild-type infection. This suggested that pnk activity was required to process and repair the cleavage products. Biochemical analysis of the cleavage termini revealed that pnk catalyzes conversion of 2':3'-cyclic phosphate and 5'-OH termini into 3'-OH and 5'-phosphate ends, creating substrates for RNA ligase. In vitro assays confirmed this, showing that only after incubation with purified pnk could the tRNA fragments be ligated by RNA ligase. Phosphatase digestion assays, thin-layer chromatography, and RNase T1 mapping of terminal groups provided direct evidence of enzymatic activity, specifically hydrolysis of 2':3'-cyclic phosphates and phosphorylation of 5'-OH ends, demonstrating the dual phosphatase and kinase roles of the pnk protein in tRNA repair. MKKIILTIGCPGSGKSTWAREFIAKNPGFYNINRDDYRQSIMAHEERDEYKYTKKKEGIVTGMQFDTAKSILYGGDSVKGVIISDTNLNPERRLAWETFAKEYGWKVEHKVFDVPWTELVKRNSKRGTKAVPIDVLRSMYKSMREYLGLPVYNGTPGKPKAVIFDVDGTLAKMNGRGPYDLEKCDTDVINPMVVELSKMYALMGYQIVVVSGRESGTKEDPTKYYRMTRKWVEDIAGVPLVMQCQREQGDTRKDDVVKEEIFWKHIAPHFDVKLAIDDRTQVVEMWRRIGVECWQVASGDF rna_ligase RNA_ligase_1 5TT6 rna_ligase.pdb PDB entry synthesises and restores essential molecules depleted by bacterial defence Ligates the broken ends of tRNA (a 5'-phosphate and 3'-OH). Escherichia coli PrrC PrrC https://defensefinder.mdmlab.fr/wiki/defense-systems/prrc Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1002/j.1460-2075.1987.tb02532.x pnk PF20819;PF09511 rna_ligase_model.html Enzymatic activity assay evidence supports ligase activity (ECO_0000005). MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHRLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNVRENETGEYISYDDIYKDATLRPYLVERYEIDSPKWIEEAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY bgt DNA modifying beta-glucosyltransferase 2BGT_A bgt.pdb PDB entry modifies phage molecules to avoid recognition Glucosylates hydroxymethyl-dCMP residues Escherichia coli RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1002/j.1460-2075.1994.tb06646.x dnmp;dcmp_hm PF09198 bgt_model.html Glucosylation activity is supported by structural determination evidence (ECO_0005031). MKIAIINMGNNVINFKTVPSSETIYLFKVISEMGLNVDIISLKNGVYTKSFDEVDVNDYDRLIVVNSSINFFGGKPNLAILSAQKFMAKYKSKIYYLFTDIRLPFSQSWPNVKNRPWAYLYTEEELLIKSPIKVISQGINLDIAKAAHKKVDNVIEFEYFPIEQYKIHMNDFQLSKPTKKTLDVIYGGSFRSGQRESKMVEFLFDTGLNIEFFGNAREKQFKNPKYPWTKAPVFTGKIPMNMVSEKNSQAIAALIIGDKNYNDNFITLRVWETMASDAVMLIDEEFDTKHRIINDARFYVNNRAELIDRVNELKHSDVLRKEMLSIQHDILNKTRAKKAEWQDAFKKAIDL stp Stp _ stp.cif AlphaFold 3 binds and inhibits host defence system Expression of Stp alleviates the restriction, and it is proposed that it binds to EcoPrrI. It triggers PrrC defence, and both inhibition and activation depend on the same residues within the N-proximal 18-residue region of Stp. Escherichia coli type IC RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1006/jmbi.1995.0343 PF08133 Ectopic expression evidence supports anti-RM activity (ECO_0000017) MSNFHNEHVMQFYRNNLKTKGVFGRQ abc2 Abc2 _ abc2.cif AlphaFold 3 binds and inhibits host defence system Binds to the RecC subunit of RecBCD and promotes its recombination activity. Escherichia coli RecBCD RecBCD _ Bacteriophage P22 https://www.ncbi.nlm.nih.gov/nuccore/AF217253.1 10.1006/jmbi.1999.3486 PF11043 Co-purification (ECO_0000022) and electrophoresis showed binding to the RecBCD complex (DOI: 10.1016/S0021-9258(17)31676-9), and genetic interaction evidence (the overexpression of RecC suppresses the phenotype caused by Abc2, ECO_0000011) supports interaction with RecC. MPAPLYGADDPRRCSGNSVSEVLDKFRKNYDLIMSLPQETKEEKEFRHCIWLAEKEERERIYQTAIRPFRKATYTKFIEIDPRLRDYRSRYGAISNN ral Ral _ ral.cif AlphaFold 3 uncategorised Enables bacteriophage λ to utilize the host EcoK methyltransferase to methylate its own genome, thereby enhancing modification and alleviating restriction. Ral likely interacts with the HsdM or HsdS subunits of the EcoK complex, altering their conformation to increase methylation efficiency. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Phage λreverse 10.1016/0022-2836(86)90071-9 PF11058 Ectopic expression evidence (ECO_0000017) MTTTIDKNQWCGQFKRCNGCKLQSECMVKPEEMFPVMEDGKYVDKWAIRTTAMIARELGKQNNKAA mom Methylcarbamoylase_mom 8BV8_A mom.pdb PDB entry modifies phage molecules to avoid recognition Recognises the sequence 5'-(C or G)-A-(C or G)-N-(C or T)-3' and performs methylcarbamoylation of adenine, preventing recognition of phage DNA by type I (EcoKI and EcoBI) restriction nucleases. Escherichia coli type I and II RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Escherichia phage Mu https://www.ncbi.nlm.nih.gov/nuccore/AF083977.1 10.1016/0042-6822(76)90232-4;10.1016/0378-1119(85)90108-8;10.1093/nar/gkaa319 Ectopic expression of the gene renders phages resistant to numerous restriction enzymes (ECO_0000017). The modification (methylcarbamoylation) was identified using liquid chromatography–electrospray ionisation mass spectrometry (ECO_0001582). MPASIPRRNIVGKEKKSRILTKPCVIEYEGQIVGYGSKELRVETISCWLARTIIQTKHYSRRFVNNSYLHLGVFSGRDLVGVLQWGYALNPNSGRRVVLETDNRGYMELNRMWLHDDMPRNSESRAISYALKVIRLLYPSVEWVQSFADERCGRAGVVYQASNFDFIGSHESTFYELDGEWYHEITMNAIKRGGQRGVYLRANKERAVVHKFNQYRYIRFLNKRARKRLNTKLFKVQPYPK abc1 Abc1 _ abc1.cif AlphaFold 3 unknown _ Salmonella Typhimurium RecBCD RecBCD _ Bacteriophage P22 https://www.ncbi.nlm.nih.gov/nuccore/AF217253.1 10.1016/0042-6822(87)90017-1 PF08281;PF04545 Deletion mutation phenotypic evidence supports anti-RecBCD activity (ECO_0001038). MRRLNITTAEMESVCGRMVACRAAEHLGLNINQFYYIAKKLSLKTAFIKPRWSDDEDKRMQTLISSGYTQRNVAKILGRSEESVKSRLSRLRKK dara DarA _ dara.cif AlphaFold 3 binds and protects the component targeted by the host defence binds and protects phage DNA. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage P1 https://www.ncbi.nlm.nih.gov/nuccore/NC_005856.1 10.1016/0042-6822(87)90324-2;10.1111/mmi.13705 hdf;ddrb;darb;ddra;ulx PF18789;PF18788 Genetic deletions (deletion mutation phenotypic evidence, ECO_0001038) result in high sensitivity to RM systems, while ectopic expression (ECO_0000017) restores plating efficiency. MEQFNINKGMTIKPGLDVLPPPVTDDEYRALMAGEDRYLMTESNTLEEIEATFFYDTPIHWCATDLLEAISSTRLQLHRTMQAFVRALNQKLNGTGISAGSDKTGDVAQSGARAIGGAEIGRARNVNGLPVLPAIIPLSDGQTISILFHSPTAENRITNSDTLVAFQFLLNKKDVTHTVAPMSGRDMTLAQVTMKLANLAEKNSAKFQRAQKKKKALVDEITQLQADSDQKEDAMSDLADQVAAVEGQKADLEQKINAVASEADSLYEENERLQGEIDRLNRTGGRDTIAPAGMTGGHSRALTDRLASIKNRMHMDGEATLSNGASMKQFIGDGEGYIQLTDPDGSVYMIKAKSIQGVDMADAIGKLFKAYKAGNVSEYLVQPEEHKPENVEPESAEDTGSSSPEPEVSVGAYRYALQMRPAAPGAIPEGNKAILPRPDEGDPYYEYARYGIATYDTPLSDQQMSEYDLKLLPREDSFDFLAKTLTNGPFGKYAQKALELATNSPDEFRVMLKTQFQKTFPNIAFPGGAGTEKMVQSMINALQAEVGEITQPEPAPAQPDETVSEADAEANKAIEYLNNVMDMQSTDMAEIRNARGNVREAIAALQTAGRFEENEELVNGAARHLADLLVAIQKAGVAA darb DarB _ darb.cif AlphaFold 3 modifies phage molecules to avoid recognition contains a methyltransferase domain, likely modifying phage. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage P1 https://www.ncbi.nlm.nih.gov/nuccore/NC_005856.1 10.1016/0042-6822(87)90324-2;10.1111/mmi.13705 dara;hdf;ddrb;ddra;ulx PF07669;PF00271;PF02384;PF04851;PF00176 darb_model.html Genetic deletions (deletion mutation phenotypic evidence, ECO_0001038) result in high sensitivity to RM systems, while ectopic expression (ECO_0000017) restores plating efficiency. MNKLSMGVFRCSSVSEILKYIRAITSHRAPIKYGVEKVEGKSYDRLRREANQKAIDLLNSLVDGATLTDEQRQILAGYTGEGGIGGSVSEYYTPKPIAEGVWEIMKLYGADVGNTLEPSAGTGVFNETKPVGTVMTATEISSVSGRINQLLHPEDSVQISPFEQLAVSTPNDSFDHVVGNVPFGGRDNTRNIDKPYAEETDMGSYFMLRMLDKIKPGGFMCVIVPPSIVSGSNMKRLRLRLSRKAEFLGAHRLPTGTFDANGTSTVVDVVLMRKHPAEMAEKIPLVHESTLESANVLWPTFISGKWFEKDGRRFVHGTQEKGFQGRIEVRADGQIDNQALKAKLIHCFESRIDWYLLDMAEPSPTADVVDEGEMRLINGVWQKYAGGRWIESDAGKELKIDVASYGADSWEALQRNLTTTEGRLGMTFTQMANVRDKYTTSISDDMVQLVDWINSQPEKYRERLYRGAMIGRMLIEYQDMKAAGHSAEQIEQQRLSLVSRLQAEIDRFGNPGRGPIAKLSGSGARAWFAFRGAIKLDGTISDELTGKLVTHDSSASYDSTSYQDTLRYLYSDLTRDPIQLDDFRLAFTGELPASDDELLNLLASTPGIAVSPYGGIVPFARATSGDINEIVAPKQEFLATLTDGPVKNNVLNQLAAIEEKRIKTPAENIRFKLNSRWFDRSVILEFLQENGYPDLRYVQSVQLEGDEMVSDTYHGGDGLFVGHRYGVVQRKDKETGEIRYEWDRKSGENATGFPAQLEKYLNGARIGGKDSATANGYREQMALLEDQFNKWIKTHDRYDELVAKYNDVFNSNIPYEHSGDPLGLKGLSGKRQPFDYQNSEVRRLSEDGRGILGFGTGLGKTTTALALEAFNYENGRSTRTAYVVPKSVLENWYYEAKEFLSEEAFSNYLFVGLDVLMDGDQIRQVPVLDENGKPVLGTDGTPVMRDALKLADEATITARMNAIPHSNYRAVVFTKEQYARIPLRDDTVDEHAQDMLYDFVAAGRVASAMDSDSHRKEAARRRVLSEYSDTGTEKAEKYPYFEDMGFDSVIADEGHNYRNSYKNGREASQLAYLPTSAVAQSARDMAIKNAYLMKKNGGRGPVLLTATPVVNTPIDAYNMLSHVLPKEYWQKMGIYGPDDFVKFFGKTRLETVQKISGEVEEKMALVGFENLDALRGIFHRWTTLKTAEDVKDTVEIPELDEHQQDAPLTEEQLAAYEELRQQAEAAAKANNGVTTSVNEDGVIEHEKARPIFSIIRDMDRVCTDMDLYYRRITYRFLPEYADAVQQLADSLPKQATSEDDDSDDSITQQSQYSLIDKGEFIQLQVPEAFEQEVNKRLARFGIDEQTVTHPVTPKYAKLIATLKEFFPEGKQIIFTDEKTQHQKLKRIICNALNLEPSKVGILNAQTVAEAGKTGKKLKAVKPPKELPDEPTDAQIAKYNEQMALYDAYIAQQNEMSLGGLEKIAADFQEGRTPIIICNKKAEVGINLHRGTTDIHHLTLPWTPASIAQRNGRGARVGSNRASVRVHYYCGKGSFDEYRLKTLKRKAGWISDILRSDKSEMENADANDMIEMQMYTAKDDGERLAMMQVQMDKAKAAQRARQKEQATIDLQNYIKAQHAAGEDVEVLTAELERSKAELEKTTAEVAKFKQAVMAKAADNADWKARWGSVHHTDRMLLAQYRASLKSAIQRKANISQAISPYEKLLNRTQKAATDIKRLRPLVEDAINKGILDVDPDLVNHASEFLVIGDRSWRVGQYYDCAGDIVRIKSLDFDSQRADVEIIFTFKGTKSGNWDVKTLDKQVDVTPDEDAVMQKISGGVSIAGINDIISCDDFYRFQQRGMIKITDSYGVQTTESGYSIDFVGTYTDPLKHAVYPDRRDGALKSSIAKWVLGMMSEGNNRQVRLAEVFLTELFGSNYGDVIASYGDTLSPEAIQEKIADAIARMPEKTSQGATRNGDSELEVTNAIFGTHEFRASDYEITTAQFGTIGIYSNKAEIKQAMDAASARIAAEREANLNHAVAALTQSWVTAIREAATTGKITPAIADVVNDGSKFMDAYKMDAVQLPSAYGQLSYRMTYNLVSMFSDLAILGLVDLNEVTPELLSMRKNHVEILQRINTVLAGRTDEEKQADADRINLALGNITEEEIAARNEKQEELSSIQGDATSIAQSLGLNYRVSTADLKMMYAPKFAAGEVFGLQEASGMKGVLFRAKDAIKTKFGARWLPAKAKNSDFPGNWWIIETKHNVADVLAVIQQYA ddra DdrA _ ddra.cif AlphaFold 3 binds and protects the component targeted by the host defence binds and protects phage DNA. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage P1 https://www.ncbi.nlm.nih.gov/nuccore/NC_005856.1 10.1016/0042-6822(87)90324-2;10.1111/mmi.13705 dara;hdf;ddrb;darb;ulx Genetic deletions (deletion mutation phenotypic evidence, ECO_0001038) result in high sensitivity to RM systems, while ectopic expression (ECO_0000017) restores plating efficiency. MTLSAIELMDLSDKLDALMSKAATASGMELLDISDEIDQIMQQMGYGASGDGSGEEKQPSEHDGVPKLVADFLADKFVDQSTDAFIGTLQDLSQYVGIYIDLDQVKQHTAAWIAANIKEAA ddrb Ddrb _ ddrb.cif AlphaFold 3 unknown negative regulator of the P1 antirestriction system. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage P1 https://www.ncbi.nlm.nih.gov/nuccore/NC_005856.1 10.1111/mmi.13705 dara;hdf;darb;ddra;ulx PF18763 Genetic deletions (deletion mutation phenotypic evidence, ECO_0001038) result in high sensitivity to RM systems, while ectopic expression (ECO_0000017) restores plating efficiency. MSLSDQVVMATSIETLIELLKNLPDFGRVSYVVTAKGDEVKTAFDIVDASALLVSNTLDGKINPDYPQELQPRDRTRASSLLQVNQISKDLRPAQLTDSGLSSHGAPIIGEDNAVESGNGRTMGIIKAYQDGNADRYREYLIDHATEFGIPPEKVESMTAPVLVRRRLTKVDRVQFAKDSNISDLQEMAASEKAFVDADSITPAMMALFNPSESGDLLSRSNDAFIRGFMTQVGATQAAGLVTEDGRPTRQLVDRIQNAIFAKAYKDARLVRMVAEEPDPDMRNVLTALNAAANDFVQMQALSGEAHKQAVTTIVDGIETADSLDKKALAALKDAVDLVRQSKESGQHITDVIAQGDMFSETAPEVKALALFIVANNRSAKRMATAFKLMAQRINDELQHQGQALGDMFGGGDVSLQDILRQVSQELENEGMQGISGGLFESVSGGSYNGVAPYTSLLLHRASGIKDIIHLIRLLSRTDPHDEQLVQVLAHFVRMPVADVKKWCRLFGISNSLLRGLLNHASSLGRDGFDEIAQAIKNGDMPPAIDWFSIRPTRVKAFLSAAHSASSLAEMVQRLSLIFTDHTALGDLTLDEMKDASIQWADQQNEVNSDFLTAFRKAVSKADDARGILKAFKALQSRVNKHVGDIDGVTAEGRDILKEHGITPEFIDEIRTDMQREVVSSLQIVARALADANPKSAAIVNRVIGDIEASEGMGVLKLFLSRAFNPNGNILPGIIGEAKKYVSEEELEQLDQLLKRFSYNPQTRWQMNQRSMGSVHEKVLSAMNSAIANSYVSEEKALEWADSFITEEVEEVRAGQNGGIDLRKELADIYRLTGGKISTLSKVVHHQGRAYANLNGVVAVNLNDENASALWHELGHHLEYSNPGLLEKARSFLKANVEGDKPSFVNIGGRGKPEWCFRSRLSNIYMAKVYPPASVSNTGKIRQKSPTISKTSATEVFSMALQLYHDKEAAAASLMNGDGLLELLLGVAKELNNAD ulx Ulx _ ulx.cif AlphaFold 3 binds and protects the component targeted by the host defence binds and protects phage DNA. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage P1 https://www.ncbi.nlm.nih.gov/nuccore/NC_005856.1 10.1111/mmi.13705 dara;hdf;ddrb;darb;ddra Genetic deletions (deletion mutation phenotypic evidence, ECO_0001038) result in high sensitivity to RM systems, while ectopic expression (ECO_0000017) restores plating efficiency. MATLSDTIKPNKTYLEAVLRTALLGKTEDEYVDFFLSGLRGRLLKNPRLYRSYGPYWPEIKKLLLERGYGNFGRLVDRDVRKFYRYDRPALTLIAATLYSHERFDNGQIYSAWHLLPVPEEVDDQDYEFESYDLEVEALAQAGEKT hdf Hdf _ hdf.cif AlphaFold 3 binds and protects the component targeted by the host defence binds and protects phage DNA. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage P1 https://www.ncbi.nlm.nih.gov/nuccore/NC_005856.1 10.1016/0042-6822(87)90324-2;10.1111/mmi.13705 dara;ddrb;darb;ddra;ulx PF18788 Genetic deletions (deletion mutation phenotypic evidence, ECO_0001038) result in high sensitivity to RM systems, while ectopic expression (ECO_0000017) restores plating efficiency. MCQMTKNKYATVDFDQVNEKGLKSLITAINKTGVTVIEVDSSNRATTKDGVKVKTAKLVLSDGQILAIQVNDTGDISSVKLNGKAIPNAQSPDIKTLGTVMGQAARKNSAKFQKSLIAKAKRVANPVDKKPAVKSNFQRLQEAKQRNAQVVAAYKSAQNSVSFNQQQITDLRAKLDKETGRLNNEKARNGELKRRLKQLKAGN dam DNA adenine methylase 1YFJ_A dam.pdb PDB entry modifies phage molecules to avoid recognition recognises the sequence 5'-GATC-3' and methylates adenine, preventing recognition of phage DNA by the MboI restriction nuclease. Triggers Dazbog and retron Sen2 defences. Escherichia coli RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1016/0378-1119(83)90098-7 PF02086 dam_model.html Ectopic expression of the T4 dam⁺ gene in E. coli dam⁻ cells methylates plasmid DNA at GATC sites, rendering it resistant to MboI digestion (ECO_0000017). MLGAIAYTGNKQSLLPELKSHFPKYNRFVDLFCGGLSVSLNVNGPVLANDIQEPIIEMYKRLINVSWDDVLKVIKQYKLSKTSKEEFLKLREDYNKTRDPLLLYVLHFHGFSNMIRINDKGNFTTPFGKRTINKNSEKRFNHFKQNCDKIIFSSLHFKDVKILDGDFVYVDPPYLITVADYNKFWSEDEEKDLLNLLDSLNDRGIKFGLSNVLEHHGKENTLLKEWSKKYNVKHLNKKYVFNIYHSKEKNGTDEVYIFN aca3 Aca3 _ aca3.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1016/j.cell.2016.11.017 PF12844;PF01381 _ MKKFEAPEIGYTPANLKALRKQFGLTQAQVAEITGTKTGYSVRRWEAAIDAKNRADMPLVKWQKLLDSLK acriic1 AcrIIC1 5VGB_B acriic1.pdb PDB entry binds and inhibits host defence system binds to the Cas9 HNH nuclease domain and prevents cleavage of the nucleic acid sequence (allows DNA binding, blocks DNA cleavage). Neisseria meningitidis type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Brackiella oedipodis and Neisseria meningitidis 10.1016/j.cell.2016.11.017;10.1016/j.cell.2017.07.037 Binding was determined by size-exclusion chromatography (ECO_0001184, doi: 10.1016/j.cell.2017.07.037). MANKTYKIGKNAGYDGCGLCLAAISENEAIKVKYLRDICPDYDGDDKAEDWLRWGTDSRVKAAALEMEQYAYTSVGMASCWEFVEL acriic2 AcrIIC2 6J9M_B acriic2.pdb PDB entry binds and inhibits host defence system binds Cas9 through interactions with the positively charged bridge helix, thereby preventing crRNA loading. Neisseria meningitidis type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Neisseria meningitidis 10.1016/j.cell.2016.11.017 Binding is supported by x-ray crystallography evidence (ECO_0005670). MSKNNIFNKYPTIIHGEARGENDEFVVHTRYPRFLARKSFDDNFTGEMPAKPVNGELGQIGEPRRLAYDSRLGLWLSDFIMLDNNKPKNMEDWLGQLKAACDRIAADDLMLNEDAADLEGWDD acriic3 AcrIIC3 6JHV_A acriic3.pdb PDB entry binds and inhibits host defence system interacts with the HNH domain of Cas9 and induces Cas9 dimerisation (hindering DNA binding). Neisseria meningitidis type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Prophages in Neisseria meningitidis 10.1016/j.cell.2016.11.017;10.1016/j.cell.2017.07.037 Binding was supported by gel-filtration (ECO_0001049) and electron microscopy (ECO_0005033) evidence. MFKRAIIFTSFNGFEKVSRTEKRRLAKIINARVSIIDEYLRAKDTNASLDGQYRAFLFNDESPAMTEFLAKLKAFAESCTGISIDAWEIEESEYVRLPVERRDFLAAANGKEIFKI acriia1 AcrIIA1 5Y6A acriia1.pdb PDB entry binds and inhibits host defence system binds Cas9, triggering its degradation during lysogeny. Listeria monocytogenes type II-A and type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Listeria monocytogenes prophage 10403S 10.1016/j.cell.2016.12.009;10.1093/nar/gkx1181 PF13443 Genetic deletions of acrIIA1 (deletion mutation phenotypic evidence, ECO_0001038) in the prophage result in restored CRISPR-Cas9 function, as demonstrated by regained plasmid-targeting ability in Listeria monocytogenes. Conversely, ectopic expression of gene (ECO_0000017) in a CRISPR-active, prophage-cured background inhibits CRISPR-Cas9 activity, restoring transformation/plating efficiency with a targeted plasmid. Binding is supported by x-ray crystallography evidence (ECO_0005670). MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSILRSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEFELYCLIKEFESANIEVLPFTFNRFENEEHVNIKKDVCKALENAITVLKEKKNELL acriia2 AcrIIA2 6MCB_C acriia2.pdb PDB entry binds and inhibits host defence system prevents Cas9 binding to DNA by occluding the protein residues required for DNA binding. Listeria monocytogenes type II-A and type II-C CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Listeria monocytogenes prophage 10403S 10.1016/j.cell.2016.12.009;10.1016/j.molcel.2018.11.011 Genetic deletions of acrIIA2 (deletion mutation phenotypic evidence, ECO_0001038) in the prophage result in restored CRISPR-Cas9 function, as demonstrated by regained plasmid-targeting ability in Listeria monocytogenes. Conversely, ectopic expression of gene (ECO_0000017) in a CRISPR-active, prophage-cured background inhibits CRISPR-Cas9 activity, restoring transformation/plating efficiency with a targeted plasmid. Binding is supported by x-ray crystallography evidence (ECO_0005670). MTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKNEKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVETNDIDDLEIATDEDEMKSGNQEIILKSELK acriia3 AcrIIA3 _ acriia3.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas2 (https://www.ncbi.nlm.nih.gov/protein/ALU77912.1) and AcrIIA3 (ipTM = 0.7, pTM = 0.72). Listeria monocytogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Listeria monocytogenes prophage ΦSLCC2482 10.1016/j.cell.2016.12.009 Ectopic expression (ECO_0000017) of acrIIA3 in L. monocytogenes inhibits CRISPR-Cas9 function in a plasmid targeting assay. Binding it supported by computational structure modeling evidence (ECO_0006368). MFNKAEIMKQAWNWFNDSNIWLSDIEWVSYTDKEKSFSVCLKAAWSKAKEEVEESKKESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVKDETKINFGLSVWACAMKAVKLHNDLFPQTAA acriia4 AcrIIA4 5XN4 acriia4.pdb PDB entry binds and inhibits host defence system inhibits Cas9 enzymatic activity and DNA unwinding, and blocks the movement of HNH, thereby preventing DNA binding. Listeria monocytogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Listeria monocytogenes prophage ΦJ0161b 10.1016/j.cell.2016.12.009;10.1038/nature22377 PF24304 Ectopic expression (ECO_0000017) of acrIIA4 in L. monocytogenes, E. coli, and HEK293T cells effectively inhibits CRISPR-Cas9 and dCas9 activity. Binding is supported by x-ray crystallography evidence (ECO_0005670). MNINDLIREIKNKDYTVKLSGTDSNSITQLIIRVNNDGNEYVISESENESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN acriiib1 AcrIIIB1 _ acriiib1.cif AlphaFold 3 binds and inhibits host defence system Directly interacts with the Cmr-a complex Sulfolobus islandicus LAL14/1 type III-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Sulfolobus islandicus rod-shaped virus 2 https://www.ncbi.nlm.nih.gov/nuccore/NC_004086.1 10.1016/j.cell.2019.09.003 Genetic deletion of acrIIIB1 (deletion mutation phenotypic evidence, ECO_0001038) from the SIRV2 viral genome results in loss of viral infectivity in Sulfolobus islandicus strains with subtype III-B CRISPR-Cas systems, demonstrating restored CRISPR activity. Conversely, ectopic expression of acrIIIB1 (ECO_0000017) in a CRISPR-active host rescues viral infectivity, indicating inhibition of CRISPR function. Binding to the Cmr-a effector complexes is supported by co-purification in pull-down assays (co-purification evidence, ECO_0006074). MEVKQIKKLNNLPWVFLDTYLNKFALDKNFVNCAYYSSRSGMTQEGCVQVMQVGDNFKVDTMREVHGIYFTPHASIISLIYRQKGIRSIDDLKEILGSLNLSKVSPKHYQLLVKYSNYTIEIYDIYFKGHIYEFPLVSQQGHLNVYNVPEPRNVYLIYYENNEEKKELNKDLFNEVSEFMIYNHRVTFEKPVLEFKNLQITPGGGALVYVPESMYVKLESSDHQSVEFRPSRDDWLLFSHPRPRRSGND acb2 Acb2 8H2X_A acb2.pdb PDB entry degrades or sequesters molecules utilised by host defence systems binds and sequesters CBASS signalling molecules (3′,3′-cGAMP, c-di-AMP, 3′,3′-c-di-UMP, 3′,3′-cUA and 3′,3′-cUG). Triggers the Panoptes defence system (DOI: 10.1038/s41586-025-09557-z) by binding 2′,3′-c-di-AMP, a signalling molecule synthesised by the defence system to keep it inactive. Pseudomonas aeruginosa type II CBASS system CBASS https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass Pseudomonas phage PaMx33 https://www.ncbi.nlm.nih.gov/nuccore/1021470655 10.1016/j.cell.2022.12.041 PF24729 Genetic deletion of acb2 (deletion mutation phenotypic evidence, ECO_0001038) from the phage genome results in a loss of viral infectivity. Acb2 binding to the signaling molecules is supported by isothermal titration calorimetry evidence (ECO_0001825) and X-ray crystallography evidence (ECO_0005670). MDNQHRKIAGYRELTQDDIDLMNRVKAVGAELLALQAALAGRLSTDLEVKQAAAKASKLAPEHESSPECVELRRFLAAEPLRWAAIAKTDIQTGVMALVRAIAQPEGC t5_orf015 T5_ORF015 _ t5_orf015.cif AlphaFold 3 unknown _ Escherichia coli type bNACHT01 NLR/bNACHT NLR/bNACHT https://defensefinder.mdmlab.fr/wiki/defense-systems/nlr Escherichia phage T5 https://www.ncbi.nlm.nih.gov/nuccore/NC_005859.1 10.1016/j.cell.2023.04.015 Ectopic expression (ECO_0000017) of orf015 in E. coli inhibits bNACHT-mediated antiphage defense in a plaque formation assay. MIRNVSLARSKGFKLVDVNTFEREDCKIEYVARNKNAFRVTEKKFDKRGNVIAETVKHFATFYAAFRGVL acb3 Acb3 _ acb3.cif AlphaFold 3 binds and inhibits host defence system binds to the bacterial CD-NTase enzyme, which is involved in the synthesis of cyclic nucleotides in the CBASS response; also inhibits the human cGAS protein, a key component of the innate immune response to viral infections. E. coli KTE188 type III CBASS CBASS https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass Ga0194137_1000084820 (IMG/VR) 10.1016/j.cell.2024.12.035 Ectopic expression (ECO_0000017) of acb3 in E. coli inhibits CBASS-mediated antiphage defense in a plaque formation assay. MKEVYKHEFNYWQQLIAMLTTMWRINKDSIDFKWGYFAPRFGLELRLNRGGYFDPYYAIAFCFIWGKFHIKLPFKTSLGEGCDLPKYGFTVSSNTLMLYWGGKFDNSIGQTNSRLKCWDLPFISYVFEHHKVLNKQGTWEDGTASYDNQNINRESYPYTYVLKSGEIQQRTATCFVEERQWHRKWLPWVKLVKPTISIEFSSEVGERVGSWKGGVTGCGYDLLPDETIEECLKRMELTRKFT tad3 Tad3 9B7D tad3.pdb PDB entry binds and inhibits host defence system binds to ThsB (Thoeris type I) Bacillus cereus MSX-D12 type I Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Ga0172379_1000020175 (IMG/VR) 10.1016/j.cell.2024.12.035 Ectopic expression (ECO_0000017) of tad3 in Bacillus cereus inhibits Thoeris-mediated antiphage defense in a plaque formation assay. Binding is supported by co-purification in pull-down assays (co-purification evidence, ECO_0006074) and x-ray crystallography evidence (ECO_0005670). MNSITHAEFEFSLLENVKYETEDEVPIVLEYKEEIINLIKKFSNSGQSGMSAPITASIITNCIKNLMAFKPIGPLVGNEEEWNYNSDDSFQNNRLSAVFKTGLNGKPYYLDAITFVGEEEYDTFHGHVEGISSRQYLKGFPFFPKTFYINVYKDFENKDENNLCSGDDGEYTYRIKYPEQLEEVFNYYDKFTKE tad4 Tad4 _ tad4.cif AlphaFold 3 binds and inhibits host defence system binds to the bacterial ThsB (Thoeris type I) and also inhibits plant (Brachypodium distachyon, BdTIR) and human (Sterile alpha and TIR motif containing 1, SARM1) TIR proteins involved in the innate immune response. Bacillus cereus MSX-D12 type I Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Ga0307375_1001427612 (IMG/VR) 10.1016/j.cell.2024.12.035 Ectopic expression (ECO_0000017) of tad4 in Bacillus cereus inhibits Thoeris-mediated antiphage defense in a plaque formation assay. Binding is supported by co-purification in pull-down assays (co-purification evidence, ECO_0006074). MRLKDAEGWQKSREANQDPYGKAGLDYAERWAEMMEQWIPEDSTEKFITQQIENVAERCSHVADTEGITGFMYGCAVGLLSQVWEHGDALRRWHNLDCQIGTEGEEANESGKILNPAILDIK tad5 Tad5 _ tad5.cif AlphaFold 3 binds and inhibits host defence system binds to ThsB (Thoeris type I) Bacillus cereus MSX-D12 type I Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Ga0224422_1102149568 (IMG/VR) 10.1016/j.cell.2024.12.035 Ectopic expression (ECO_0000017) of tad5 in Bacillus cereus inhibits Thoeris-mediated antiphage defense in a plaque formation assay. Binding is supported by co-purification in pull-down assays (co-purification evidence, ECO_0006074). MKRLLTLRNMIKSTTGVITVMAPGYKPRNIQAIVEYIIKYFNENCSDQEGYWKSNKSVMFYIDSDNIYDFVDKMLKGCKEFNELNLSQYEVDRGITVDDSSRPAWVIGGTSTGDHLKEYYDFIDIDACVRNISGELYWGFLDNDLYEGKVEIVNISEAAE tad6 Tad6 _ tad6.cif AlphaFold 3 binds and inhibits host defence system binds to ThsB (Thoeris type I) Bacillus cereus MSX-D12 type I Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Ga0105013_100020934 (IMG/VR) 10.1016/j.cell.2024.12.035 Ectopic expression (ECO_0000017) of tad6 in Bacillus cereus inhibits Thoeris-mediated antiphage defense in a plaque formation assay. Binding is supported by co-purification in pull-down assays (co-purification evidence, ECO_0006074). MKMYVKAVLSVRRYKAGYEVREELVAGNQFDMDDIKVKTAYTPDGHYIGDSKTAYRLCKKRGIKPEPIDSEHNVCSIGFCEKEQKWYGWSHRAIYGFGIGSTCKKGDCHYVPTSFEEIQVDCYAKEEDDCVANCTVALEPVNPDEPERVQRAIPDSEEGRCVCAQENCVFEVGRGEWVAKTLDDAKQMAVDFAKSVA tad7 Tad7 _ tad7.cif AlphaFold 3 binds and inhibits host defence system binds to the bacterial type II Thoeris protein, ThsB Bacillus amyloliquefaciens Y2 type II Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Ga0114343_100093911 (IMG/VR) 10.1016/j.cell.2024.12.035 Ectopic expression (ECO_0000017) of tad7 in Bacillus amyloliquefaciens inhibits Thoeris-mediated antiphage defense in a plaque formation assay. METKEIVELMLKSSDHNPYTGMLSKKDNLIAAIDLAKLCKDFADNGQTDEAMGIPSEQWDEVIDELNNL tad8 Tad8 _ tad8.cif AlphaFold 3 binds and inhibits host defence system binds to the bacterial type II Thoeris protein, ThsA Bacillus amyloliquefaciens Y2 type II Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Ga0080708_10004799 (IMG/VR) 10.1016/j.cell.2024.12.035 Ectopic expression (ECO_0000017) of tad8 in Bacillus amyloliquefaciens inhibits Thoeris-mediated antiphage defense in a plaque formation assay. MKLFRNKETNKEIAERLIKLTDGKDYGIFAPPMKAQVAVDELCRYFLGEDWYSVNPISNEQINTEIVYEIECRFKKIKRG aris AriS _ aris.cif AlphaFold 3 unknown suppresses the SOS response by targeting RecA, Listeria monocytogenes strain 10403S SOS response SOS stress response _ Prophage in listeria monocytogenes strain 10403S 10.1016/j.celrep.2022.110723 AriS suppresses expression of SOS Gene (ECO_0000270), which is reversed by RecA overexperssion (ECO_0000012) MSNLQVIANDMLPVMENEKGEKFVNARELHQSLQVGKKFATWITDKFNNYGFSKDEDYFPILGESTFGRPRTEYLLTLDTAKELAMVQNNEMGRSIRKYFIEVEKQARKLATEYPTFSYMIEDPVARAKKWIEEQQEKQEALKQLEEQKPKVVFAEAVQTSENTILVKDLATILKQKGLDIGQNRLFEWLRCSGYLLNKGAYYNKPSQKAMNLGLFEQKTHIHTDRNGLMITTYTPRVTGKGQIYLLNKLLEEHNQVII ntase NTase _ ntase.cif AlphaFold 3 uncategorised synthesises cyclic dinucleotides that bind to and competitively inhibit host STING immune sensors. Sphingobacterium faecium TIR-STING effector CBASS;CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass;https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Bacillus phage Bcp1 https://www.ncbi.nlm.nih.gov/nuccore/NC_024137.1 10.1016/j.celrep.2023.112305 ntase_model.html Mass spectrometry evidence (ECO_0001096) for production of cyclic nucleotide. The NTases inhibited the NADase activity of TIR-STING from Sphingobacterium faecium in a dose-dependent manner, measured using a fluorescent ε-NAD substrate assay (ECO_0000184). MKTVDLSVTGMSWMEERTILLTPYGSRLYGTDTENSDWDFKGVCIPPKEYFLGLETFNEYNNTGGKTFKNTKDDVDINIIHVSKFVKDAMHGVPNNIEVLFAREQDYIILTELGQVLRDNRHLFLSKQIITKFGGYTRSLTNKLKNGAGRQELVEEFGYDTKNFMQGVRLQLSAIEILETGDYSTYRPERDFLLGCRNGEYTREQALALVESYDERLQVAHENSKLPEKPDYNKINGMLMAINEDALKFGIHS atd1 Atd1 _ atd1.cif AlphaFold 3 degrades or sequesters molecules utilised by host defence systems depletes the starvation alarmones (p)ppGpp. TIR response (in vitro) gp29/gp30 https://defensefinder.mdmlab.fr/wiki/defense-systems/mmb_gp29_gp30 Pectobacterium phage PcCB7V https://www.ncbi.nlm.nih.gov/nuccore/1950483712 10.1016/j.celrep.2023.112305 Pyrophosphatase assay measuring ppGpp-degrading activity of Atd1 confirms its function as a nucleotide pyrophosphohydrolase (ECO:0000005). MEDKPGYHLDKIKKRRFGSLGKIQEEVEELVDAHRQGSHVMILVEMSDLYGAMQGFLEENYPGFKMEDLKKFSGITQRAFKNGYRG acriia10 AcrIIA10 _ acriia10.cif AlphaFold 3 binds and inhibits host defence system (putative) binding affinity to SpCas9 was demonstrated using biolayer interferometry, but detailed MoA is unknown. Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Metagenome 10.1016/j.chom.2019.01.003 A synthetic E. coli strain was engineered with a genetic circuit where SpCas9 targeted a plasmid-borne chloramphenicol resistance gene. If an anti-CRISPR protein inhibits Cas9, the strain retains chloramphenicol resistance and survives antibiotic selection (ECO_0007003). In vitro cleavage assay evidence (a biochemical assay where purified Cas9 protein and guide RNA are incubated with target DNA and a candidate ACR protein outside of any living cell) further supported anti-CRISPR activity. Direct binding is supported by biolayer Interferometry evidence (ECO_0006350). MDNKFKLRKAINGIEELNFAFDKLTAIDYKTICRIERKMNGLSVDALADSIIASAGTRKTSSEFRIACAWVAAVKGTDGLTVDDYDQLSLDDLLELETFGLLFFVGSLE acriia8 AcrIIA8 _ acriia8.cif AlphaFold 3 unknown binding affinity to SpCas9 was demonstrated using biolayer interferometry, but detailed MoA is unknown. Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Metagenome 10.1016/j.chom.2019.01.003 A synthetic E. coli strain was engineered with a genetic circuit where SpCas9 targeted a plasmid-borne chloramphenicol resistance gene. If an anti-CRISPR protein inhibits Cas9, the strain retains chloramphenicol resistance and survives antibiotic selection (ECO_0007003). In vitro cleavage assay evidence (a biochemical assay where purified Cas9 protein and guide RNA are incubated with target DNA and a candidate ACR protein outside of any living cell) further supported anti-CRISPR activity. Direct binding is supported by biolayer Interferometry evidence (ECO_0006350). MSIFTDMIPAELLINEYKKGQSGAKHDNYVSVGRIMVAIYKNNSFKNTGTVKYQDSTHSGITMSKVFIDGKEYRIDIDTQHYEVQDFDTSGRQTTLILKRIDLYG acriia9 AcrIIA9 _ acriia9.cif AlphaFold 3 unknown binding affinity to SpCas9 was demonstrated using biolayer interferometry, but detailed MoA is unknown. Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Metagenome 10.1016/j.chom.2019.01.003 PF14058 A synthetic E. coli strain was engineered with a genetic circuit where SpCas9 targeted a plasmid-borne chloramphenicol resistance gene. If an anti-CRISPR protein inhibits Cas9, the strain retains chloramphenicol resistance and survives antibiotic selection (ECO_0007003). In vitro cleavage assay evidence (a biochemical assay where purified Cas9 protein and guide RNA are incubated with target DNA and a candidate ACR protein outside of any living cell) further supported anti-CRISPR activity. Direct binding is supported by biolayer Interferometry evidence (ECO_0006350). MKGTEHFKQTIKEYLDGRAQTDELFAVSYAKENKNLDDCITFILNQVKASGCCGMTDDEVWSLAIHYYDEDNIDVGNPISCGVVVNHKVELTEEEKAQARKEALKAYQEEEMRKIQQRHSKPKPTAKAAQSNQTELSLFDF hgmtad2 HgmTad2 8KBI hgmtad2.pdb PDB entry degrades or sequesters molecules utilised by host defence systems sequesters cyclic dinucleotides and gcADPR. Pseudomonas aeruginosa strain PAO1 type I (ThsA with SIR2) Thoeris CBASS;Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass;https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Metagenome 10.1016/j.chom.2019.01.003;10.1038/s41586-024-08122-4 PF11195 A synthetic E. coli strain was engineered with a genetic circuit where SpCas9 targeted a plasmid-borne chloramphenicol resistance gene. If an anti-CRISPR protein inhibits Cas9, the strain retains chloramphenicol resistance and survives antibiotic selection (ECO_0007003). In vitro cleavage assay evidence (a biochemical assay where purified Cas9 protein and guide RNA are incubated with target DNA and a candidate ACR protein outside of any living cell) further supported anti-CRISPR activity. However, direct binding is not supported by biolayer Interferometry evidence (ECO_0006350). Another study was not able to reproduce anti-CRISPR activity (10.1038/s41586-024-08122-4). Gel Shift Assay (ECO_0000096) was used to test binding to cyclic oligonucleotides, affinity was measured by Surface Plasmon Resonance (ECO_0001127), and there is X-ray crystallography evidence (ECO_0005670) to support the binding. MTFGQALESLKRGHLVARKGWNGKGMFIFMRPEDSLPTNMIVNQVKSLPESFKRWVANNHGDSETDRIKFTAYLCMKAADGTIVNGWLASQTDMLANDWVIVE acriia12 AcrIIA12 _ acriia12.cif AlphaFold 3 unknown _ Listeria monocytogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Listeria monocytogenes prophage φA006 10.1016/j.chom.2020.04.001 Ectopic expression (ECO_0000017) of acrIIa12 in Listeria monocytogenes inhibits Cas9-mediated targeting measured in a plaque formation assay. It inhibits CRISPRi, likely acting upstream of DNA targeting. MSKTMYKNDVIELIKNAKTNNEELLFTSVERNTREAATQYFRCPEKHVSDAGVYYGEDFEFDGFEIFEDDLIYTRSYDKEELN acriiib2 AcrIIIB2 _ acriiib2.cif AlphaFold 3 binds and inhibits host defence system blocks the dissociation of cleaved target RNA from Cmr-α, inhibiting Cmr-α turnover and thereby suppressing Cas10 activities. Sulfolobus islandicus type III-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Sulfolobus islandicus rod-shaped virus 3 isolate SIRV3 https://www.ncbi.nlm.nih.gov/nuccore/KX712143.1 10.1016/j.chom.2023.10.003 The gene gp40 from SIRV3 (AcrIIIB2) was used to replace the known Acr gene (acrIIIB1) in SIRV2. The recombinant virus SIRV2ΔacrIIIB1+gp40 was able to infect S. islandicus LAL14/1, which has a functional type III-B CRISPR-Cas system. This restored infectivity demonstrated that gp40 compensates for the loss of acrIIIB1, confirming its role as an anti-CRISPR protein. Pull-down assays with HA-tagged AcrIIIB2 revealed direct interaction with the Cmr-a complex, a type III-B effector (ECO_0006074). MSSTCNPITSFGKLLNKIAKQSLISSQLWNDIIQDLYTAYSVYKYINTSLQYQFQYEFSYGNLNTLNSLFNNLYFYIFNQKPYQFVPLINASPGLPLTVNYMNNLIKAITKLANENNITLAKPLNFVQSNEIVKSRKFNDIIYVINQFLTFDFNSYFLLDCYGSIFSNLVNSQSAFLNILIDNPSQNISTNNIYIKNLIINYLKTVLNFYGNSAIDNFLIGNHQPPYLAVVGSSIYLYNNSAINNLIIYENYGYIQLNDNSYIQNLIISINNATIELNDNAIIENLICKQNYGQIQINGNAQIINNNCQ zadi_1 ZadI-1 _ zadi_1.cif AlphaFold 3 unknown _ Zorya type I Zorya https://defensefinder.mdmlab.fr/wiki/defense-systems/zorya/ Pseudomonas phage vB_PaeM_FBPa35 https://www.ncbi.nlm.nih.gov/nuccore/ON857938.1 10.1016/j.chom.2025.06.010 Ectopic expression (ECO_0000017) of ZadI-1 in Pseudomonas aeruginosa inhibits Zorya type I-mediated antiphage defense in plaque formation and liquid culture collapse assays. Alanine substitutions at conserved lysines K64 and K68 abolish the anti-defense activity (ECO_0001038). MTSSKWTIGRNDTIEVEAVNSREDFRWNGKIRVIHYSAGQIVNIIEFYHHDLDWAIKNFGIKLKAVSKGLEILHTCYFGKYVK dadiii DadIII-1 _ dadiii.cif AlphaFold 3 unknown _ Druantia type III Druantia https://defensefinder.mdmlab.fr/wiki/defense-systems/druantia Pseudomonas phage vB_PaeM_FBPa21 https://www.ncbi.nlm.nih.gov/nuccore/ON857942.1 10.1016/j.chom.2025.06.010 Ectopic expression (ECO_0000017) of DadIII-1 in Pseudomonas aeruginosa inhibits Druantia type III-mediated antiphage defense in plaque formation and culture collapse assays. MPSSSKRVDPSFIRESLRLLCGLFGPREPADGRKDQDRTLNSLGSGAAVLWAIAMSIEVYPVVVAAPELDRSGSSLEHHRAFVSKNMFRHRMFLYNVQLCFTSAQEEYTIS tadiii_1 TadIII-1 _ tadiii_1.cif AlphaFold 3 unknown AF3 predicts an interaction between TadIII-1 molecules and the ThcB1 dimer, but the co-purification experiment did not detect it Thoeris type III Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Pseudomonas phage vB_PaeM_FBPa12 https://www.ncbi.nlm.nih.gov/nuccore/ON857930.1 10.1016/j.chom.2025.06.010 An interaction between two TadIII-1 molecules and the ThcB1 dimer was predicted (computational structure modeling evidence, ECO_0006368). MNATYQALKTLRDSCEAAKDEKGTINGNKLNALRNKAVKEMEAGGETYSDAIAMAHDLIKKYRKQSPARFAGP bdi1 Bdi1 _ bdi1.cif AlphaFold 3 unknown inhibit a broad range of nucleic-acid-targeting defense systems, but MoA is unknown Druantia type I;Zorya type I;Hypnos;RADAR Druantia;Zorya;Hypnos;RADAR https://defensefinder.mdmlab.fr/wiki/defense-systems/druantia;https://defensefinder.mdmlab.fr/wiki/defense-systems/zorya/;_;https://defensefinder.mdmlab.fr/wiki/defense-systems/radar Pseudomonas phage Nemo https://www.ncbi.nlm.nih.gov/nuccore/KT372694.1 10.1016/j.chom.2025.06.010 Ectopic expression (ECO_0000017) of Bdi1 in Pseudomonas aeruginosa inhibits Zorya type I, RADAR, Hypnos, and Druantia type I antiphage defense systems in plaque formation and culture collapse assays. Site-directed mutagenesis of residues D14A, L33E, and W54A abolishes Bdi1-mediated inhibition of Zorya type I (ECO_0001038). MAKVVRVDELKTGDEILIKLRADAAARNKAIVLSVECWRDEITLELTCPAGDYWEDWRGKYRAYDKVVLLKRD bdi2 Bdi2 _ bdi2.cif AlphaFold 3 unknown inhibit a broad range of nucleic-acid-targeting defense systems, but MoA is unknown Druantia type I;Zorya type I;Hypnos;RADAR Druantia;Zorya;Hypnos;RADAR https://defensefinder.mdmlab.fr/wiki/defense-systems/druantia;https://defensefinder.mdmlab.fr/wiki/defense-systems/zorya/;_;https://defensefinder.mdmlab.fr/wiki/defense-systems/radar Pseudomonas phage vB_PaeM_FBPa10 https://www.ncbi.nlm.nih.gov/nuccore/ON857929.1 10.1016/j.chom.2025.06.010 Ectopic expression (ECO_0000017) of Bdi2 in Pseudomonas aeruginosa inhibits Zorya type I, RADAR, Hypnos, and Druantia type I antiphage defense systems in plaque formation and culture collapse assays. MAKILMACELLVGDEILTHVDVNDPVRRRAIVLRSGAAPHSKVSIEVSALIGADWVDFKLKLAGTILPSAYMTSTRSALAALPNRRTPWQKL ipi_ IPI* 2JUB ipi_.pdb PDB entry binds and inhibits host defence system binds and inhibits the GmrS/GmrD complex (glucose-modified hydroxymethylcytosine restriction endonuclease). Escherichia coli type IV RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1016/j.jmb.2007.10.064 PF11634 Ectopic expression (ECO_0000017) of IPI* in Escherichia coli rescues infection by T4 and other glucosyl-HMC-modified T-even phages from restriction by the GmrSD type IV endonuclease in plaque formation assays. Purified IPI* inhibits GmrSD nuclease activity in vitro (ECO_0000184). Site-directed mutagenesis of residue A40T abolishes IPI*-mediated inhibition of GmrSD (ECO_0001038). MKTFKEFTSTTTPVSTITEATLTSEVIKANKGREGKPMISLVDGEEIKGTVYLGDGWSAKKDGATIVISPAEETALFKAKHISAAHLKIIAKNLL gp4_5 Gp4.5 _ gp4_5.cif AlphaFold 3 unknown inhibits the Lon Protease activity (it degrades the antitoxin) Escherichia coli type II TA SanaTA TA https://defensefinder.mdmlab.fr/wiki/defense-systems/sanata Escherichia phage T7 https://www.ncbi.nlm.nih.gov/nuccore/NC_001604.1 10.1016/j.molcel.2013.02.002 PF17574 Ectopic expression of Gp4.5 in E. coli restored susceptibility to phage in strains otherwise resistant due to the presence of the sanaTA system (ECO_0000017). Co-immunoprecipitation supports direct interaction with the Lon protease (ECO_0006030). MSNVAETIRLSDTADQWNRRVHINVRNGKATMVYRWKDSKSSKNHTQRMTLTDEQALRLVNALTKAAVTAIHEAGRVNEAMAILDKIDN acrvia1_plus AcrVIA1+ _ acrvia1_plus.cif AlphaFold 3 binds and inhibits host defence system binds to Cas13a. Leptotrichia wadei type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Leptotrichia wadei 10.1016/j.molcel.2020.03.033 Ectopic expression (ECO_0000017) of AcrVIA1 in rescues phage from Cas13a interference in plaque assays. Purified AcrVIA1 inhibits Cas13a RNase activity in vitro (ECO_0000184). Co-immunoprecipitation confirms direct interaction with LwaCas13a (ECO_0005644). MEKIKLICLRINNDELITTDKDEWLKFIKRHRGKVSSIEQFNWKIPGNKLQKALEYSFDELYKFKQKENRRETD acrvia2_plus AcrVIA2+ 7XMW acrvia2_plus.pdb PDB entry binds and inhibits host defence system binds to the Leptotrichia wadei Cas13-crRNA, inhibiting its activity. Leptotrichia buccalis type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Leptotrichia wadei 10.1016/j.molcel.2020.03.033 Ectopic expression (ECO_0000017) of AcrVIA2 in restores growth under Cas13a-mediated targeting in both phage and plasmid interference assays. Purified AcrVIA2 inhibits Cas13a RNase activity in vitro (ECO_0000184). Binds specifically to the Cas13a-crRNA complex (ECO_0005644). MWKCKKCGCDRFYQDITGGISEVLEMDKDGEVLDEIDDVEYGDFSCAKCDNSSSKIQEIAYWDEINGKNKTYLSKDK acrvia3_plus AcrVIA3+ _ acrvia3_plus.cif AlphaFold 3 binds and inhibits host defence system binds to the Leptotrichia wadei Cas13-crRNA, inhibiting its activity. Leptotrichia wadei type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Leptotrichia wadei 10.1016/j.molcel.2020.03.033 Ectopic expression (ECO_0000017) of AcrVIA3 protects against Cas13a-mediated interference in plaque and plasmid transformation assays. In vitro assays show AcrVIA3 inhibits Cas13a enzymatic activity (ECO_0000184). Co-immunoprecipitation indicates selective binding to the Cas13a-crRNA complex (ECO_0005644). MFKEFLEKCLRYGNLYILEETGDRKKVKRISKRHGKVTEASVLLFDSGTKRTTINEIYLNSQGYFIIRDQKRLKLEKFK acrvia4_plus AcrVIA4+ _ acrvia4_plus.cif AlphaFold 3 binds and inhibits host defence system binds to the Leptotrichia wadei Cas13a. Leptotrichia buccalis and Leptotrichia wadei type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Leptotrichia wadei 10.1016/j.molcel.2020.03.033 Ectopic expression (ECO_0000017) of AcrVIA4 in E. coli inhibits Cas13a-based interference in plaque assays. In vitro inhibition of Cas13a cleavage confirmed (ECO_0000184). Co-immunoprecipitation confirms binding to LwaCas13a (ECO_0005644). MDKANRCLKAKDKILNILEKEEITLDEFNNISKDIAKEYVEKAVLKPKDIAERIINMVKNAKSISFDELASEISEE acrvia5_plus AcrVIA5+ _ acrvia5_plus.cif AlphaFold 3 binds and inhibits host defence system binds to the Leptotrichia wadei Cas13a. Leptotrichia buccalis type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Leptotrichia wadei 10.1016/j.molcel.2020.03.033 Ectopic expression (ECO_0000017) of AcrVIA5 abolishes Cas13a interference in bacterial plaque and plasmid targeting assays. Purified AcrVIA5 strongly inhibits Cas13a cleavage in vitro (ECO_0000184). Co-immunoprecipitation confirms binding to Cas13a (ECO_0005644). Mutation studies show key residues required for inhibition (ECO_0001038). MERNFKKVTENTGRKEVFKVMHDKVEIINDFNTNEKREARIIFHDQKIYVILYQNLNFEELKWLNFYILIYGNQSYGKNTFFEFKLNKNNLIYHLQVWNIIENKKFKSKSISLLVKALSSKAGV acrvia6_plus AcrVIA6+ _ acrvia6_plus.cif AlphaFold 3 binds and inhibits host defence system binds to the Leptotrichia wadei Cas13a. Leptotrichia wadei type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Rhodobacter capsulat R121 10.1016/j.molcel.2020.03.033 Ectopic expression (ECO_0000017) of AcrVIA6 from Rhodobacter capsulatus rescues phage infection from Cas13a in E. coli. In vitro assays confirm inhibition of Cas13a RNA cleavage (ECO_0000184). Co-immunoprecipitation confirms direct interaction with Cas13a (ECO_0005644). MADKVKSIQPGPIFYDVFLVYLRVIGTNLKDWCAPHGVTATNAKSAATGGWNGTKARALRQKMIDEVGEETFLRLYTERLRREAA acrvia7_plus AcrVIA7+ _ acrvia7_plus.cif AlphaFold 3 unknown _ Leptotrichia wadei type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Leptotrichia buccalis 10.1016/j.molcel.2020.03.033 Ectopic expression (ECO_0000017) of AcrVIA7 from Leptotrichia buccalis rescues cells from Cas13a activity in plaque assays. Purified AcrVIA7 inhibits Cas13a RNase activity in vitro (ECO_0000184). MRIIKLYERIIPKTSSTSYISRWEALNIPDENRNTAAWHPRTYLFSYDKDKAINLYNTTNVLGNSGIKKRIIDYPSKREVYIANFPRAIADLVLTMKDYQLSSLHNCCNDFFNEDETEQLYQYLRSIKDNRRVDEFLKYEFTVRYFNDKKF u56 U56 _ u56.cif AlphaFold 3 unknown _ Escherichia coli type II Retron (Eco1) Retron https://defensefinder.mdmlab.fr/wiki/defense-systems/retron Escherichia phage ukendt https://www.ncbi.nlm.nih.gov/nuccore/NC_052661.1 10.1016/j.molcel.2024.05.001 PF08719 u56_model.html Ectopic expression of U56 rescues phage from Retron-Eco1 in plaque assays (ECO_0000017). Purified U56 binds ADPr with micromolar affinity as shown by isothermal titration calorimetry (ECO_0001825). MRITDEYVFFFSHKDVFSNWYIAPFTETEPGYNETFCCVEQYMMWRKALLFKDHAIAAAILDHSTIRDNERKQAYYKRMGRAVSGFNNDMWEENRERIVMRGLCLKYVQNPDLYADLHLYQYKTFVEASPYDKVYGIGMGMYEPGVLNPATWKGQNLLGKYHNKLIEILFQDKIPQRRYL acrvib1 AcrVIB1 _ acrvib1.cif AlphaFold 3 binds and inhibits host defence system binds to the Riemerella anatipestifer Cas13b. Prevotella buccae type VI-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Riemerella anatipestifer 10.1016/j.molcel.2022.05.003;10.1016/j.molcel.2025.01.020 Ectopic expression (ECO_0000017) of AcrVIB1 abolishes Cas13b interference in bacterial plasmid transformation and phage infection assays. Cell-free TXTL assays show potent inhibition of Cas13b RNA cleavage activity (ECO_0006064). Timing experiments indicate inhibition occurs upstream of ribonucleoprotein complex formation (AcrVIB1 is most effective when added before RNP (Cas13b:gRNA) complex formation). MKDLDLSKLKGEEIAQWLLNNKKATAIQLSSERTDTDDGFMHILVHKDEYVEIIYSYLKIDEDDVMQNFTIYSKRWGNIDNSYFELQTFEGEIFTGESDKILCGVLSLGDLTTLK ugi Ugi _ ugi.cif AlphaFold 3 binds and inhibits host defence system binds specifically and reversibly to the host uracil-DNA glycosylase, preventing removal of uracil residues from PBS2 DNA by the host uracil-excision repair system. Bacillus subtilis uracil-DNA glycosylase DNA repair _ Bacillus phage PBS2 10.1016/S0021-9258(19)70472-4 PF18880 Biochemical evidence of inhibition of UDG activity using a [³H]-poly(dU) substrate (ECO_0000184). MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML ocr Ocr 1S7Z ocr.pdb PDB entry binds and inhibits host defence system binds to the DNA-binding groove of the type I DNA restriction/modification complex with higher affinity than DNA and blocks it. Triggers Paris defence. Escherichia coli type I RM and type I BREX RM;BREX https://defensefinder.mdmlab.fr/wiki/defense-systems/rm;https://defensefinder.mdmlab.fr/wiki/defense-systems/brex Escherichia phage T7 https://www.ncbi.nlm.nih.gov/nuccore/NC_001604.1 10.1016/0022-2836(75)90083-2;10.1016/s1097-2765(02)00435-5;10.1093/nar/gkaa290;10.1038/s41467-025-57006-2 PF08684 Wild-type bacteriophage T7 is not subject to restriction by the Escherichia coli B and K restriction systems, but T7 mutants that are susceptible to such restriction have been isolated. These mutants are all defective in gene 0.3 (ECO_0000016), which encodes the Ocr protein. Ocr deletion mutants are unable to plate on restricting hosts (ECO_0001038). Moreover, T7 Δ0.3 mutants were blocked by BREX, while wild-type T7 (with intact Ocr) overcame BREX-mediated restriction (ECO_0000016). Complementation with plasmid-encoded Ocr restored infectivity (ECO_0000017), and Ocr was shown to physically interact with the BrxX methyltransferase (x-ray crystallography evidence, ECO_0005670). MAMSNMTYNNVFDHAYEMLKENIRYDDIRDTDDLHDAIHMAADNAVPHYYADIFSVMASEGIDLEFEDSGLMPDTKDVIRILQARIYEQLTIDLWEDAEDLLNEYLEEVEEYEEDEE arn Arn 3WX4 arn.pdb PDB entry binds and inhibits host defence system binds and inhibits the Rgl enzyme (restriction of non-glucosylated DNA) through DNA mimicry. Triggers Paris defence Escherichia coli type IV RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1038/260454a0;10.1074/jbc.M114.590851 PF22134 MIIDSQSVVQYTFKIDILEKLYKFLPNLYHSIVNELVEELHLENNDFLIGTYKDLSKAGYFYVIPAPGKNIDDVLKTIMIYVHDYEIEDYFE aca1 Aca1 7FA3_A aca1.pdb PDB entry unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1038/nature11723 _ MRFPGVKTPDASNHDPDPRYLRGLLKKAGISQRRAAELLGLSDRVMRYYLSEDIKEGYRPAPYTVQFALECLANDPPSA acrif1 AcrIF1 5uz9_I acrif1.pdb PDB entry binds and inhibits host defence system directly binds Cas7, sterically occluding target DNA and preventing its hybridisation to the crRNA. Pseudomonas aeruginosa PA14 type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage JBD30 https://www.ncbi.nlm.nih.gov/nuccore/NC_020198.1 10.1038/nature11723 PF20829 Phage JBD30 evades type I-F CRISPR-Cas immunity in P. aeruginosa PA14 through expression of AcrIF1 (gene 35). A frameshift mutation in gene 35 abolished infectivity on CRISPR-proficient hosts (ECO_0007269). Complementation with plasmid-expressed AcrIF1 restored phage replication (ECO_0000017). MKFIKYLSTAHLNYMNIAVYENGSKIKARVENVVNGKSVGARDFDSTEQLESWFYGLPGSGLGRIENAMNEISRRENP acrif3 AcrIF3 5GQH_B acrif3.pdb PDB entry binds and inhibits host defence system binds to the Cas3 nuclease and locks it in the ADP-bound form, thus preventing it from binding to the effector-DNA complex and cleaving the target DNA. Pseudomonas aeruginosa PA14 type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage JBD5 https://www.ncbi.nlm.nih.gov/nuccore/NC_020202.1 10.1038/nature11723 PF21401 Phage MP29 expresses AcrF3 (gene 29), which blocks CRISPR interference in P. aeruginosa PA14. Plasmid-based expression of AcrF3 restored transformation of targeted plasmids and increased phage plaquing efficiency (ECO_0000016, ECO_0000017). MSNTISDRIVARSVIEAARFIQSWEDADPDSLTEDQVLAAAGFAARLHEGLQATVLQRLVDESNHEEYREFKAWEEALLNADGRVASSPFADWGWWYRIANVMLATASQNVGVTWGSRVHGRLMAIFQDKFKQRYEEQA acrif4 AcrIF4 7YHS_J acrif4.pdb PDB entry binds and inhibits host defence system binds to Cascade, the RNA-guided Csy complex, blocking DNA binding. Pseudomonas aeruginosa PA14 type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage JBD24 https://www.ncbi.nlm.nih.gov/nuccore/NC_020203.1 10.1038/nature11723 AcrF4 (gene 30) was cloned from phage D3112 and shown to counteract CRISPR immunity in P. aeruginosa. Plasmid expression of AcrF4 in PA14 enhanced replication of CRISPR-sensitive phages (ECO_0000017). MMTISKTDIDCYLQTYVVIDPVSNGWQWGIDENGVGGALHHGRVEMVEGENGYFGLRGATHPTEKEAMAAALGYLWRCRQDLVAIARNDAIEAEKYRAKA acrif5 AcrIF5 7F45 acrif5.pdb PDB entry binds and inhibits host defence system binds to the Csy–dsDNA complex, destabilising the helical bundle domain of Cas8f and thus preventing subsequent Cas2/3 recruitment. Pseudomonas aeruginosa PA14 type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage JBD5 https://www.ncbi.nlm.nih.gov/nuccore/NC_020202.1 10.1038/nature11723 The AcrF5 protein, encoded by gene 33 of phage JBD88a, enables escape from CRISPR interference in type I-F systems. Expression in P. aeruginosa PA14 permitted replication of targeted phages and efficient transformation of CRISPR-targeted plasmids (ECO_0000017). MSRPTVVTVTETPRNPGSYEVNVERDGKMVVGRARAGSDPGAAAAKAMQMAMEWGSPNYVILGSNKVLAFIPEQLRVKM gp54 Gp54 _ gp54.cif AlphaFold 3 unknown _ Mycobacterium phage Phrann gp29/gp30 gp29/gp30 https://defensefinder.mdmlab.fr/wiki/defense-systems/mmb_gp29_gp30 Mycobacterium phage Tweety https://www.ncbi.nlm.nih.gov/nuccore/EF536069.1 10.1038/nmicrobiol.2016.251 A gain-of-function mutants of gp54 (in the DEM 200-series) acquire the ability to bypass the defence (ECO_0000042), but deletion of gp54 (Tweety Δ54) restores sensitivity to the Phrann defence system (ECO_0001038), demonstrating that gp54 is not essential for lytic growth, but its presence (in variant forms) enables escape from prophage-mediated inhibition. MSIDLDRITHPLRLAKGSHQPGSGKGCAMNVISYINGDTKITDYPECSARPLAALVQMCNDQLAGPDGFLSAENSVLVLDLGWQTVGTAGVSDAVHALWIADMLDSPEWGVIRFADEVGAVAIREIADLHRQAAAGQVPFAWAAWSAAGYAAWSAAWSAAQSAAGYAAGSAAGYAAGSAAGSAAGSAAGYAAGSAAGSAAGSAAWSAAWYAAGSAAWYAAGSAAQSAAGYAAGSAAGSAAGSAAGSAAGSAAGYAAGSAAWYAAGSAAWYAAGSAAQSAAGSAAGSAAGSAAGSAAGSAAWSAAGSAAGSAAGSAAWSAAGSAAWSAAGSAAGSAALIEFTRQAIARWRELAGLDLETEIDAADINAALARING aca2 Aca2 7EZY_A aca2.pdb PDB entry unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1038/nmicrobiol.2016.85 PF08965;PF13560 _ MTHYELQALRKLLMLEVSEAAREIGDVSPRSWQYWESGRSPVPDDVANQIRNLTDMRYQLLELRTEQIEKAGKPIQLNFYRTLDDYEAVTGKRDVVSWRLTQAVAATLFAEGDVTLVEQGGLTLE acrif10 AcrIF10 6ANW acrif10.pdb PDB entry binds and inhibits host defence system binds to Cas5f and Cas8f, occupying the DNA duplex binding site. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Vibrio cyclitrophicus 10.1038/nmicrobiol.2016.85 Ectopic expression (ECO_0000017) of AcrIF10 in Pseudomonas aeruginosa resulted in inhibition of type I-F CRISPR-Cas system activity, as demonstrated by restored replication of CRISPR-targeted bacteriophages in plaque assays. MTTFRIENVRIETINDFDMVKFDLVTDLGRVELAEHVNYDSEGDFKSVEYTDSNIRYNMVDELCSVFDLTDKPSLMPAIDYVTFAEIIEAVEEMLEA acrif6 AcrIF6 6vqx_A acrif6.pdb PDB entry binds and inhibits host defence system binds at the junction between Cas7.6f and Cas8f to inhibit DNA duplex splitting. Pseudomonas aeruginosa type I-F and I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa 10.1038/nmicrobiol.2016.85 Ectopic expression (ECO_0000017) of AcrIF6 in Pseudomonas aeruginosa resulted in inhibition of type I-F CRISPR-Cas system activity, as demonstrated by restored replication of CRISPR-targeted bacteriophages in plaque assays. MKVPAFFAANILTIEQIIEAINNDGSAMTSAPEIAGYYAWDAATDALESENDLEQLTEDDFVAHLEVLEERGAKIDRDAAIAVALQFQAAAVNDLHSGDE acrif8 AcrIF8 6VQW_A acrif8.pdb PDB entry binds and inhibits host defence system binds to the Csy spiral backbone, occupying the cavity surrounded by Cas5f, Cas7.4–7.6f, and Cas8f, to prevent DNA hybridisation. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pectobacterium phage ZF40 https://www.ncbi.nlm.nih.gov/nuccore/NC_019522.1 10.1038/nmicrobiol.2016.85 Ectopic expression (ECO_0000017) of AcrIF8 in Pseudomonas aeruginosa resulted in inhibition of type I-F CRISPR-Cas system activity, as demonstrated by restored replication of CRISPR-targeted bacteriophages in plaque assays. MARIAPNEDSTMSTAYIIFNSSVAAVVDTEIANGANVTFSTVTVKEEINANRDFNLVNAQNGKISRAKRWGNEASKCEYFGREINPTEFFIK acrif9 AcrIF9 7C78 acrif9.pdb PDB entry binds and inhibits host defence system binds to the effector complex, triggering sequence-non-specific dsDNA binding. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Vibrio parahaemolyticus 10.1038/nmicrobiol.2016.85 Ectopic expression (ECO_0000017) of AcrIF9 in Pseudomonas aeruginosa resulted in inhibition of type I-F CRISPR-Cas system activity, as demonstrated by restored replication of CRISPR-targeted bacteriophages in plaque assays. MKAAYIIKEVQNINSEREGTQIEATSLSQAKRIASKEQCFHGTVMRIETVNGLWLAYKEDGKRWVDCQ acriia6 AcrIIA6 6EYX acriia6.pdb PDB entry binds and inhibits host defence system binds allosteric centre of the Cas9 protein inducing its dimerisation and reducing its DNA binding affinity Lactococcus lactis type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Streptococcus phage D4276 https://www.ncbi.nlm.nih.gov/nuccore/NC_070686.1 10.1038/s41467-018-05092-w Ectopic expression (ECO_0000017) of AcrIIA6 from a plasmid in Streptococcus thermophilus with a type II-A CRISPR-Cas system completely abolished immunity against the CRISPR-sensitive virulent phage D5842. MKINDDIKELILEYMSRYFKFENDFYKLPGIKFTDANWQKFKNGGTDIEKMGAARVNAMLDCLFDDFELAMIGKAQTNYYNDNSLKMNMPFYTYYDMFKKQQLLKWLKNNRDDVIGGTGRMYTASGNYIANAYLEVALESSSLGSGSYMLQMRFKDYSKGQEPIPSGRQNRLEWIENNLENIR dpda DpdA _ dpda.cif AlphaFold 3 modifies phage molecules to avoid recognition inserts the 7-deazaguanine base into phage genomic DNA, with 2′-deoxy-7-deazaguanine modifications protecting phage DNA from host restriction enzymes. Type II RM (in vitro) RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage 9g https://www.ncbi.nlm.nih.gov/nuccore/NC_024146.1 10.1038/s41467-019-13384-y dPreQ0 was found in strains expressing dpdA (ECO_0001096). Phages with modifications (e.g., dG+, dPreQ0, dPreQ1) showed varied resistance to various restriction enzymes. MNVFMAAVYTNGYMPGQQRYEKLSEHEKNITRNLPHILESYHYVGRQKYVDQMRADGAKVFLDSGAFSAHSLGAHIDIVEYCEYIKRNKDILRVEDGAVMASVLDGIGDPLQTYRNQLEMEARGAKPLPCFHFGEDFRYLEYYMKNYEYITIGGMVGRSTDTLKTWLDRMWDKYICDGSGRAKIKLHAFGITSTTIMERYPWYSCDSSSWIQAAAFGSVVTPPWGPMRVSDKSPDRHHFGKHVSTLTEIEQDAVLKYLEQNGFTYERLSTIYESRAAFNLWAYGVIAANINATHDGTFRDRVMELF fole FolE _ fole.cif AlphaFold 3 modifies phage molecules to avoid recognition converts GTP into dihydroneopterin triphosphate (the first step of synthesis of 7-cyano-7-deazaguanine, a precursor in the pathway that leads to various DNA modifications found in phages). Type II RM (in vitro) RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage 9g https://www.ncbi.nlm.nih.gov/nuccore/NC_024146.1 10.1038/s41467-019-13384-y quec;qued;quee PF01227 fole_model.html Viral genes (e.g., folE, queD, queE from phage 9g) were expressed in corresponding E. coli deletion mutants (ΔfolE, ΔqueD, ΔqueE), restoration of queuosine (Q) modification in tRNA confirmed that these viral enzymes are functional analogs of their bacterial counterparts (ECO_0000017). Phages with modifications (e.g., dG+, dPreQ0, dPreQ1) showed varied resistance to various restriction enzymes. MIGFLKSWDGFGIKCMKTQLSTMFDLYRNFIHLFMIIKEEYKMKIEHLDKIGNVLGRENGWASLKPDEIVTLDNTEAAVQRLFGLLGEDAERDGLQDTPFRFVKALAEHTVGYREDPKLHLEKTFDVDHEDLVLVKDIPFNSLCEHHLAPFVGKVHIAYIPKDKITGLSKFGRVVEGYAKRLQVQERLTQQIADAIQEVLNPQAVAVIVEAEHTCMSGRGIKKHGATTVTSTMRGLFQDDASARAELLQLIKK quec QueC _ quec.cif AlphaFold 3 modifies phage molecules to avoid recognition catalyses the ATP-dependent conversion of 7-cyano-7-deazaguanine (preQ0) into 7-carboxy-7-deazaguanine (CDG). Type II RM (in vitro) RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage 9g https://www.ncbi.nlm.nih.gov/nuccore/NC_024146.1 10.1038/s41467-019-13384-y fole;qued;quee PF06508 quec_model.html dG+ was significantly present only when both dpdA and gat-queC were expressed (ECO_0001096). Phages with modifications (e.g., dG+, dPreQ0, dPreQ1) showed varied resistance to various restriction enzymes. MKSVVLLSGGVDSATCLAIEVDKWGSKNVHAIAFNYGQKHEAELENAANVAMFYGVKFTILEIDSKIYSSSSSSLLQGKGEISHGKSYAEILAEKEVVDTYVPFRNGLMLSQAAAYAYSVGASYVVYGAHADDAAGGAYPDCTPEFYNSMSNAMEYGTGGKVTLVAPLLTLTKAQVVKWGIDLDVPYFLTRSCYESDAESCGTCATCIDRKKAFEENGMTDPIHYKEN qued QueD _ qued.cif AlphaFold 3 modifies phage molecules to avoid recognition converts dihydroneopterin triphosphate (H2NTP) into 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) (the second step of synthesis of 7-cyano-7-deazaguanine, a precursor in the pathway that leads to various DNA modifications found in phages). Type II RM (in vitro) RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage 9g https://www.ncbi.nlm.nih.gov/nuccore/NC_024146.1 10.1038/s41467-019-13384-y quec;fole;quee PF01242 Viral genes (e.g., folE, queD, queE from phage 9g) were expressed in corresponding E. coli deletion mutants (ΔfolE, ΔqueD, ΔqueE), restoration of queuosine (Q) modification in tRNA confirmed that these viral enzymes are functional analogs of their bacterial counterparts (ECO_0000017). Phages with modifications (e.g., dG+, dPreQ0, dPreQ1) showed varied resistance to various restriction enzymes. MRVSKTLTFDAAHQLVGHFGKCANLHGHTYKVEISLAGGTYDHGSSQGMVVDFYHVKKIAGTFIDRLDHAVLLQGNEPIALANAVDTKRVLFGFRTTAENMSRFLTWTLTELMWKHARIDSIKLWETPTGCAECTYYEIFTEDEIEMFKNVTFIDKDEKITVREILEQEQDNG quee QueE _ quee.cif AlphaFold 3 modifies phage molecules to avoid recognition catalyses the conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) into 7-cyano-7-deazaguanine (preQ0) (the third step of synthesis of 7-cyano-7-deazaguanine, a precursor in the pathway that leads to various DNA modifications found in phages). Type II RM (in vitro) RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage 9g https://www.ncbi.nlm.nih.gov/nuccore/NC_024146.1 10.1038/s41467-019-13384-y quec;fole;qued PF13353;PF04055 Viral genes (e.g., folE, queD, queE from phage 9g) were expressed in corresponding E. coli deletion mutants (ΔfolE, ΔqueD, ΔqueE), restoration of queuosine (Q) modification in tRNA confirmed that these viral enzymes are functional analogs of their bacterial counterparts (ECO_0000017). Phages with modifications (e.g., dG+, dPreQ0, dPreQ1) showed varied resistance to various restriction enzymes. MVNQYNQPERGKIRINVRDPEKMPIMEIFGPTIQGEGMVIGQKTIFIRTGGCDYHCNWCDSAFTWNGTTEPEYITGKEAASRILKLAFNDKGEQICNHVTLTGGNPALINEPMAKMISILKEHGFKFGLETQGTRFQEWFKEVSDITISPKPPSSGMRTNMKILEAIVDRMNDENLDWSFKIVIFDENDLAYARDMFKTFEGKLRPVNYLSVGNANAYEEGKISDRLLEKLGWLWDKVYEDPAFNNVRPLPQLHTLVYDNKRGV acric10 AcrIC10 _ acric10.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas7c (https://www.ncbi.nlm.nih.gov/protein/UEM35122.1) and AcrIC10 (ipTM = 0.75, pTM = 0.78). P. aeruginosa strains LL77 type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Xanthomonas translucens 10.1038/s41467-020-17652-0 Ectopic expression (ECO_0000017) of AcrIC10 in P. aeruginosa strains expressing type I-C CRISPR-Cas system restored plating efficiency of CRISPR-sensitive phages JBD8 and DMS3m. Binding it supported by computational structure modeling evidence (ECO_0006368). MTKINPEWLAFNNLINEGGEGFNPHPKYISATATAQAPIVANSAGKVYRDSRGMPIDPLAQIADAETRLARVTDPFGRELIERSIANYRKMLEA acric9 AcrIC9 8G9T_A acric9.pdb PDB entry binds and inhibits host defence system mimics the shape and charge distribution of double-stranded DNA (dsDNA), effectively occupying the site where the PAM interacts with the Cascade. Pseudomonas aeruginosa strains LL77 type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Rhodobacter phage RcNL1 https://www.ncbi.nlm.nih.gov/nuccore/NC_020489.1 10.1038/s41467-020-17652-0;/10.1016/j.molcel.2023.12.034 Ectopic expression (ECO_0000017) of AcrIC9 in P. aeruginosa strains expressing type I-C CRISPR-Cas system restored plating efficiency of CRISPR-sensitive phages JBD8 and DMS3m. METKMTSFYKITAYNSQALYFWGTDADVDRYVDWLNRDREINVYAAEAIPEAEWAQYEGRDDVLSGEECGWDDFMSAEA aca9 Aca9 _ aca9.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1038/s41467-020-19415-3 PF13560;PF01381 _ MKNTRLVDAQSKLGFSKAEMARALSVHYNTYDKWERGEQKPQAAVYTAVDMLLFMHAKGILTEWMSRSE acrie8 AcrIE8 _ acrie8.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a high-confidence interactions between CasB (https://www.ncbi.nlm.nih.gov/protein/QZE32572.1) and AcrIE8 (ipTM = 0.85, pTM = 0.87). Pseudomonas aeruginosa type I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Klebsiella pneumoniae 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIE8 increased the plating efficiency of phages that are normally targeted by active type I-E CRISPR–Cas systems. Binding it supported by computational structure modeling evidence (ECO_0006368). MTTITINTYDPEARFNMSGEEAKEFFAFVEEQAKVSGFDVYYDSCTYVDEESERFVEKCFQNY acrif15 AcrIF15 _ acrif15.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas8f/Csy1 (AHH51210.1) and AcrIF15 (ipTM = 0.70, pTM = 0.61). Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Klebsiella michiganensis 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF15 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. Binding it supported by computational structure modeling evidence (ECO_0006368). Prevents CRISPRi, indicating inhibition occurs upstream of DNA binding. MTTITIAYEVSNDKVETIKTMVESQQIHNVNFNGEEFTIERGDFTSIDKDEAEHVKLLNKIQDIIHGYS acrif16 AcrIF16 _ acrif16.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pectobacterium parmentieri 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF16 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. Does not prevent CRISPRi, suggesting it inhibits after DNA binding. MSLSDKKEQKEAYLDALRIAPLDRGVLKRIHAVNDNTLDKWLYVADRYPTFRACWELWMFQRKRRVLISRKLHVLINRSTNRTIEAFEKTYPPEERVVGKSYRDLVTEKGERSANMYIINGEVVGAKDASILLGYSSYNTLYAKMKRLGIQPGDDISHLKPEKRGRKKECS acrif17 AcrIF17 _ acrif17.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pectobacterium carotovorum 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF17 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. Does not prevent CRISPRi, suggesting it inhibits after DNA binding. MFSEIKFSSLSDHALAELIKMAMEEFQQRLIKPGVNTIKTVDVEPVVLHAPSDNEMVFINNCLKKRRAGEYIHASMKDKYRDLTRKYPQWFSVKAYPDDLRGSVSKHYVDYFTKKE acrif18_ AcrIF18* _ acrif18_.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas8f/Csy1 (AHH51210.1) and AcrIF18* (ipTM = 0.71, pTM = 0.61). Pseudomonas aeruginosa type I-F and I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Serratia marcescens 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF18 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. Prevents CRISPRi, indicating inhibition occurs upstream of DNA binding. Binding it supported by computational structure modeling evidence (ECO_0006368). MTTIKAAYISKDQNWNDGTTTYWFDVNGETFGVVHGGESWNAKVVDCDGAPSDQYTVDQFNITEDMIAE acrif19 AcrIF19 _ acrif19.cif AlphaFold 3 unknown _ Pectobacterium and Serratia type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pectobacterium carotovorum 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF19 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. MKPLHTMNYDNNQMSLVYESYDEYGFEYSVKLKISVRDYRGIDVSAFNAFPEWEDTLRMRDRVMSVEEIENAMISRYKSLFIAPPDCTYEFDI acrif20 AcrIF20 _ acrif20.cif AlphaFold 3 unknown _ Pectobacterium and Serratia type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pectobacterium parmentieri 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF20 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. MTKSEIFKFAWVDAHYLATTLGGNAVEYFAECLKKSHMINRTTAVSFEQKEYAVDVAYAAITILADGAVL acrif21 AcrIF21 _ acrif21.cif AlphaFold 3 unknown _ Pectobacterium and Serratia type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pectobacterium carotovorum 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF21 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. MTSIKTIEYKGNTLTLQKDSDADCISYEILLRDPIKHWVGRDVQILNIDSSTSVDVFYKKFSDSELASMMQREYDDCFPLQVTATLESVIEAGDINTKWFVVNDETGALSGKYGSVYTESGWYICNESGEKIENKLTENQISSIRVAMDRGDTATYSWEFDD acrif22_ AcrIF22* _ acrif22_.cif AlphaFold 3 unknown _ Pectobacterium and Serratia type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pectobacterium parmentieri 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF22 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. MSKKFLEIVGNASTSATFNGKLIGHNVNASAYEKDGEIIIHLETNGSRWKSSPEVRMTKEEYDSFCEKQSRPLFVRGIELFGAEALLS acrif23 AcrIF23 7FIA acrif23.pdb PDB entry binds and inhibits host defence system interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, inhibiting its DNA cleavage activity. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Prophage in Pseudomonas aeruginosa 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF23 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. MTNFQTWLDSADIPVQQNGQWIDLETGIAYDPSYNYAANTRRASLSPRGIDARAVAKTFGGRALTGTARQKEWAEKIRAEKVQQMNQDQAEMACDPSGLLTAAKFWIENRNDSAQEIAGFVMQQKALLAQHRSAKAAGQADKVAKIAAEYNALTARWGF acrif24 AcrIF24 7XI1 acrif24.pdb PDB entry binds and inhibits host defence system forms a dimer that binds to the Csy complex in the type I-F CRISPR-Cas system, blocking target DNA hybridisation to crRNA and inducing the binding of non-sequence-specific dsDNA, which interferes with the CRISPR-mediated DNA cleavage process. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Prophage in Pseudomonas aeruginosa 10.1038/s41467-020-19415-3 Ectopic expression (ECO_0000017) of AcrIF24 increased the plating efficiency of phages that are normally targeted by active type I-F CRISPR–Cas systems. MNAIHIGPFSITPAARGLHYGGLPHHQWTLYYGPREMAIKTLPDSYTSSEVRDEFSDIIAEFVIDARHRYAPDVLELVNSDGDAVLARVAVSRLPEALSGCIPDDRFPYWLLTASRPRLGLPVTLNEYTALAVELSAPPLAWITGLLPGEVLTHDAEEWRPPTSWELRHVVGEGSFTGVSGAAAAALLGMSATNFRKYTAGDSAANRQKISFAAWHYLLDRLGVKRAS acrva5bsp AcrVA5Bsp _ acrva5bsp.cif AlphaFold 3 adds a post-translational modification and deactivates bacterial defence inhibits Cas12a via acetylation, similar to its remote structural homolog, AcrVA5. Lachnospiraceae bacterium type V-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Bacteroidota phage (IMG_VR genome, ID: Ga0247610_10000168) 10.1038/s41467-024-45068-7 acrva5bsp_model.html In vitro assay showed ability to inhibit DNA cleavage by Cas12a (ECO_0000183). MLEDRSSLFKIEWYASLDGIMCFNAYDAEHNWVHLGKVIVDVKNVEYERYKHLCNGNKLAKIVRVETDSEYCGWGVATELLKEVIRIYKDFNLYLLCHPMPRGHYDESHKTVKDLRRFYGKLGFVPCGELLPTMIRKAPLPTLGE adg_17 ADG.17 _ adg_17.cif AlphaFold 3 binds and inhibits host defence system acts as a Phd-like antitoxin, binding to and inhibiting the Doc toxin (a kinase that targets EF-Tu) Sulfolobus islandicus type II TA system PhD-Doc TA _ Sulfolobus islandicus rod-shaped virus 10.1038/s41467-024-48074-x MARVHKLSAKQKKIIKRMHNRIDYILEKYKEYLDALAEFDRTGVLKIHGKVIYERKYNDQKK sifv2_gp15 SIFV2 gp15 _ sifv2_gp15.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between the helicase Cas3 (https://www.ncbi.nlm.nih.gov/protein/WP_014513746.1) and SIFV2 gp15 (ipTM = 0.79, pTM = 0.81). Sulfolobus islandicus CRISPR-Cas subtype I-A CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Sulfolobus islandicus filamentous virus 2 10.1038/s41467-024-48074-x Binding it supported by computational structure modeling evidence (ECO_0006368). Ectopic expression of sifv2_gp15 (ECO_0000017) restores infectivity of susceptible SIRV2MΔgp45–47 in Sulfolobus strain with an active subtype I-A system. MVEVKQKTLSYKLKINTRDYSITLEAELKAVINVKGNDLVYEDKQQKFVGYIETISSYETKNAKENADEILNERFEKYANGLKVLEQTAEAINAEIEIE acriia5 AcrIIA5 6LKF acriia5.pdb PDB entry binds and inhibits host defence system binds Cas9, inhibiting its activity. Streptococcus thermophilus type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Streptococcus phage D4276 https://www.ncbi.nlm.nih.gov/nuccore/NC_070686.1 10.1038/s41564-017-0004-7;10.1016/j.celrep.2019.10.078 Ectopic expression (ECO_0000017) of AcrIIA5 increased the plating efficiency of phage D5842 that is normally targeted by active type II-A CRISPR–Cas systems. Binding to Cas9-DNA complex is supported by Electrophoretic Mobility Shift Assays (ECO_0000096). MAYGKSRYNSYRKRSFNRSNKQRREYAQEMDRLEKAFENLDGWYLSSMKDSAYKDFGKYEIRLSNHSADNKYHDLENGRLIVNIKASKLNFVDIIENKLDKIIEKIDKLDLDKYRFINATNLEHDIKCYYKGFKTKKEVI aca8 Aca8 _ aca8.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1038/s41564-018-0120-z _ MKRQVRVGNTIYYVESEDDLVSVTHELARQGIKIEKIAYLLGVSVRKVRQYLESC acrid1 AcrID1 6EXP acrid1.pdb PDB entry binds and inhibits host defence system binds as a dimer to the Cas10d. Sulfolobus islandicus LAL14/1 type I-D CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Sulfolobus islandicus rudivirus 3 isolate SIRV3 https://www.ncbi.nlm.nih.gov/nuccore/1063656926 10.1038/s41564-018-0120-z PF07118 A mutant virus, SIRV2M, with a 4 kb deletion (ECO_0001038) including the acrID1 gene, lost the ability to infect Sulfolobus islandicus LAL14/1, which has active CRISPR–Cas systems. Reintroduction (using homologous recombination) of SIRV3 gp02 (acrID1) alone into SIRV2M restored infectivity, indicating gp02 is necessary and sufficient for overcoming CRISPR immunity. Pull-down assays (ECO_0006249) and gel filtration chromatography (ECO_0001049) demonstrated that AcrID1 binds directly to Cas10d, the large subunit of the I-D effector complex. MNYKELEKMLDVIFENSEIKEIDLFFDPEVEISKQEFEDLVKNADPLQKVVGDNYITETFEWWEFENQYLEFELDYYVKDEKIFVLEMHFWRKIRKLE acriia16 AcrIIA16 _ acriia16.cif AlphaFold 3 unknown _ Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Prophage in Listeria monocytogenes 10.1038/s41564-020-0692-2 Ectopic expression (ECO_0000017) of AcrIIA16 in a P. aeruginosa strain with a heterologous SpyCas9 CRISPR system restores replication of sensitive phage. AcrIIA16's expression inhibit CRISPR Interference, suggesting inhibiting of SpyCas9 at the step of target DNA binding or at an upstream stage. Co-immunoprecipitation (ECO_0005644) showed binding to Cas9. MGYIGTKRSERSQDAIEDYEVPLNHFNKDLIQAFIDENEAYDTLKTKKVRLWKFVAPRAGATSWHHTGTYYNKTDHYSLEKVADELLQNGDEWEEQFKAYVKEEQETATSEPVFLSVIKVQIWGGSMKRPKLVGHEVVMGVKKEGWLHAVSKATQSKYKLSANKVEMQKHYSLEDYSALTKDFPEFKAQKRAINKKMKEMYN acriia17 AcrIIA17 _ acriia17.cif AlphaFold 3 unknown _ Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Enterococcus faecalis 10.1038/s41564-020-0692-2 Ectopic expression (ECO_0000017) of AcrIIA17 in a P. aeruginosa strain with a heterologous SpyCas9 CRISPR system restores replication of sensitive phage. AcrIIA16's expression inhibit CRISPR Interference, suggesting inhibiting of SpyCas9 at the step of target DNA binding or at an upstream stage. Co-immunoprecipitation (ECO_0005644) showed binding to Cas9. MAILNNKGEKISIDCADLISEVEEDILIFGGTFLVYAICSWREIEQVEYISDYVHADNPESYKDELTTKEYAELKEIYEKDLEELKITKNKQMNLNELLSILTIQNSIT acriia18 AcrIIA18 7VLM acriia18.pdb PDB entry degrades or sequesters molecules utilised by host defence systems cleaves the single guide RNA (sgRNA), disrupting the ability of Cas9 to target DNA. Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus macedonicus 10.1038/s41564-020-0692-2 Ectopic expression (ECO_0000017) of AcrIIA18 in a P. aeruginosa strain with a heterologous SpyCas9 CRISPR system restores replication of sensitive phage. AcrIIA16's expression inhibit CRISPR Interference, suggesting inhibiting of SpyCas9 at the step of target DNA binding or at an upstream stage. Co-immunoprecipitation (ECO_0005644) showed binding to Cas9. MKIDTTVTEVKENGKTYLRLLKGNEQLKAVSDKAVAGVNLFPGAKIGSFLVRQDNIVVFPDNKGEFDLDFFNLLNDNFETLVEYAKMADCLDIAFDINEKSYFNMIMWLMKNIDENWSQSPYGESFYSSKDIDWGYKPEGSLRVSDHWNFGQDGEHCPTAEPVDGWAVCKFENGKYHLIKKF acriia19 AcrIIA19 _ acriia19.cif AlphaFold 3 unknown _ Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Staphylococcus simulans 10.1038/s41564-020-0692-2 Ectopic expression (ECO_0000017) of AcrIIA19 in a P. aeruginosa strain with a heterologous SpyCas9 CRISPR system restores replication of sensitive phage. AcrIIA16's expression inhibit CRISPR Interference, suggesting inhibiting of SpyCas9 at the step of target DNA binding or at an upstream stage. Co-immunoprecipitation (ECO_0005644) showed binding to Cas9. MKLIVEVEETNYKNLVNYTKLTNESHNILVNRLISEYITKPYELRLDLSERYSNRDLIEFKFMLIEYCKEALQDIKELANSDEAYETDEAFEAVFRQLFEEVISNPDTVLKAFHSYTSFLEENK acriia23 AcrIIA23 _ acriia23.cif AlphaFold 3 unknown _ Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Streptococcus phage phiAp1.1 https://www.ncbi.nlm.nih.gov/nuccore/MW168838 10.1038/s41564-021-00996-8 In a heterologous phage assay, ectopic expression (ECO_0000017) of AcrIIA23 in Staphylococcus aureus restored infection of a Cas9-targeted phage. In S. pyogenes, inducible expression of AcrIIA23 restored transformation of a plasmid carrying a CRISPR-targeted sequence, demonstrating inhibition of CRISPR interference. MFIYVIRRNKMEYGNKIFEIYNKPFKYRNSSSTNYNKVRASGIEPNTKFVVNKTANINCAVYPRHGSIEKVFYWGDRKITQATAEKRCGYFKG adfa AdfA (anti-DarT factor A) _ adfa.cif AlphaFold 3 unknown _ Escherichia coli type IV TA system DarTG TA https://defensefinder.mdmlab.fr/wiki/defense-systems/dartg Enterobacteria phage RB69 https://www.ncbi.nlm.nih.gov/nuccore/NC_004928.1 10.1038/s41564-022-01153-5 PF23813 RB69 phages were serially passaged on E. coli cells containing DarTG1 to select for escape mutants, mutations in gene 61.2 (renamed adfA) were found in resistant phages. Ectopic expression of mutant gp61.2(adfA) or T4 homolog increased plaquing efficiency on DarTG1-expressing cells. ELTA assays showed inhibition of DarT1 toxin activity (no ADP-ribosylation of DNA) when RB69 expressed evolved adfA (ECO_0005801). MHKNAPFKYGKFPNAQCYNITPNENNNGYHIGVIFVIVKDNEIVAWADFKGTTYDVNPVPFTYYNIMDLAYDYNWFNHDTLAHIEGVGFDISYSSYSLCPMSRAHGKDASYLSIRKRVNFKRSTEYVGGLFVKDNKITRISYPLSVSQKDVDVDLDLTENNINRIASVYFDIDEKIVVCGYELPPEEKAEAIEVELEISVDDQIFNAFMNRG mga47 mga47 (DNA polymerase) _ mga47.cif AlphaFold 3 uncategorised mutated DNA polymerase capable of replicating DNA modified (ADP-ribosylated) by the host DarTG system. Escherichia coli type IV TA system DarTG TA https://defensefinder.mdmlab.fr/wiki/defense-systems/dartg Phage SECϕ18 10.1038/s41564-022-01153-5 SECϕ18 phages were serially passaged on E. coli cells containing DarTG2 to select for escape mutants, mutations in mga47 were found in resistant phages. Overexpression of mga47 (wild-type and mutant) showed it does not trigger DarT2 activity. ELTA assays demonstrated that even in presence of mutant mga47, DarT2 was still active and ADP-ribosylated phage DNA, suggesting the polymerase mutations allowed phage to tolerate or bypass DarT2’s modification (ECO_0005801). MKDFERLFLDTETFSDVGLKKVGAYAYAEHPSTEIMICTYAIDEGRVQTWDATESPTMPRELRKALRRVSRKKAKIVMANGLLFDRLVIREKWGIDLPVSQIEDTMIMAFRHALPGSLDMQCQVLGVDAEHAKDKAGKALIKRFCKPTPKTYKIRRYTRETHPEEWAKFLRYAALDIVAMREVYWRIPDWGNSPKEDEILLIDQIINDRGFYVDVDLANAAIKAVRAHKEELKEEAWERFGGKLTGNDFLPILRDIAPAFTIHNAQKSTLNDLLEDPDFPDEGKALIEMRLGASSTASTKYNPLVNGLSADGRRRGCIQYGGAKRTLRWAGKGFQPQNLARGEYSDDHEGKIKRREGESDVSFWVRSHMLTNGINSLLRGTAHWAYDISKLTASTVRGCIIPAKGKKFVVADYSNVEGRGLAWIAGEKTALMVFKAGRDIYCETAGKMFGLDPDYIKANRKDLRQIGKACELGLGYGGGVAAFLQFAKNLGLDLYTMADVMKGTFPDHIWAAAKRGYEYARINEAKRPPKPGKKDERPTYILPKNVWLTCDAIKRMWREAHPKTVAFWAELEDAVLCAIRNPGKAYWAGANVRPDGRKALKIVRTKAKHDPTFDEERDDPNAAGWWLKIELPSGRIMSYPGIALSVTTEIDEDTGKKRTSTRIKYQGENQTTRQWGFQYTYGGKLTENIVQALCRDILAWSMPGVEAAGYEIVLSVHDELVTEVPDTDDYTTEELCALMCDLPIWAKGFPLAAEGDCMYRYRK p0020 P0020 _ p0020.cif AlphaFold 3 unknown _ Vibrio crassostreae type ABCDEFGH Dnd Dnd https://defensefinder.mdmlab.fr/wiki/defense-systems/dnd Vibrio phage 44E38.1 https://www.ncbi.nlm.nih.gov/nuccore/2085581620 10.1038/s41564-022-01157-1 A transposon mutagenesis screen identified that insertions in p0020 (ECO_0005509) abolished the ability of phage P5_14 red variants to infect V5red Vibrio crassostreae strains, indicating p0020 is essential for overcoming Dnd-mediated host defence. A targeted gene deletion of p0020 (ECO_0001038) confirmed this result, as the mutant phage lost infectivity. Reintroduction of p0020 into a blue phage background restored infectivity on hosts with Dnd defence, demonstrating p0020 is necessary and sufficient for anti-Dnd activity (ECO_0000017). MARARNIKPAFFDNDELADNDPLGRLLFIGLWTIADCNGNLEWRSKRVKKQLLAYDECCIDSLAINLDKSGFVRFYSDGDKIYLNVINFDKHQNPHKNEKAKGTEIPEYCEEYRQAIDLNKLAINRDLSGLKPNDSDSNPADSLNLIPDSLNLIPDTVTEQKPSPAKAGSDVLEIFNYWKEVMKKGGTTRLNKKREKLISDRLKEGYQVDEFKTAIFNCSMSPFHMGQNDNQTKYNDIELICRPDKFEQFRDNVGQQAQPRQMSKATERTFNNIIDVELD p0021 P0021 _ p0021.cif AlphaFold 3 unknown _ Vibrio crassostreae type ABCDEFGH Dnd Dnd https://defensefinder.mdmlab.fr/wiki/defense-systems/dnd Vibrio phage 44E38.1 https://www.ncbi.nlm.nih.gov/nuccore/2085581620 10.1038/s41564-022-01157-1 Deletion of p0021 (ECO_0001038) alone did not abolish infectivity, but the double deletion of p0020 and p0021 reduced phage infectivity more than deletion of p0020 alone, suggesting that p0021 enhances the anti-defence function of p0020. MAQHVKVNPTTDEMLSKLSEKRKRENSFIRTKQDIAAEAIVALYKKEMKNG dsad1 DSR anti-defence 1 _ dsad1.cif AlphaFold 3 binds and inhibits host defence system competes with the tail tube protein, DSR2's trigger, by binding DSR2 and inhibiting its NADase activity. Bacillus subtilis 29R type II DSR DSR https://defensefinder.mdmlab.fr/wiki/defense-systems/dsr Bacillus phages SP-beta and phi3T 10.1038/s41564-022-01207-8 Homologous recombination allowed transfer of the DSAD1 gene from DSR2-resistant phages (SPbeta or phi3T) to DSR2-sensitive SPR phages, rendering the hybrid SPR phages resistant to DSR2 defense. Co-expression of DSAD1 with DSR2 in B. subtilis inhibited DSR2-mediated protection against SPR infection (ECO_0000017), confirming DSAD1’s role as an anti-defense protein. Pulldown assays (ECO_0006249) demonstrated a direct physical interaction between DSAD1 and DSR2. MIEIFKDTGATHDLVYHSKINTFVWDVEFDIVLSDSKELNKCYFVKCFNPYRINGKCDFAVSSIDIFSEGKRLLIENEFNFKITKAVHVATSKDVTEIVLHLSERISSPFPIVKEVVYLD jss1_004 JSS1_004 _ jss1_004.cif AlphaFold 3 adds a post-translational modification and deactivates bacterial defence phosphorylates Dnd, CRISPR-Cas, QatABCD, SIR2+HerA and DUF4297+HerA defence systems. Salmonella enterica type ABCDEFGH Dnd, the type I-E CRISPR-Cas system, QatABCD, SIR2+HerA and DUF4297+HerA. Dnd;CRISPR-Cas;QatABCD;SIR2+HerA;DUF4297+HerA https://defensefinder.mdmlab.fr/wiki/defense-systems/dnd;https://defensefinder.mdmlab.fr/wiki/defense-systems/cas;https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_qat;https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_her;https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_her Salmonella phage JSS1 https://www.ncbi.nlm.nih.gov/nuccore/2337562262 10.1038/s41564-024-01851-2 jss1_004_model.html Deletion of JSS1_004 (JSS1Δ004) completely abolished JSS1’s ability to evade Dnd-mediated immunity (ECO_0001038), resulting in up to six orders of magnitude reduction in efficiency of plating (EOP) on Dnd+ strains. Quantitative phosphoproteomic analysis identified 480 phosphorylation sites across 333 proteins during JSS1 infection, including multiple sites on the DndFGH complex. Purified JSS1_0041–262 (containing only the kinase domain) was shown to phosphorylate DndFGH in vitro. Phosphomimetic mutations (e.g., T22E, T23E, Y1510E) in DndF and DndH mimicked phosphorylation and abolished the antiphage activity of the DndFGH complex. To test activity against other defence systems (QatABCD, SIR2+HerA, DUF4297+HerA), the authors performed phage plaque assays in bacterial strains expressing these systems. JSS1Δ004 showed reduced EOP on these strains, indicating active defence. In contrast, wild-type JSS1 overcame these defences, demonstrating that JSS1_004 enables immune evasion beyond Dnd. MNYTDIQARLAIIKSLPISELDKRQPLLVALAADIVNGETSDGNDTDGSNGLEYQDWWHTLGALMRDAGFRMLGNGHFSAAYSHELLPGRVIKVGFKKEDSGAAYTAFCRMHQGRAGIPNVYHVARHAGCYTVVLDRLQSCDRYANDVHAKYASAAQEFIECTGGDADWYAQYVSNEFIETCKMIREFFHGIASFDMHSGNIMFDDNDVPYITDPVSFSHDREREDGFPLDPEALLAEVEAVAQERMIERCRNRKAKCDPNGTFQVNRKAAMKRRKRNRKLRAKVAERDRLHFMAIRRERGVIERNERRAEMLMGSAWHDFWLRNGNATVRKIDQVNGLKWQLGDRLAIQAGLPLNIDKVLDAHLMG acriii_1 AcrIII-1 2X4I_A acriii_1.pdb PDB entry degrades or sequesters molecules utilised by host defence systems binds to cyclic tetra-adenylate and cleaves it into two linear diadenylates. Sulfolobus islandicus M.16.4 type III-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Sulfolobus islandicus rudivirus 1 https://www.ncbi.nlm.nih.gov/nuccore/55770953 10.1038/s41586-019-1909-5 PF08960 Ectopic expression of AcrIII-1 (SIRV1 gp29) enables infection of a sensitive phage SSeV a host with active type III-B CRISPR immunity (ECO_0000017). Plasmid encoding gp29 (AcrIII-1 gene) can transform in a host with active cA4 (Csx1), but not cA6 (Csm6). LC-MS results show AcrIII-1 cleaves cA4 to ApA>P and ApA (ECO_0001096). AcrIII-1 is crystallised with cA4 (ECO_0001823) MNKVYLANAFSINMLTKFPTKVVIDKIDRLEFCENIDNEDIINSIGHDSTIQLINSLCGTTFQKNRVEIKLEKEDKLYVVQISQRLEEGKILTLEEILKLYESGKVQFFEIIVD acb1 Acb1 7T26 acb1.pdb PDB entry degrades or sequesters molecules utilised by host defence systems serves as a metal-independent phosphodiesterase and hydrolyses the host's cyclic di- and trinucleotide CBASS signals, such as 3'3'-cGAMP, 3'3'-cUA and 3'3'3'-cAAA. Escherichia coli KTE188 type III CBASS CBASS https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass Escherichia phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1038/s41586-022-04716-y PF23474 Cell lysates from E. coli infected with phage T4 degraded CBASS signals such as 3′3′-cGAMP, cUA, and cAAA. Purified T4 Acb1 protein selectively cleaved these CBASS cyclic dinucleotides and trinucleotides. A deletion mutant of T4 (Δacb1) failed to degrade 3′3′-cGAMP and showed over 300-fold reduced replication in CBASS-expressing E. coli compared to wild-type T4 (ECO_0001038). The crystal structure of Acb1 bound to 3′3′-cGAMP (ECO_0001034) revealed the mechanism of substrate recognition and metal-independent hydrolysis. In vitro, Acb1-treated 3′3′-cGAMP failed to activate CBASS effector proteins such as Cap5, confirming functional inhibition of immune signaling. MMEFKDFSTGLYVAAKFSELTLDALEELQRSLRVPNPVPREKIHSTICYSRVNVPYVPSSGSFEVASSGHLEVWKTQDGSTLVLVLDSEYLRCRHMYARALGATHDFDDYTPHITLSYNVGPLSFSGDVQIPVVLDREYKEPLKLDWADDLK apyc1 Apyc1 7T28 apyc1.pdb PDB entry degrades or sequesters molecules utilised by host defence systems serves as a metal-dependent cyclic NMP phosphodiesterase, degrading cyclic pyrimidines used in signalling. E. coli KTE188 type III CBASS Pycsar https://defensefinder.mdmlab.fr/wiki/defense-systems/pycsar Bacillus phage SBSphiJ https://www.ncbi.nlm.nih.gov/nuccore/1315671043 10.1038/s41586-022-04716-y PF23023 apyc1_model.html Cell lysates from Bacillus subtilis infected with SBSphiJ-family phages degraded Pycsar signals like cCMP and cUMP. Purified Apyc1 protein from SBSphiJ cleaved these cyclic pyrimidine mononucleotides with broad specificity. The crystal structure of Apyc1 (ECO_0001034) revealed a metal-dependent phosphodiesterase with a metallo-β-lactamase (MBL) fold and coordination of Zn²⁺ ions. Mutations in catalytic residues abolished activity. In vitro, Apyc1-treated cUMP failed to activate the Pycsar effector PycTIR. Expression of Apyc1 (ECO_0000017) in Pycsar-expressing E. coli rescued infection by phage T5, demonstrating Apyc1's role in subverting Pycsar-mediated defense. MLHTTQIRMVGTGSAFSKKFYNNSALVTFTNGYNLLIDCGHSVPKGLHDADIPLESIDGILITHTHADHIGGLEEVALYNKFVLGGRKIDLLVPNTLVESLWENSLKGGLRYSDTYDDLSLSDYFTVRSLKTFTSGAARTQLEENIAIKLYPTFHVSHMASYAVGLEDRGEDKVFYSSDTIFDEYLIDYALTYSWVFHDCQFFTGGVHASLDELLNYIPEEDQDRVFLMHYGDNMEDFFTKTGRMRFALQGRTYIL racc RacC _ racc.cif AlphaFold 3 unknown _ Salmonella enterica type II Retron (Sen2) Retron https://defensefinder.mdmlab.fr/wiki/defense-systems/retron Metagenome 10.1038/s41586-022-05091-4 A high-throughput reverse genetics approach (Toxin Inhibition by Conjugation, TIC) was used to identify RcaT inhibitor. The TIC (Toxin Inhibition by Conjugation) platform is a high-throughput genetic screen designed to identify blockers of toxin activity. It involves conjugating an E. coli overexpression library into a strain expressing the toxin gene (rcaT) under an inducible promoter. Strains that regain growth upon co-induction of the toxin and a library gene are considered to carry candidate RcaT inhibitors. MITNYEATVVTTDDIVHEVNLEGKRIGYVIKTENKETPFTVVDIDGPSGNVKTLDEGVKKMCLVHIGKNLPAEKKAEFLATLIAMKLKGEI tad1 Tad1 7UAV_A tad1.pdb PDB entry degrades or sequesters molecules utilised by host defence systems binds the signalling molecule (1′′–3′ gcADPR) Bacillus subtilis BEST7003 type I Thoeris;Pseudomonas aeruginosa type II-A and type III-C CBASS Thoeris;CBASS https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris;https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass Bacillus phage SBSphiJ7 https://www.ncbi.nlm.nih.gov/nuccore/2228685031 10.1038/s41586-022-05375-9 PF24729 Gene was identified based on differential sensitivity to the Thoeris defence systems — among closely related phages infecting Bacillus subtilis, all phages were sensitive to the Thoeris defence systems, apart from SBSphiJ7. Ectopic expression (ECO_0000017) of Tad1 into B. subtilis cells that also express the Thoeris system from Bacillus cereus MSX-D12 rendered phages usually sensitive to Thoeris resistant to the defence system. Silencing of tad1 in the phage SBSphiJ7 using dCas9 restored Thoeris defense, proving that Tad1 is necessary for immune evasion. To detect signalling molecules, lysates from Thoeris-expressing cells infected with phage lacking Tad1 and triggered ThsA NADase activity, but co-expression with Tad1 abolished this activity, indicating that Tad1 interferes upstream of ThsA activation. Adding purified Tad1 to signalling-containing lysates eliminated the ThsA activation signal, indicating Tad1 directly binds and sequesters the signalling molecule. Tad1 was crystallised (ECO_0001823) in complex with the signalling molecule (1′′–2′ gcADPR). MRELKHELLPTRHTQVFHEDKDKMEFNAPHHFVVVPAGSPLVDQQIHYGRGGNSKKVTVKDFAGKLATVNFQLGPVTEHGANGVMNEDLIAMVITRLQYFQNSEFNCRENAMAITKLEEALMWLNKRTAEREQRGVEGTHEK vs_4 Vs.4 7UQ2_A vs_4.pdb PDB entry degrades or sequesters molecules utilised by host defence systems binds the signalling molecule cGAMP. Escherichia coli type II CBASS CBASS https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1038/s41586-023-05862-7 PF24729 Deletion of vs.4 (ECO_0001038) in the T4 phage reduced its ability to infect bacteria lacking functional Cap2, indicating that Vs.4 helps evade CBASS immunity. Complementation with wild-type vs.4 restored phage infectivity, supporting its role as a CBASS antagonist. Isothermal titration calorimetry (ECO_0001825) revealed that Vs.4 binds cGAMP with high affinity (Kd ≈ 30 nM). Crystal structure (ECO_0001034) showed that Vs.4 forms a hexamer, each binding three cGAMP molecules. Mutations in Vs.4 that disrupt cGAMP binding or hexamerization (e.g., Y8A, F82A, A77I) abolishes CapV inhibition (ECO_0000015). MIEDIKGYKPHTEEKIGKVNAIKDAEVRLGLIFDALYDEFWEALDNCEDCEFAKNYAESLDQLTIAKTKLKEASMWACRAVFQPEEKY gad1 Gad1 8U7I_E gad1.pdb PDB entry binds and inhibits host defence system binds the GajAB complex, forming an octameric web around it that inhibits DNA recognition and cleavage. Bacillus cereus VD045 Gabija Gabija https://defensefinder.mdmlab.fr/wiki/defense-systems/gabija Bacillus phage phi3T https://www.ncbi.nlm.nih.gov/nuccore/KY030782.1 10.1038/s41586-023-06855-2 B. subtilis cells expressing Gabija show phage resistance, but co-expression of Gad1 with Gabija allowed phage SPβ to replicate, showing loss of defence. Mutation of Gad1 abolished this immune evasion. Electrophoretic mobility shift assays (ECO_0000096) and DNA cleavage assays demonstrated that GajAB normally cleaves sequence-specific phage DNA, but when Gad1 is present, GajAB is no longer able to bind DNA or perform cleavage. Pull-down (ECO_0006249) assays and cryo-EM structural analysis (ECO_0006181) show that Gad1 binds directly to the bacterial Gabija GajAB complex. MKLIGIKTSNCFLVSDNIEGKRYFHSQLDELLFDGKRATETYKSDWFKLEKEPSVIEKQMPAKKINHRYELKEGFQESELTPKVIKASYIGEDSEYYEVKGLYDLKFEEIPQQNEKIEFEMNVIEEIDGELKLQSHNFNLNYNLLDRIQTHPMLLETKPCYLSQEESYKIIRNHIKANINPKFARITSDYDFCLTVVKVLELYKPHEYIVDLNAMYKRRKPKLEKRFQTKREVEIYKVAPKAYQSYPIVEPFSGKDVEDLKSNIKKFLDDLMAKINEPLVECKCCKGRGVILNEN gad2 Gad2 _ gad2.cif AlphaFold 3 unknown _ Bacillus cereus VD045 Gabija Gabija https://defensefinder.mdmlab.fr/wiki/defense-systems/gabija Bacillus phage SPbetaL6 and SPbetaL7 https://www.ncbi.nlm.nih.gov/nuccore/2517172319 10.1038/s41586-023-06869-w gad2_model.html Comparative genomics of closely related phages with differential sensitivity to the Gabija defence system identified Gad2 as a factor conferring resistance. Deletion of Gad2 from phage SPβL7 (ECO_0001038) rendered it susceptible to Gabija-mediated defence. MSYQFEKNKLYAYLGEELVEALKRNEAIIAGGAITSLFNNKEINDVDIYFRSDKKACSFLEECWNSNVYVTSHTKKATLFIKKRLKLQMIHFKFFSDAESIFNTFDFTVCMGAFDFKTEAFTLHEDFLKHNSQRILKFNSQTAFPIVSLLRVQKYTDKEYTISKPEFIRIVLTCMDLTINTYEELKDQMGGMYGINYDKLFEDEKDEDFNLREAVDKIADMVLDEDYFKEPVNLEFNDLDDLLNDINKSPVMTLKINDDQYRIGLDGFLKESVSAPCTEIKLDTKDFFDKTNFYKFVRKQNGKLTSFYDKNFEYVIGEEAKAEGVIDSWSNSGKLFFNEKAAIEQSTYYGKEDGVLIEVKIKEKDFVDADNGKVEATACQVIREVSKDEWKEYISANNSK had1 Had1 8TTO_A had1.pdb PDB entry unknown _ Bacillus cereus B4087 Hachiman Hachiman https://defensefinder.mdmlab.fr/wiki/defense-systems/hachiman Bacillus phage SBSphiJ4 https://www.ncbi.nlm.nih.gov/nuccore/2228684288 10.1038/s41586-023-06869-w Comparative genomics of closely related phages with differential sensitivity to the Hachiman defence system identified Had1 as a factor conferring resistance. Deletion of Had1 from phage SBSphiJ4 (ECO_0001038) rendered it susceptible to Hachiman-mediated defence. Ectopic expression of Had1 in B. subtilis (ECO_0000017) with Hachiman allowed otherwise blocked phages to infect. MEEFKMTVWTNGKAIRKYTGQDKHPDTNPSKRMEWFKATAIIKPDTGDNNERD tad2 Tad2 8SME tad2.pdb PDB entry degrades or sequesters molecules utilised by host defence systems sequesters gcADPR Bacillus subtilis type I Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Bacillus phage SPO1 https://www.ncbi.nlm.nih.gov/nuccore/NC_011421.1 10.1038/s41586-023-06869-w PF11195 Comparative genomics of closely related phages with differential sensitivity to the Thoeris defence system identified Tad2 as a factor conferring resistance. Deletion of tad2 rendered phages sensitive to Thoeris defence (ECO_0001038) and inserting tad2 into a sensitive phage conferred resistance.. Ectopic expression of tad2 (ECO_0000017) blocked Thoeris-mediated defence in bacterial cells. Biochemical evidence supports that Tad2 binds 1″–3′ gcADPR (KD ≈ 23.3 nM), preventing activation of the NADase effector ThsA. Crystal structure further supports sponge function (ECO_0005671). SMKTKMSFGEALEVLKQGMQVYRSGWNGKNMFLFLKSSDALASDFGFGFGEYINEPVFGNIIFIKTADNKIHAWVPSQTDVLAEDWDIVS acrib3 AcrIB3 _ acrib3.cif AlphaFold 3 binds and inhibits host defence system replaces Cas5 (shares sequence similarity to Cas proteins) in a defective Cascade interference complex that fails to engage target DNA. Listeria seeligeri type I-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE and prophages in Listeria genomes 10.1038/s41586-024-07923-x PF09704 Ectopic expression of AcrIB3 in L. seeligeri LS1 (ECO_0001038) blocked Cas3-mediated degradation of a target plasmid. Expression of AcrIB3 blocks CRISPR interference. AcrIB3 co-purified with the Cascade complex (Cas6–3×Flag), indicating it integrates into the complex (ECO_0000085). MKAIKLNVYLETANFRNPMSFQSKESYPLPPFSTVIGMVHVACGFKSYHAMDVSVAGNSFSTVHDLASRYEFNPTTKYESARHQMKVYSPQKDKMIGITQGISHIHLITDLHLQLHIIPEDQSEVYFIESKLKNPSQFLSLGRHEDVMMIKDVKVIDVQEETLPSNRELTKATYVPVSYKIGGAFFRLNKNYELVEQKKKWYRKFSKQEVLYAGEGTIIPKDSLIWVDEDGEVLFPV acrib4 AcrIB4 _ acrib4.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas8a1 (https://www.ncbi.nlm.nih.gov/protein/WWV39459.1) and AcrIB4 (ipTM = 0.91, pTM = 0.76). Listeria seeligeri type I-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE and prophages in Listeria genomes 10.1038/s41586-024-07923-x Binding it supported by computational structure modeling evidence (ECO_0006368).Ectopic expression of AcrIB4 (ECO_0000017) abolished interference against target plasmid. Expression of AcrIB4 blocks CRISPR interference. AcrIB4 did not disrupt Cascade assembly (immunoprecipitation). MESVEKQAYEAGVTYRKKQLVSEGNYQTLVYKLTSIIKKGSKEAFVETLLDYSKVKRKQIPSVFQEDVMNEEKTFKSSAYAFVIGLTQ acrib5 AcrIB5 _ acrib5.cif AlphaFold 3 unknown _ Listeria seeligeri type I-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE and prophages in Listeria genomes 10.1038/s41586-024-07923-x PF24304 Ectopic expression of AcrIB5 (ECO_0000017) abolished interference against target plasmid. MAGFIKKYLDSRGWTIYQLGNATGLAHQTIRSADSKTVDQLSAKNVRLIAEVFHFTAGEILDEFYEIEEEITNDEILKELTTVFEKHGHNTDEISSELLDGETIKLDMTDDNITKLADAVNATEHFTAYLDSSTDYMIVEATQ acrib6 AcrIB6 _ acrib6.cif AlphaFold 3 unknown _ Listeria seeligeri type I-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE and prophages in Listeria genomes 10.1038/s41586-024-07923-x Ectopic expression of AcrIB6 (ECO_0000017) abolished interference against target plasmid. MGKTYWYNEGTDTLLTEKEYKELMEREAKALYEEVQEEEKDFESSEKTSFEEFLKTCYENESDFVLSDNEGNKLEEW acrib7 AcrIB7 _ acrib7.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas5b (https://www.ncbi.nlm.nih.gov/protein/WWV39457.1) and AcrIB7 (ipTM = 0.79, pTM = 0.78). Listeria seeligeri type I-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE and prophages in Listeria genomes 10.1038/s41586-024-07923-x Ectopic expression of AcrIB5 (ECO_0000017) abolished interference against target plasmid. Binding it supported by computational structure modeling evidence (ECO_0006368). MRTFKMLLKKYNVEIKDVIQKGGIDKSSIYTLANKEKSKPDSSRITGAMLQSVALTVGIDPGTVINDLLYLEQEVGILAEEMQNTLVNVIQTEGKEAALKFLPGFIFKVVEQEEFKEEFERYYSEFILAYKG acrib8 AcrIB8 _ acrib8.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between the endonuclease Cas2 (https://www.ncbi.nlm.nih.gov/protein/WWV39453.1) and AcrIB8 (ipTM = 0.77, pTM = 0.77). Listeria seeligeri type I-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE and prophages in Listeria genomes 10.1038/s41586-024-07923-x Ectopic expression of AcrIB5 (ECO_0000017) abolished interference against target plasmid. Binding it supported by computational structure modeling evidence (ECO_0006368). MKTIDRDEIAKDINACIKGLGRDIRTNWELGFEEGQIITLERYESWTTGGDFTVCNDCPVEYHFEIENEVPCHVVDYNNKEEVIALGAEDCEDENEVLLPAGTKLEVVYGEHEDDNEEMGFYTVIFKYLEEEK acrib9 AcrIB9 _ acrib9.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas8a1 (https://www.ncbi.nlm.nih.gov/protein/WWV39459.1) and AcrIB9 (ipTM = 0.81, pTM = 0.8). Listeria seeligeri type I-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE and prophages in Listeria genomes 10.1038/s41586-024-07923-x Ectopic expression of AcrIB5 (ECO_0000017) abolished interference against target plasmid. Binding it supported by computational structure modeling evidence (ECO_0006368). MNKFAFENDKYLERNIKAVVEKIARDFNLHLKSKYSKDCEFTVVADNSFDNIENSTIFLEIKRNDGKACQDHHIYAEYECDEDDNEYIALTVKFYGSSASNQINTVQGIKSSKYASCIVSDTDNQLSKSIHELNLKKEKEQQEAWNKKEAEYARKKQAYVSQSQREKYEDIFDLPFDFYDYIDKKEQGLI acriic7 AcrIIC7 _ acriic7.cif AlphaFold 3 unknown _ Listeria seeligeri type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Listeria seeligeri 10.1038/s41586-024-07923-x PF13443;PF01381 Ectopic expression of AcrIIC7 (ECO_0000017) abolished interference against target plasmid. MNVIEAEKFLKPKTNTTTFKFLVDEKLTANLTGMKPIGRIDTQRFLESHRASEFKILLDNPLKELLNYYGLTQYYLQKEVGLSQSTVSKLISDNRPIGSFQFAVLLKIAEATNRSVGEVADQLKEFNDLRDA acriic8 AcrIIC8 _ acriic8.cif AlphaFold 3 unknown _ Listeria seeligeri type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Listeria seeligeri 10.1038/s41586-024-07923-x Ectopic expression of AcrIIC8 (ECO_0000017) abolished interference against target plasmid. MDVTLFLVRALSSQIYEKDINNLFNKHALEAYHEAQSSQFWDSKAIQELPPADEIRARKILGLVCLSVKQEALRDELTTIFFRKIGFVLQRYIVKADTKEYYDTHLEEMKQQINALPQRVLDYVLILLDGNGVEKEQTLNYVYTLLKHWDQEGDIFLGLSEKEKTNTLQLLEEYFPQKKFTSYYDLLKVLYTEKKIDKAHIGAFQYFFISEKISTEEYATQIDIRKAELRQLFAIAVQKELPREIMPFYVISGIESLIVAKSYNKLRNSLLKSDLELVEVKHQVQQNDEVKWKRENALLKQENKRLQERIKFLEGEQHSWNKEEIKTRDETIFILEQLLEKKVNELDQPSNLDMEEEVNTTEEVVLDGLKIAIVGGYPKLNLDLKKEIPDLAIFTTIDRLNKSLQQFDYVFLLTSYSSHAMKMKLDSLKVDYFYLASMTTQSVVKELKKQIQAKAK acriic9 AcrIIC9 _ acriic9.cif AlphaFold 3 unknown _ Listeria seeligeri type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Prophage in Listeria 10.1038/s41586-024-07923-x Ectopic expression of AcrIIC9 (ECO_0000017) abolished interference against target plasmid. MTNLIMQKIVESEITNNYTSKKAFAEKYLGVSNVSLSRYLSGEQGLKAETMNRVEALFTHYELCIIKKMLLASMHTPEFRENPVGEFNRLKLEIAKKWVSYNRSFLDAGTSELPCELKIQYQGPSIDPSYQKWSSAVMAVKLVHDNPMMNDVITLRLPGVSKNRASAVPAGKKNRQEWFEKHIDQEFNAEI acrvia2 AcrVIA2 _ acrvia2.cif AlphaFold 3 unknown inhibits type VI-A CRISPR immunity by causing crRNA degradation (may degrade crRNAs directly, but there is a possibility that AcrVIA2 prevents loading of crRNAs into Cas13). Listeria seeligeri type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Listeria seeligeri 10.1038/s41586-024-07923-x PF22590;PF00270;PF00271;PF04851 acrvia2_model.html Ectopic expression of AcrVIA2 (ECO_0000017) abolished Cas13-dependent interference against a plasmid target in L. seeligeri, as demonstrated by plasmid-targeting assays. The same ectopic expression restored infection by a Cas13-sensitive phage (ϕLS59), indicating functional suppression of CRISPR immunity. A DEAD-box mutant (DEFD→AAFD) of AcrVIA2 (ECO_0000015) was expressed at similar levels but failed to inhibit Cas13 activity, confirming that the helicase motif is essential for function. MKNIHQKIQLNKLQVKTVQNKGKDLLINAPTGSGKTEASLLAVSDASKSVSYLLPTVVSTNVMYLRLKRDYKLNLSVQTSTKKEISNFAEGVHIKLECPDFALIDFIKTGKKTLGDTIICDEFDHYPEMVKSALMEYKHTFSETQIIFVSATLNKESLMGIDLEEIALDTEKNLIKYKVYPNDDFRMDDIINNGKAYGKKIGIIFNSISQLECFIKPGEDFYDDHFSKFKKGENDYIIHSQVDDYDKALAENAIVNNDFSVLIGTDSISYSIDVNFDILIMMASSEMATNIQRLGRCNRLNKHVTDYNLYFFGSYLSDLKAPFINENVAFNNLERITSSHLCISRKNINEIKKELPVSEIMEYIEVKKHVLDEEESLRPIPFKVRRGIEKEVVKFNAKGLKQTKVIKTYQTFNMMDLKYAFCEEYYYDKKNSRALDVIQQFDFENDWFDRGDFTVKLYNLKTEQQALKQLLLKLEEYIEPEAPDETDEDFYYRNPDILLKYTDYDKLFIKGWTYSILSIDGKTIYIA adps NARP1_Adps (ADPR-PP synthetase) _ adps.cif AlphaFold 3 synthesises and restores essential molecules depleted by bacterial defence adds a pyrophosphate group from ATP to ADPR to create ADPR-PP Bacillus subtilis type I Thoeris, DSR1, DSR2 and SEFIR. Escherichia coli SIR2–HerA Thoeris;DSR;SIR2-HerA;SEFIR https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris;https://defensefinder.mdmlab.fr/wiki/defense-systems/dsr;https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_her;https://defensefinder.mdmlab.fr/wiki/defense-systems/sefir Escherichia phage JohannRWettstein (Bas63) https://www.ncbi.nlm.nih.gov/nuccore/MZ501086.1/ 10.1038/s41586-024-07986-w namat PF14572;PF00156 Expression of Adps homolog from phage SpβL1 produced ADPR-PP from ADPR and ATP in vitro. Mass spectrometry (MS/MS) confirmed the identity of the reaction products (ADPR-PP and ADPR-cyclic phosphate, ECO_0001096). Mutation of a key active site residue (K162A) abolished activity, showing the importance of this residue (ECO_0000015). MKTVIEAVVTAQEKFFHTEKFNIIQFPSGEIGGNFSEDFVKFTERNAGKIDNVIITVQGYDKDTLFALALAKDAVDSLVPQKSAMKTIVFGFLPNARYDRHMFKGDAAALKVFANLVNAMGFDAVCALDPHSNVAENLFKCFQSMKQKDVAVHFASDPRIDFLVAPDAGAAKKTEDTAKEVDKPYITMSKVRNLKTGEITGMRILDDVDLTDKTVMILDDICDGGRTFVEAAKHLREAGAKRVELYVTHGIFSKGVENLLDNGIDHIYTTNTLGEAKDRGLTHYGQVTVATID namat NARP1_Namat (nicotinamide ADPR transferase) _ namat.cif AlphaFold 3 synthesises and restores essential molecules depleted by bacterial defence synthesises NAD+ from ADPR-PP and nicotinamide Bacillus subtilis type I Thoeris, DSR1, DSR2 and SEFIR. Escherichia coli SIR2–HerA Thoeris;DSR;SIR2-HerA;SEFIR https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris;https://defensefinder.mdmlab.fr/wiki/defense-systems/dsr;https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_her;https://defensefinder.mdmlab.fr/wiki/defense-systems/sefir Escherichia phage JohannRWettstein (Bas63) https://www.ncbi.nlm.nih.gov/nuccore/MZ501086.1/ 10.1038/s41586-024-07986-w adps Expression of Namat homolog from phage SpβL1 catalyzed the formation of NAD⁺ from ADPR-PP and nicotinamide in vitro. LC–MS and MS/MS confirmed NAD⁺ as the product (ECO_0001096). Mutation of the predicted active site residue (R306G) abolished activity, indicating its essential catalytic role (ECO_0000015). MTKSLYAVPAGLNADAYKSGHVYQYPSATEYLMFNLTPRSDKWFNSPLAIDGVVAFGIQRFVKDYLIDHWNATFFERDKKEAIDEILEVMNGVLGKDAIGREHWEALHDLGYLPVEVYAVEEGTVVPMRVPMIVFQNTVSGFHWVAGYLEDAFSAEIWKACTIATIALHYKRICKKWADLTCDNDLHLPYQCHDFAMRGMSGFTDDAFNAVGHLTSFKGTDSFPAVYTAKRIYGQSYPISDIGSSVPATEHSVMCANIAWEGGNELIEEERRFKGELQTFRRFLTETYPTGIASIVSDTYNFWRTVSEILPALRKEIMERDGKLVIRPDSGDPVHIVTGYKAIHLECAKKAYYEHLSKLEASDTMLDAVLNMKLENISYGIAGWLLSEGYEMVVDREDFEVADTVMLKNAYMVGSANVVTRPVAEIDGAIKTLYNIFGGTTNSKGFKVLDEHIGLIYGDSITLERANEILKRLYEMGFASSNVVFGVGSYTYQYMTRDTFAFAVKATLASIGGKEIMLAKDPKTDSGVKKSAFGGVSPMWDGDKLKAVDGYGFQSFADVLDHPACALRLVFSDSEQFGYTTLGDIRNNIDKQL nampt NARP2_nampt (nicotinamide phosphoribosyltransferase) _ nampt.cif AlphaFold 3 synthesises and restores essential molecules depleted by bacterial defence synthesises nicotinamide mononucleotide from nicotinamide and phosphoribosyl pyrophosphate Bacillus subtilis type I Thoeris, DSR1, DSR2 and SEFIR. Escherichia coli SIR2–HerA Thoeris;DSR;SIR2-HerA;SEFIR https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris;https://defensefinder.mdmlab.fr/wiki/defense-systems/dsr;https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_her;https://defensefinder.mdmlab.fr/wiki/defense-systems/sefir Vibrio phage KVP40 https://www.ncbi.nlm.nih.gov/nuccore/NC_005083.2 10.1038/s41586-024-07986-w nmnat PF18127 Expression of Nampt from Vibrio phage KVP40 produced NMN from PRPP and nicotinamide in vitro. LC–MS confirmed NMN as the primary product (ECO_0001096), and the enzyme showed a strong substrate preference for PRPP over ADPR-PP. MLNLNQNIAIATDSYKVSHWSQFPRGLEYSQYYVESRGGKFDKIMVDGMAYMCRILEKGVSMNDVKRAKRLFKKHFGSEVFNDKGWDIIVNELKGKLPIKIRAVKEGTVVPVKHPILTIENTDPRFGWLPGYLETFILRALWYPTTVATISFEVKKIIRQFMKKTVDDERIAEQEPFKLHDFGSRGVSSGESAAIGGSAHLKNFLGTDTVEALVAVEELYAEDVEDFIAGFSIPAREHSTTTIYKEAGEDQAFLNSIEQWGAALYACVMDSYDYEAAMNRVSTGRFKELIISKGGTFVARPDSGVPVDVVMKGLEILGKNVGYTINSKGYKVLHPSYRIIQGDGVNIEEIRRILSYMESKGWSAENIAFGMGGGLLQQLDRDTQRFAMKMSAAIINGEYVSVFKMPKTDPTKASKAGFLDLIAVDADNPNPAARGYVTFSSEDYDNRVHPKSVMQTIFEDGVTVADFSLEEARKLSDVQADFLNEGEWKIAKKIQTA nmnat NARP2_nmnat (nicotinamide mononucleotide adenylyltransferase) _ nmnat.cif AlphaFold 3 synthesises and restores essential molecules depleted by bacterial defence transfers adenyl group from ATP to nicotinamide mononucleotide and form NAD+ Bacillus subtilis type I Thoeris, DSR1, DSR2 and SEFIR. Escherichia coli SIR2–HerA Thoeris;DSR;SIR2-HerA;SEFIR https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris;https://defensefinder.mdmlab.fr/wiki/defense-systems/dsr;https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_her;https://defensefinder.mdmlab.fr/wiki/defense-systems/sefir Vibrio phage KVP40 https://www.ncbi.nlm.nih.gov/nuccore/NC_005083.2 10.1038/s41586-024-07986-w nampt PF01467;PF00293 nmnat_model.html Expression of Nmnat from Vibrio phage KVP40 enabled the conversion of NMN to NAD⁺ in vitro in the presence of ATP. Co-expression with Nampt reconstituted NAD⁺ from PRPP and nicotinamide, validating functional linkage (ECO_0001096). MSHAIFIGRFRPFHNGHLSAITQAFDALDLDKMTILIGSSNRHRSVKNPFVFEEVRDMMGVALPDHIRSKVRFVPLGDYAKDDVWQSNVRSRARGATHIVGYDKDESSYYLKLFPELKLFQPEPVKMYNKVISATDFRELYFSEILLNHPVMSGLIPKETMMFLDNWSKTEFFTEMKAEYDSSVREIEKFKDYPYQGHLNIACADNVVTCAGHVLLVERKFNPGKGCLALPGGHKHEKETFLDAAIRELQEETNIKVPEKVLRGSLVGEKMFDNPNRSYPHTRITMAYHLKVHPNPDNTFPKVKPADDAVSAKWYPLSEVRDMQERLYDDHYQIIQYFTGI card_protein CARD domain-containing protein _ card_protein.cif AlphaFold 3 binds and inhibits host defence system disrupts the interaction between the inflammasome complex and caspase, preventing activation of the caspase. Lysobacter Enzymogenes Gasdermins Gasdermin https://defensefinder.mdmlab.fr/wiki/defense-systems/gasdermin Acinetobacter phage 133 https://www.ncbi.nlm.nih.gov/nuccore/NC_015250.1 10.1038/s41586-024-08498-3 The evidence comes from a preprint (DOI: 10.1101/2023.05.28.542683), which later formed the basis of a peer-reviewed publication, though the specific experiments discussed here may were not included in the final paper. Co-expression of the CARD-like protein and the gasdermin system in cells led to increased phage sensitivity, implying inhibition of the defense. Infected cells expressing the CARD-like protein showed reduced gasdermin cleavage—a key step in gasdermin activation—supporting functional inhibition. Engineering the CARD-only gene into phage T4 under a native promoter resulted in T4 phages that were partially resistant to gasdermin-based immunity, confirming anti-defense activity in a different phage context. MIKVDRENIDAFAARIKNFELKAGESFTDYFMDSEVFAGSWGFWLIGKGYVERGNAIINAYNKANKKHWSDQELCFAVNASPLRWDAASNEFYPWGLDQGINLDLAINADYKLWAEFLISSDRYYEPFLKYLENFEAGGEY orf35 ORF35 _ orf35.cif AlphaFold 3 unknown _ Escherichia coli Tmn Tmn https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_tmn Phage ΦSMS22 10.1038/s42003-025-07730-8 Deletion of ORF35 from ΦSMS22 (ECO_0001038) resulted in reduced infectivity against E. coli expressing the Tmn defense system. ORF35 (anti-Tmn) from ΦSMS22 was cloned into a plasmid and co-expressed with Tmn in E. coli. When challenged with phages, only ΦSMS22-derived anti-Tmn restored infectivity of phages that were otherwise blocked by Tmn (e.g., ΦKSS9), confirming its inhibitory activity against Tmn (ECO_0000017). MKILQEEKNYWAAQCLEAREQVERVSALADTNQALYEGEKKVRQEIEERFELLFELMGIMQRTGYTTSVALNRITADGLREHCKRAEEYVRPKGRRSSIDRKDLVLVAWRANGSYYSDCRPLGKDEMARVQRVLTRGKGEKIALYALDPQNDGE acriia13 AcrIIA13 8K4M acriia13.pdb PDB entry binds and inhibits host defence system AlphaFold 3 predicts a direct interaction between SauCas9 and AcrIIA13 (ipTM = 0.9, pTM = 0.79), which is supported by a PDB entry without a corresponding publication (https://www.rcsb.org/structure/7ENI). Staphylococcus aureus type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Staphylococcus schleiferi 10.1073/pnas.1917668117 AcrIIA13 was cloned into a plasmid and tested in a TXTL assay, where it restored GFP expression otherwise suppressed by SauCas9 (ECO_0000017), confirming inhibition. In vitro cleavage assays showed that AcrIIA13 blocked SauCas9-mediated DNA cleavage, primarily by preventing DNA binding. RNA EMSA confirmed it did not interfere with RNP formation (ECO_0001807). Binding it supported by computational structure modeling evidence (ECO_0006368). MEVMNKSIEIKDQNNIVLIDSLGQFFTDIENDNNGRYNIDYVLLNEVEHDNGNTYYEVGMYRTEEVPFSDKVTQDNVELLEDKWLQIDQQGESYVESIFFENEEDAREYIKLVLKGHETFEETAKAIGVIK acriia14 AcrIIA14 7ENM acriia14.pdb PDB entry unknown _ Staphylococcus aureus type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Staphylococcus simulans 10.1073/pnas.1917668117 AcrIIA14 was discovered through guilt-by-association analysis using genes co-occurring with an aca-like gene near AcrIIA13. AcrIIA14 was cloned and tested in a TXTL assay, where it restored GFP expression suppressed by SauCas9, indicating anti-CRISPR activity (ECO_0000017). In vitro cleavage assays showed complete inhibition of SauCas9 activity, despite RNA and DNA EMSAs showing no interference with RNP formation or DNA binding (ECO_0001807). This suggests AcrIIA14 blocks cleavage post-DNA binding. Deletion of the N-terminal domain reduced but did not eliminate activity (ECO_0007379). SMKSVKYISNMSKQEKGYRVYVNVVNEDTDKGFLFPSVPKEVIENDKIDELFNFEHHKPYVQKAKSRYDKNGIGYKIVQLDEGFQKFIELNKEKMKENLDY acriia15 AcrIIA15 8JFO_B acriia15.pdb PDB entry binds and inhibits host defence system binds to Cas9, blocking the PAM recognition sites. Staphylococcus aureus type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Staphylococcus delphini 10.1073/pnas.1917668117 AcrIIA15 was also identified by guilt-by-association with genes adjacent to the same aca-like region. AcrIIA15 was cloned and expressed in TXTL, where it restored GFP expression in the presence of SauCas9 (ECO_0000017). In vitro, AcrIIA15 inhibited SauCas9 cleavage only when added prior to sgRNA, indicating it blocks RNP formation by binding Cas9 before guide RNA association. RNA EMSA confirmed it disrupted RNP formation, and DNA EMSA showed inhibition of target binding if added early (ECO_0001807). The C-terminal domain alone retained inhibitory activity, while the N-terminal HTH domain was dispensable for Cas9 inhibition. MRKTIERLLNSELSSNSIAVRTGVSQAVISKLRNGKKELGNLTLNSAEKLFEYQKEMEKVDTWIVYRGRTADMNKSYIAEGSTYEEVYNNFVDKYGYDVLDEDIYEIQLLKKNGENLDDYDVDSDGINNYDKLDEFRESDYVDLEDYDYRELFENSSSQVYYHEFEITHE gp1_2 Gp1.2 7U66 gp1_2.pdb PDB entry binds and inhibits host defence system binds and inhibits Dgt Escherichia coli dGTPase dGTPase https://defensefinder.mdmlab.fr/wiki/defense-systems/dgtpase Escherichia phage T7 https://www.ncbi.nlm.nih.gov/nuccore/NC_001604.1 10.1073/pnas.2123092119 Gp1.2 was structurally characterized by NMR and shown to bind directly to Dgt, as confirmed by cryo-EM of the Dgt–Gp1.2 complex with or without bound (d)GTP (ECO_0006181). Biochemical assays showed Gp1.2 is a mixed-type inhibitor, reducing both Vmax and increasing KM, with an IC50 of 160 nM. GTP alone competitively inhibited Dgt (KI ~120 µM), but in combination with Gp1.2, inhibition was synergistic, reducing Gp1.2’s IC50 to 27 nM and GTP’s IC50 to 310 nM. GSFTMGRLYSGNLAAFKAATNKLFQLDLAVIYDDWYDAYTRKDCIRLRIEDRSGNLIDTSTFYHHDEDVLFNMCTDWLNHMYDQLKDWK gam Gam 2UUZ gam.pdb PDB entry binds and inhibits host defence system binds to the RecBCD complex and may bind to KiwaB. triggers retrons Se72 and Ec48, as well as Old nuclease. Escherichia coli RecBCD and Kiwa RecBCD;Kiwa _;https://defensefinder.mdmlab.fr/wiki/defense-systems/kiwa Enterobacteria phage lambda https://www.ncbi.nlm.nih.gov/nuccore/NC_001416.1 10.1073/pnas.70.8.2215;10.1016/j.cell.2025.07.002 PF06064 Purified Gam protein inhibits all catalytic activities of RecBC DNase (measured using using radiolabeled DNA substrates), including its ATPase (measured by tracking hydrolysis of [γ-³²P]ATP) and exonuclease functions. Two-hybrid (ECO_0000068) and pull-down (ECO_0006249) assay showed binding between KwaB and Gam. MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYYIQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITRAFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV rexb RexB _ rexb.cif AlphaFold 3 uncategorised stabilises the antitoxin MazE by affecting the ClpP proteases responsible for its degradation. Escherichia coli type II TA system MazEF TA https://defensefinder.mdmlab.fr/wiki/defense-systems/mazef Enterobacteria phage lambda https://www.ncbi.nlm.nih.gov/nuccore/NC_001416.1 10.1073/pnas.95.26.15481 In P1 phd‑doc tests, cells expressing wild-type rexB survived plasmid loss at high temperature (ECO_0000017), whereas rexB mutants (nonsense or deletion) did not. Induction of ppGpp—or amino acid starvation via serine hydroxamate—to activate mazEF leads to ~90% killing; but rexB, either from plasmid or a λ lysogen, restores survival up to ~60–100% depending on the setup. Pulse-chase experiments demonstrated that λRexB extends the half-life of the antitoxins Phd (from P1) and MazE (from E. coli), both substrates of ClpP proteases (ClpPX and ClpPA, respectively) MRNRIMPGVYIVIIPYVIVSICYLLFRHYIPGVSFSAHRDGLGATLSSYAGTMIAILIAALTFLIGSRTRRLAKIREYGYMTSVVIVYALSFVELGALFFCGLLLLSSISGYMIPTIAIGIASASFIHICILVFQLYNLTREQE pina PinA _ pina.cif AlphaFold 3 binds and inhibits host defence system binds to the Lon protease, which degrage some antitoxins Escherichia coli RM Lon Protease TA https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1074/jbc.273.1.518 PF10465 PinA inhibits ATP-dependent degradation of casein by Lon protease, as measured by the release of trichloroacetic acid-soluble peptides from [³H]-labeled casein. PinA forms a stable complex with Lon, shown by co-elution during gel filtration chromatography (ECO_0001049). MITVDKWFRINRADTGLCNYWPELSAGTVFKVRELVKECEDDIEPDTGIIEIELSDGKIINIYDKPITYWCLWNTESVENGEIEEVVERTNQVVQKPKADFQGERISYALAKLAAQENNDGYEGNLMQAAAEYIEWLETQISFSDRMIQQYKRLHQMFYNT dcmp_hm Deoxycytidylate hydroxymethylase 1B5E_A dcmp_hm.pdb PDB entry modifies phage molecules to avoid recognition converts 2′-deoxycytidylate (or 2′-deoxycytidine-5′-monophosphate, dCMP) into 5-hydroxymethyl-dCMP (a step in DNA hypermodification). Escherichia coli RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1093/emboj/18.5.1104 dnmp;bgt PF00303 dcmp_hm_model.html Crystal structure of T4 deoxycytidylate hydroxymethylase with dCMP shows the direct binding (ECO_0001034). Substitution of Asp179 to Asn drastically reduced activity on dCMP (~15,000-fold decrease in k_cat/K_M), but increased activity on dUMP, suggesting Asp179 is crucial for dCMP specificity and catalysis. MISDSMTVEEIRLHLGLALKEKDFVVDKTGVKTIEIIGASFVADEPFIFGALNDEYIQRELEWYKSKSLFVKDIPGETPKIWQQVASSKGEINSNYGWAIWSEDNYAQYDMCLAELGQNPDSRRGIMIYTRPSMQFDYNKDGMSDFMCTNTVQYLIRDKKINAVVNMRSNDVVFGFRNDYAWQKYVLDKLVSDLNAGDSTRQYKAGSIIWNVGSLHVYSRHFYLVDHWWKTGETHISKKDYVGKYA psia PsiA _ psia.cif AlphaFold 3 unknown _ Escherichia coli SOS response SOS stress response _ Escherichia coli plasmid 10.1093/nar/18.15.4597 Deletion (ECO_0007379) or nonsense (ECO_0007161) mutations in the psiB gene abolished SOS inhibition, indicating that PsiB protein is required for the phenotype. Ectopic expression (ECO_0000017) of PsiB alone (12 kDa protein) is sufficient for inhibiting SOS functions. MSVRSQALVPLSTEQQAAWRAVAETEKRRHQGNTLAEYPYAGAFFRCLNGSRRISLSDLRFFMPSLTAEELHGNRLQWLYAIDVLIETQGEVCLLPLPGDAAERLFPSVRFRVRERSRHKSALVMQKYSRQQAREAEQKARAYQALVAQAEIELAFHSPETVGSWHARWSDRVAEHDLETLFWQWGERFPSLAGMERWQWQDMPFWQVIAEASLAAREAGHAVREMERWMVPNKLREAA acriia20 AcrIIA20 _ acriia20.cif AlphaFold 3 unknown _ Streptococcus iniae type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus iniae 10.1093/nar/gkaa219 In vitro DNA cleavage assays with SpyCas9, SinCas9, SauCas9, and AsCas12a showed Strong inhibition of SinCas9, weak inhibition of SpyCas9 and no inhibition of SauCas9 or AsCas12a. Competition binding assay with AcrIIA2 shows ML1 binds Cas9–sgRNA and blocks AcrIIA2 binding. MKNYEVTNEVKNLNTQVETIGQAVDLYKEYGSNTIVWSIDKNEDLIDEVTELVAEYAEKGTVIK acriia21 AcrIIA21 _ acriia21.cif AlphaFold 3 unknown _ Streptococcus pyogenes, Streptococcus aureus, and Streptococcus iniae type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus agalactiae GB00548 10.1093/nar/gkaa219 PF13545;PF01047;PF06970 In vitro DNA cleavage assays with SpyCas9, SinCas9, SauCas9, and AsCas12a showed strong inhibition of SinCas9, weak inhibition of SpyCas9, SinCas9, and SauCas9 and no inhibition of AsCas12a. MDYDNENYLIPKILLQDDFYSSLSAKDILVYAVLKDRQIEALEKGWIDTDGSIYLNFKLIELAKMFSCSRTTMIDVMQRLEEVNLIERERVDVFYGYSLPYKTYINEV vcrx091 Vcrx091 (SSB) _ vcrx091.cif AlphaFold 3 synthesises and restores essential molecules depleted by bacterial defence involved in repairing double-strand DNA breaks via recombination between short sequence repeats (single-strand-binding protein). Vibrio cholerae type I CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Plasmid pVCR94 10.1093/nar/gkaa518 vcrx092;vcrx093 PF00436 Deletion of any of these genes (vcrx091, vcrx092, and vcrx093) caused a 6–200 fold drop in conjugation efficiency in the presence of CRISPR targeting (ECO_0007379). Evasion was RecA-independent, consistent with single-strand annealing-type repair. The effect was rescued by complementation in the recipient only, showing the repair happens post-entry. Repaired plasmids showed specific deletions between short direct repeats, confirming Bet/Exo-mediated recombination. MSKGVNKVILVGNLGSDPEIRYMPSGTAVANFNVATTDTWRDKQSGEQREHTEWHRVVLKGRLAEVAGEYLKKGSQVYLEGSNRTRKWTDNQQIERYTTEVHCFEMQMLGGRGNAPQDNSQRAAPQQGQRTGAGTQSAPVQQSAPQGGMGGGYGPAPDGWDDDIPFMRLHHLAGG vcrx092 Vcrx092 (Bet) _ vcrx092.cif AlphaFold 3 synthesises and restores essential molecules depleted by bacterial defence involved in repairing double-strand DNA breaks via recombination between short sequence repeats (single-strand-annealing recombinase). Vibrio cholerae type I CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Plasmid pVCR94 10.1093/nar/gkaa518 vcrx091;vcrx093 PF03837 Deletion of any of these genes (vcrx091, vcrx092, and vcrx093) caused a 6–200 fold drop in conjugation efficiency in the presence of CRISPR targeting (ECO_0007379). Evasion was RecA-independent, consistent with single-strand annealing-type repair. The effect was rescued by complementation in the recipient only, showing the repair happens post-entry. Repaired plasmids showed specific deletions between short direct repeats, confirming Bet/Exo-mediated recombination. MVSPNRSFVNWRNTMSDNKSLVTRIASRFGVDTRKFYETLKATAFKQRDGSAPTDEQMMTLLIVAEQYGLNPFTREIYAFPDKQNGIIPVVGVDGWSRIINEHPQYDGVEFVYSDKMVRMQGAKVECPEWIECVIYRKDRSRPIRIKEFIDEVYREPFQGQGRNGAYTVDGPWQTHTKRQLRHKSLIQCSRVAFGFSGIYDQDEAERIREMEQASAINPAIANLPSPSQVHSQEPLAIEHKELDPILTKLANRAIAEKAWSAAHEYVKGRYEGSELQYATQFLREKEMDQMEPPKPDYQESHEQESAAGGSANAELGAEEMPPLSDEDMIPVMEEEGAEGSYY vcrx093 Vcrx093 (Exo) _ vcrx093.cif AlphaFold 3 synthesises and restores essential molecules depleted by bacterial defence involved in repairing double-strand DNA breaks via recombination between short sequence repeats (double-strand exonuclease). Vibrio cholerae type I CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Plasmid pVCR94 10.1093/nar/gkaa518 vcrx091;vcrx092 PF09588 vcrx093_model.html Deletion of any of these genes (vcrx091, vcrx092, and vcrx093) caused a 6–200 fold drop in conjugation efficiency in the presence of CRISPR targeting (ECO_0007379). Evasion was RecA-independent, consistent with single-strand annealing-type repair. The effect was rescued by complementation in the recipient only, showing the repair happens post-entry. Repaired plasmids showed specific deletions between short direct repeats, confirming Bet/Exo-mediated recombination. MKIVNLSQREEDWLDWRRQGVTATDAAILLNRSPYKTRWRLWAEKTGYAREVDLSLNPLVRRGIENEDAARRAFEEKYDDMLLPACVESVQYPLMRASLDGLRDNGEPVELKSPSATVWEDVCAEKANSKAYQLYYPQVQHQLLVTGAKQGWLVFYFEGQIQEFPILRDEAMIQEILAEAKKFWQQVVDKKEPDKDPERDLYIPQGEEVNRWIAAAEEYRLYDAEIQELKQRLSELQERQKPHLDTMKSLMGEYFHADYCGVMVTRYKAAGRVDYKKLLADKASGVKPEDVDQYREKSSERCRVTVTGSVKPRYIVDEDVLAPLDDLPEEVETFYW vcrx089 Vcrx089 _ vcrx089.cif AlphaFold 3 unknown _ Vibrio cholerae type I CRISPR-Cas RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Plasmid pVCR94 10.1093/nar/gkaa518 vcrx090 PF07728;PF08406;PF00437 vcrx089_model.html Deletion of vcrx089-090 (ECO_0007379) led to a 25-fold decrease in conjugation efficiency to V. cholerae O395 (which has an active type I R-M system). The transfer defect was rescued by deleting the recipient’s hsdR gene, confirming it’s R-M-related. Complementation in recipient cells (not donors) restored transfer efficiency, supporting their function post-DNA entry. MSQYSQFSVSKVFGMPSIPEKVTAIGYADGSNPFIPATDTNYVFRKEFLREVLAYLKEPGGDALFVTGPTGSGKTSGITEIAGRLNWPVQQITAHGRMELTDLIGHHALVAEKPGQPPVMKFMYGPLAVAMREGHLFLINEVDLADPAELAGLNDVLEGRPLVIAQNGGEIIKPHPMFRVVVTGNSTGSGDASGLYQGVMMQNLAAMDRYRFTKVGYADEEAELSILGRVTPKLPENVRKGMVRIANQVRKLFLGENGEDGQISVTMSTRTLVRWAKLSLAFRGAPNALEYALDQALLIRAAKEEREAILRVAKDVFGDQWR vcrx090 Vcrx090 _ vcrx090.cif AlphaFold 3 unknown _ Vibrio cholerae type I CRISPR-Cas RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Plasmid pVCR94 10.1093/nar/gkaa518 vcrx089 Deletion of vcrx089-090 (ECO_0007379) led to a 25-fold decrease in conjugation efficiency to V. cholerae O395 (which has an active type I R-M system). The transfer defect was rescued by deleting the recipient’s hsdR gene, confirming it’s R-M-related. Complementation in recipient cells (not donors) restored transfer efficiency, supporting their function post-DNA entry. MKKNDCLCRRYTFKDALTSETFEVFIGYKLLREPSSSGPGQFTMVKLNRTVTDGKAENWSETKLEGPFEANGPDTIPMSYKDKESQYVSQFLSQGYTFLDEVLVNAETQTVLEGGSVSAGQTASLGSLNWLLSPPSELPPGDINLFKGFVAGVFAKGAGLIGFEVARSEGSNDLLPSVLMRTDSGYELGVSTGLGENTIHPATLEGAGELRPEHGHKPLLMLVYLQQRFADDFSNVEKPLVAFCDEQGDTFDYERFDSLKPLIERFGFSYDEVRADAERLGLVSELIRLAEIDAEQEDHFF acrif7 AcrIF7 6M3N acrif7.pdb PDB entry binds and inhibits host defence system binds to the target DNA-binding site of Cas8f. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage LPB1 https://www.ncbi.nlm.nih.gov/nuccore/NC_027298.1 10.1038/nmicrobiol.2016.85;10.1093/nar/gkaa690 Ectopic expression of AcrF7 in Pseudomonas aeruginosa with a type I-F CRISPR-Cas system lead to restoration of phage replication with CRISPR-sensitive phage. AcrIF7 inhibited CRISPR interference. Electrophoretic Mobility Shift Assays (ECO_0000096) shows binding to the Csy complex. Pull-down experiments and gel filtration, indicating specific interaction with Cas8f (ECO_0006249). GHMTTFTSIVTTNPDFGGFEFYVEAGQQFDDSAYEEAYGVSVPSAVVEEMNAKAAQLKDGEWLNVSHEA aca10 Aca10 7XI5_A aca10.pdb PDB entry unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1093/nar/gkab006 PF13560 _ MSSATPDPAEILTARKAVGLSQTAAAALVHSSLRTWQQWEAGDRRMHPGLWELFLLKTQLPSPSS acric3 AcrIC3 _ acric3.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a high confidence interaction between helicase/endonuclease Cas3 (https://www.ncbi.nlm.nih.gov/protein/UEM35119.1) and AcrIC3 (ipTM = 0.91, pTM = 0.90) and a moderate confidence interaction between Cas2 (https://www.ncbi.nlm.nih.gov/protein/UEM35125.1) and AcrIC3 (ipTM = 0.7, pTM = 0.56). Pseudomonas aeruginosa type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa 10.1093/nar/gkab006 DMS3m phage was genetically engineered to carry the acrIC3 gene. This phage was then used to infect PAO1IC, a strain expressing the Type I-C CRISPR–Cas system from PaLML1 along with a crRNA targeting DMS3m. The engineered phage formed plaques on PAO1IC with an efficiency comparable to that on a non-targeting control strain, indicating strong anti-CRISPR activity. However, when Cas3 was fused to Cas8 — mimicking natural Cas3-Cas8 fusions to eliminate the normal recruitment interface — AcrIC3 no longer conferred protection, suggesting it normally blocks Cas3 recruitment to the Cascade complex. Binding it supported by computational structure modeling evidence (ECO_0006368). MSIQVTSTNGRTVNLEIELGSVVASSGQVKFMADKTDRGLESRFLVPEAGNRRIEVALTGRDLEAANALFSELAASVEATNEMYRELDAERAQINKALEG acric4 AcrIC4 8DFO_M acric4.pdb PDB entry binds and inhibits host defence system interacts with both the Cas7c and Cas8c subunits and inhibits dsDNA binding by acting as a negatively charged structural blockade at the PAM recognition site. Pseudomonas aeruginosa type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa 10.1093/nar/gkab006;10.1016/j.molcel.2023.01.024 Phage-encoded acrIC4 was introduced into DMS3m and tested on PAO1IC. The phage regained full infectivity (EOP ≈ 1), indicating strong inhibition of Type I-C immunity. AcrIC4 also relieved transcriptional repression in a CRISPRi assay, confirming it blocks DNA binding by Cascade. AcrIC4 activity was unaffected by Cas3–Cas8 tethering. Cryo-EM structure of the type I-C Cascade bound to AcrIC4 (ECO_0006181) revealed how AcrIC4 inhibits DNA targeting, specifically binding at the PAM recognition site, forming an electrostatic and steric blockade that prevents the Cascade complex from engaging with its DNA target. MDNKITPADEEKIREWLNCEEASVDNDGDVWVAVPMTGHWLSDEQKAKYIEWRGDET acric5 AcrIC5 _ acric5.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas8c (https://www.ncbi.nlm.nih.gov/protein/UEM35121.1) and AcrIC5 (ipTM = 0.92, pTM = 0.85). Pseudomonas aeruginosa type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas delhiensis 10.1093/nar/gkab006 PF22147 The acrIC5 gene was cloned into DMS3m and tested on both PAO1IC and a P. aeruginosa strain expressing a heterologous Eggerthella lenta Type I-C system. In both hosts, the phage carrying AcrIC5 formed plaques at wild-type levels (EOP ≈ 1). CRISPRi assays confirmed DNA binding was inhibited. This demonstrates AcrIC5 has broad activity across divergent I-C systems, despite only moderate Cas protein sequence identity (~35–55%). Binding it supported by computational structure modeling evidence (ECO_0006368). MSKVTLNGQQIDFDAAVNLMDAELREELHSAQEWTNDQEFLDAYVQAHAAKFDGEEFQVA acric6 AcrIC6 _ acric6.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas phragmitis 10.1093/nar/gkab006 The DMS3m phage expressing acrIC6 showed only partial restoration of infectivity on PAO1IC (EOP ≈ 0.01), suggesting weak inhibition of the Type I-C system. It also did not relieve repression in CRISPRi assays, leaving its mechanism unclear. AcrIC6 had marginal activity against the Type I-E system as well. MTESLIHLRVPAATKGRWVRASRAVGLRLTDYITQAVEAYMQQQLTRVAIPDDIEFSDLKLARDPDGAVSFDWAVIERICHASGLPLEMMRDAPEDNVASLIIGWYQAHRADGGAADPVADDLIAEAMAEDAAGQQFSHQPGRA acric7 AcrIC7 _ acric7.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas stutzeri 10.1093/nar/gkab006 Three homologs of acrIC7 (from P. stutzeri, P. citronellolis, and P. aeruginosa) were expressed from DMS3m and tested on PAO1IC and a Type I-E strain. AcrIC7Pst and AcrIC7Pci blocked both Type I-C and I-E systems (EOP ≈ 1 in both). AcrIC7Pae only blocked I-E, not I-C. CRISPRi confirmed that all three variants that inhibited systems also blocked DNA binding. MATVTKITLNGQNHYNFGSECSEADAEGYREWIAQELAENFPGAEIEINEADSTYSVVVEIDDESYYDEARGLKDDVNVFCIDAWDRCPWDWVS acric8 AcrIC8 8g9s_A acric8.pdb PDB entry binds and inhibits host defence system inactivates Cascade by trapping the PAM-recognising Cas8 subunit in a non-productive conformation, incapable of performing PAM recognition. Pseudomonas aeruginosa type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa 10.1093/nar/gkab006 DMS3m phage engineered to express acrIC8 was tested on PAO1IC and a Type I-E strain (PA4386). The phage regained full infectivity on both (EOP ≈ 1), and CRISPRi assays confirmed inhibition of DNA binding. AcrIC8 is a broad-spectrum Acr that blocks both Type I-C and Type I-E systems. SMYAIRKIQFFYGPTDKKSYVGEEAGGRRELFKTRAEAQARIEDLEEGVYYLAHNESGRPDYKIVWVRGE acrie9 AcrIE9 _ acrie9.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa type I-E CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa 10.1093/nar/gkab006 Although not a primary focus, AcrIE9 was included as a comparison. It was expressed from phage and tested on a P. aeruginosa strain with a Type I-E system. The phage formed plaques efficiently (EOP ≈ 1), and CRISPRi rescue confirmed that AcrIE9 blocks Cascade DNA binding. MEMQINSRKLGRTITFSRPGASYIFADLNGKSGTLGCQICSGGGTMGSTLSYDGDDQAQFEAICRRWYRAHVRGE acrif2_c2 AcrIF2/C2 5uz9_J acrif2_c2.pdb PDB entry binds and inhibits host defence system acts as a dsDNA mimic that blocks target recognition by competing for a critical DNA-binding site on Cas7 and Cas8. Pseudomonas aeruginosa type I-F and I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas aeruginosa phages D3112 10.1093/nar/gkab006 AcrIF2* was expressed from a DMS3m phage and tested on PAO1IC (Type I-C) and PA14 (Type I-F). The engineered phage formed plaques efficiently on both hosts (EOP ≈ 1), demonstrating dual inhibition of I-C and I-F systems. However, when both CRISPR systems were co-expressed in the host (one targeting, one non-targeting), AcrIF2*-phage infectivity dropped ~100–1000-fold at low MOI, suggesting a competition effect between the two systems for the inhibitor. Structural data and mutagenesis of eight acidic residues (to alanine) showed that AcrIF2* can tolerate major surface changes and still function, but loses robustness under competition. In CRISPRi assays, it fully rescued transcription, confirming it acts by blocking Cascade DNA binding. Its function is consistent with DNA mimicry. MATKTAQMIAQQHKDTVAACEAAEAIAIAKDQVWDGEGYTKYTFDDNSVLIQSGTTQYAMDADDADSIKGYADWLDDEARSAEASEIERLLESVEEE aca11 Aca11 _ aca11.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1093/nar/gkac099 _ MKVDTKQIEWLLKNASGYQISKMSGVAQPTISALINKKRSIENLTIETGHKLTELANQMQKTP aca12 Aca12 _ aca12.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1093/nar/gkac099 PF13443 _ MLINTSRVEMVLMNKAISAYRLAKEIGIQESSISLLRNGKKDLDKLSLEVAMRVQAWIDAGNYSFSYDYSELIEKLEADIEKGLADEYIYIVRGGYNEVMEKCMIIDYYYDPEEIAEGDIAEKVLTSSALAEMEKDNEIF aca13 Aca13 _ aca13.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1093/nar/gkac099 _ MTDELTARQRADKKWNEKNREHRNYMTKRSTARGFIRNHATKEDLLELQKLIQENLKKF acriia24 AcrIIA24 _ acriia24.cif AlphaFold 3 unknown _ Streptococcus thermophilus and Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Streptococcus phage CHPC930 https://www.ncbi.nlm.nih.gov/nuccore/NC_070681.1 10.1093/nar/gkac099 Ectopic expression of AcrIIA24 conferred resistance to CRISPR targeting in E. coli expressing St3Cas9 (ECO_0000017). AcrIIA24 was cloned into a plasmid and co-expressed with St3Cas9 and a targeting sgRNA. In plasmid interference assays, AcrIIA24 significantly restored bacterial colony formation. In vitro cleavage assays showed that AcrIIA24 did not prevent DNA binding by Cas9 but strongly inhibited cleavage. MKKAQQLLKEIKTNNVSYAIMDEDNEIYCNKETNNIMDIYGYDNENGHFYGVYGDVVDGQIDSRYFSDDAILNAIDKLLFLGDPIKRTDLPSDADFKRTFFFEE acriia25 AcrIIA25 _ acriia25.cif AlphaFold 3 unknown _ Streptococcus thermophilus and Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Streptococcus phage P7602 https://www.ncbi.nlm.nih.gov/nuccore/NC_070704.1 10.1093/nar/gkac099 Deletion of the genomic locus encoding AcrIIA25 from its native mobile element resulted in loss of inhibitory activity against SpyCas9 in E. coli (ECO_0007379). AcrIIA25 was cloned into an inducible expression plasmid and co-expressed with SpyCas9 targeting a plasmid. Plasmid interference assays showed moderate restoration of colony formation. In vitro assays showed AcrIIA25 could inhibit DNA cleavage but had minimal effect on DNA binding. Phage assays confirmed restored phage infectivity in strains expressing AcrIIA25, indicating moderate anti-CRISPR function via inhibition of the cleavage step. MKNRLLGSRYTDAIKNDCGTANKMSNIYNKLNKDSLREIHSALYGLLTAGYDISNMRNIEELEKYVNLKKSRGQLLNVSSDDIKLYHKLFVIRFGK acriia26 AcrIIA26 _ acriia26.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a high confidence interaction between Cas9 (https://www.ncbi.nlm.nih.gov/protein/QHB64844.1) and AcrIIA26 (ipTM = 0.87, pTM = 0.79) Streptococcus thermophilus and Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus sp. 10.1093/nar/gkac099 Expression of AcrIIA26 in E. coli prevented Cas9-mediated plasmid interference, particularly with SpyCas9 (ECO_0000017). AcrIIA26 was cloned and tested in interference assays, which showed full protection. In vitro cleavage reactions showed that AcrIIA26 blocked RNP–DNA binding. EMSAs confirmed prevention of RNP-DNA complex formation (ECO_0001807). In human cells, co-transfection of AcrIIA26 with SpyCas9 led to near-complete loss of indel formation, supporting a DNA-binding inhibition mechanism. Binding it supported by computational structure modeling evidence (ECO_0006368). MKKLYIQTNQFANGELQVENTSYELCDTFKELYSVASNLVDENTLNFVEDNFIEQNYKDEYNGVYENDGDTGEFVGQVFENKVTEEQFKELLEQLEITYTEFDPEEELAKCIANKNRKSEFYGNGLKVIAEYLESISHEDALAVVTYYYFYFGFGYEDQLISDIKDDQEDGVKFEHVERSETI acriia27 AcrIIA27 _ acriia27.cif AlphaFold 3 unknown _ Streptococcus thermophilus and Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus pyogenes K23866 10.1093/nar/gkac099 Cloned from a Streptococcus mobile genetic element, AcrIIA27 provided complete rescue in E. coli expressing SpyCas9 or St3Cas9 in plasmid interference assays (ECO_0000017). In vitro cleavage assays confirmed inhibition only when Acr was added prior to DNA, suggesting inhibition of binding. MKTFNIIVSESANLKEHSSELVDNIIYKVEAKNRREAFKKAREEYSFSSKWKFNMRDLTAIDNTHRRAWGRRYLRVEEA acriia28 AcrIIA28 8WRX acriia28.pdb PDB entry binds and inhibits host defence system binds to the REC3 domain of SpyCas9. Streptococcus thermophilus and Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Streptococcus phage Javan128 https://www.ncbi.nlm.nih.gov/nuccore/1608078509 10.1093/nar/gkac099 AcrIIA28 was identified from a Streptococcus phage element and tested using a plasmid interference assay in E. coli. In this system, E. coli was engineered to express SpyCas9 or St3Cas9 and an sgRNA targeting a co-transformed plasmid carrying an antibiotic resistance gene. Cas9-mediated cleavage of this plasmid resulted in loss of resistance and prevented colony formation on selective media. Co-expression of AcrIIA28 reversed this Cas9-induced growth inhibition, indicating strong anti-CRISPR activity. In vitro DNA cleavage assays confirmed that AcrIIA28 prevented formation of the Cas9–DNA complex. EMSAs further demonstrated that AcrIIA28 blocks DNA binding by Cas9 (ECO_0001807), consistent with its function as a DNA binding inhibitor. MKTIFTKKQTEELLNDISIEKQKELFNSMHDFRSQHAKEARIPGWSDKYNKLEKKMLSDFEEVTGIKYDTLESELIWDNLSNKFLYNS acriia29 AcrIIA29 _ acriia29.cif AlphaFold 3 binds and inhibits host defence system binds to SpyCas9 via the REC3 domain, inhibiting DNA loading. Streptococcus thermophilus and Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus pyogenes NS3335 10.1093/nar/gkac099 AcrIIA29 was identified from a Streptococcus prophage and tested using a plasmid interference assay in E. coli. In this setup, E. coli cells were engineered to express St3Cas9 and an sgRNA targeting a co-transformed antibiotic resistance plasmid. Cas9-mediated cleavage of this plasmid reduced colony formation on selective media. Co-expression of AcrIIA29 partially restored colony formation, indicating moderate anti-CRISPR activity. In vitro cleavage assays revealed that AcrIIA29 inhibited DNA cleavage without affecting Cas9’s DNA binding ability. MKPSQKIKWLLTATGITTYKIGKDIEESTQFLDRYKNDPEKIGGMRLEKAEKLLEYISNLRQEDVIKTNWNNQQILVQNSTEKEITKYFNSYPFAIKLNWIKPHKEMFIVNFDTTSNKTFRKYPYDLKNLYFLVDKNRDKMSQFAEFLIICGRKSHFGGSRVLYEVEGKKYQIIFSIKRPSELGPTIRLINVVETDTYRDDLVPKISEEESILRSEDLDLKGKRVSIKDSELLELMSIIDN acriia30 AcrIIA30 _ acriia30.cif AlphaFold 3 unknown _ Streptococcus thermophilus type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus gordonii NCTC7870 10.1093/nar/gkac099 AcrIIA30 was identified in a Streptococcus genomic element and shown to be a specific inhibitor of St1Cas9. In the plasmid interference assay, E. coli cells expressing St1Cas9 and a plasmid-targeting sgRNA lost the targeted plasmid and failed to grow on selective media. Co-expression of AcrIIA30 restored growth, indicating effective inhibition. In vitro cleavage assays confirmed that AcrIIA30 blocked Cas9–DNA complex formation. EMSA showed that AcrIIA30 not only prevented DNA binding but also caused a supershift (ECO_0001807), suggesting potential dimerization or complex stabilization. MITANEIVKTHKGIRLVQRKNESWEEFKERIQEVIAKQGDNYLTQTKPVHEIKNKGTRNIRRTYVNILLKEGA acriia31 AcrIIA31 _ acriia31.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Cas9 (https://www.ncbi.nlm.nih.gov/protein/CAD0136979.1) and AcrIIA31 (ipTM = 0.87, pTM = 0.62). Streptococcus thermophilus type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus sp. SR1 10.1093/nar/gkac099 Binding it supported by computational structure modeling evidence (ECO_0006368).AcrIIA31 was found in a mobile element from Streptococcus sp. and validated as a strong and specific inhibitor of St1Cas9. In plasmid interference assays, E. coli expressing St1Cas9 and a targeting sgRNA failed to maintain the targeted plasmid, leading to growth inhibition. Co-expression of AcrIIA31 rescued colony formation, confirming its inhibitory activity. In vitro DNA cleavage assays and EMSAs demonstrated that AcrIIA31 blocked Cas9–DNA interactions but did not interfere with sgRNA loading. MVTEEQLKEVLVGIYETEYKDEQTFEEYADGWDFWIDKDGDILIEGRGMKPIDGVQKVGHVDNGVIYAY acriia32 AcrIIA32 8YE6_B acriia32.pdb PDB entry binds and inhibits host defence system interacts with the WED domain of SpyCas9, where it spatially obstructs conformational changes of the WED and PI domains, thereby inhibiting SpyCas9 from recognising the protospacer adjacent motif (PAM) and unwinding double-stranded DNA. Streptococcus thermophilus and Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus uberis NZ01 10.1093/nar/gkac099;10.1007/s11427-024-2607-8 AcrIIA32 was cloned from a Streptococcus phage-associated mobile element and tested for its ability to inhibit SpyCas9 and St3Cas9. In plasmid interference assays, E. coli expressing either Cas9 ortholog along with targeting sgRNA displayed growth inhibition due to plasmid cleavage. Co-expression of AcrIIA32 restored growth, indicating effective inhibition. In vitro cleavage assays confirmed that AcrIIA32 inhibited both DNA binding and cleavage by Cas9. EMSAs showed no Cas9–DNA complex formation in the presence of AcrIIA32. Cryo-electron microscopy structure supports direct binding (ECO_0006181). MKNEDGKLVVSKAHFGNMIRNCQSVEDFKKSFERLTYYSSENRESTVRQRLKIAEKEYNFKAGVKEDLEIKNTTDKEILDYVRNELSKIDSKKQADKNWSEKNREHRNYLSKRSSARSFINNNATHEDLLELKKIIEEKLK acric11 AcrIC11 _ acric11.cif AlphaFold 3 unknown _ Xanthomonas albilineans CFBP7063 type I-C CRISPR-Cas system CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Xanthomonas albilineans 10.1093/nar/gkad1097 PF03230 AcrIC11 was identified from Xanthomonas albilineans and tested for its ability to inhibit the Type I-C CRISPR-Cas system. In E. coli Cas3 degradation assays, co-expression of Cascade, targeting crRNA, and Cas3 led to cell death due to chromosomal targeting. Co-expression of AcrIC11 restored colony formation, indicating effective inhibition. In vitro TXTL assays confirmed that AcrIC11 inhibited DNA degradation but not DNA binding by Cascade. deGFP repression assays showed no significant impact on Cascade-DNA binding, while degradation assays revealed ~60% inhibition of Cas3 activity. AlphaFold-based structural comparison with KlcA supports functional homology (RMSD = 1.467 Å), consistent with nuclease-targeting activity. MNKETQITASAVVGEDKRLEFLSKHFGVRFARRGEALVFAWLLRLAKVPIEWTRLQYYTLSNSGFYLAPRELRISECELSADAVGIVATMLTLRQLAHESAACVEADSTYPAAKLAVTASVKFAQQYHHLAAYSVKHAESINIYRAID acriia33 AcrIIA33 _ acriia33.cif AlphaFold 3 unknown _ Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus equi DSM 20561 10.1093/nar/gkad995 AcrIIA33 was discovered from Streptococcus DNA and tested using a system in E. coli designed to identify anti-CRISPR proteins. In this system, Cas9 cuts a plasmid that carries a red fluorescent gene called mCherry. Cutting this plasmid kills the bacteria because the plasmid is needed for survival. When AcrIIA33 was expressed, bacteria survived, meaning Cas9 was blocked. This showed that AcrIIA33 stops Cas9 from cutting DNA. MELNFVGQFDNGHDFYDVEKFVDVNVETGRLSDEDIIKVYDALRNEHRLRRGKGKYGNLLSFAEYGDEDVLTDTTYWYEEDILPAQDRLEEL acriia34 AcrIIA34 _ acriia34.cif AlphaFold 3 unknown _ Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Streptococcus lutetiensis AM38-2 10.1093/nar/gkad995 In E. coli, co-expression of AcrIIA34 with Cas9 allowed bacteria to grow, showing it blocked Cas9 from cutting the plasmid. Lab tests confirmed it does not block sgRNA loading, but does block Cas9 from binding DNA, as shown by DNA binding (EMSA) experiments (ECO_0001807). MKNIANEIKTIRYAFEDGRSTQKSIMRKIKALTDQFETMDDLIDSLNSYADTHYTWAITYFQLARIIISFQASNNTTSEKKIDLQSGPIEVNGKLKIRVTVDEFMADLANWEHLEDIKKLAKELA sam_amp_lyase SAM-AMP lyase _ sam_amp_lyase.cif AlphaFold 3 degrades or sequesters molecules utilised by host defence systems cleaves the signalling molecule SAM-AMP used by type III CRISPR systems CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Metagenome 10.1093/nar/gkaf655 DAN18478 was identified as a candidate phage-encoded SAM-AMP lyase from a human microbiome viral metagenome. It was recombinantly expressed in E. coli and purified to near-homogeneity. The purified protein exhibited SAM-AMP lyase activity, producing MTA-AMP. The phage enzyme functionally substituted for C. botulinum SAM-AMP lyase in a plasmid interference assay involving the B. fragilis Cmr/CorA type III CRISPR system, supporting its functional role in neutralizing CRISPR immunity. MTIQEKIKKTGEGKYKIKFSTIKRYSIEFGRTVTDYYFVEYKNNTFITETPFILLKNFGDNFSKDGSGAGYCSEIVSCERYFRENEYKNQNSRTQEFKLVIGINQGYNHNNDSSINIYELYQEVAEKIYQKYGTYISATITESKVIYSASWGCPAGGEIVYSIKGTKNPQFVDDFEKYKKATERVAKTLASELKQSTFTLTWKNLELNYFKKIDGSCK ardb ArdB 2WJ9 ardb.pdb PDB entry unknown _ Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Plasmid pKM101 10.1093/nar/gkp1144 PF03230 When expressed in E. coli, ArdB inhibited four major subfamilies of Type I restriction–modification systems (RM types IA–ID), allowing bacteriophages to infect cells that would otherwise restrict them. This shows that ArdB confers anti‑restriction protection inside living cells. However, purified ArdB did not block the restriction activity of a model enzyme (EcoKI) in test-tube assays (in vitro). That implies that ArdB doesn’t act by binding directly to the restriction enzyme’s active site. MMNVMLPAPDLYSLSFIHITRISYMKTLSQNTTSSACAPETGLQQLVATIVPDEQRISFWPQHFGLIPQWVTLEPRVFGWMDRLCENYCGGIWNLYTLNNGGAFMAPEPDDDDDETWVLFNAMNGNRAEMSPEAAGIAACLMTYSHHACRTECYAMTVHYYRLRDYALQHPECSAIMRIID klca KlcA 2KMG klca.pdb PDB entry unknown _ Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Plasmid pBP136 10.1093/nar/gkp1144 PF03230 KlcA expression in E. coli enables infection by phages even in the presence of functional Type I RM systems IA–ID. This confirms robust anti‑restriction activity in living cells. Purified KlcA also doesn’t inhibit EcoKI enzyme activity in vitro. MNTEEQPVTASLVAEAQRLDFLPTYFGPRLMMRGEALVYAWMRRLCERYNGAYWHYYALSDGGFYMAPDLAGRLEIEVNGNGFRGELSADAAGIVATLFALGQLAAEIADTDAADALIDRYHFLRGFAAGHPEAAAIYRAID hia5 Hia5 _ hia5.cif AlphaFold 3 modifies phage molecules to avoid recognition modifies adenine residues to N6-methyladenine. Diverse set of REases from type I RMs RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Prophage in Haemophilus influenzae 10.1093/nar/gkr1039 hia5_model.html Hia5 was cloned from Haemophilus influenzae biotype aegyptius ATCC 11116. Its DNA methyltransferase activity was demonstrated in multiple assays. In vitro assays using [³H]-AdoMet showed significant methyl group incorporation into λ DNA, which was abolished in the catalytically dead D194A mutant (ECO_0000315). Thin-layer chromatography (ECO_0000325) confirmed the modified base as N6-methyladenine (m6A). Restriction enzyme protection assays showed that plasmid DNA from E. coli expressing Hia5 became resistant to 30 restriction endonucleases known to be blocked by m6A methylation, indicating broad adenine modification. HPLC analysis quantified that Hia5 methylated ~61% of adenines in λ DNA. Kinetic assays using synthetic oligonucleotides showed Hia5 methylated most adenine-containing dinucleotides (e.g., CA, GA, TA) except poly(A) tracts, suggesting minimal sequence specificity (AB or BA, where B = C, G, or T) MANQNTFKQAPLPFIGQKRMFLKHFETVLNENIKGDGEGWTIIDTFGGSGLLSHAAKVIKPKAHVIYNDFDSYAERLAYINDTNALRTQIFAKIGNATPKNKRLPKSLKAEIIKIIDQFKGYKDLNCLTSWLLFSGQQVSSLDELYKKDFWHCVRLSDYPSAEGYLDGVEVIRESFHTLLPKFSDNPKALFVLDPPYLCTKQESYKQATYFDLIDFLRLVNITLPPYIFFSSTKSEFVRFIEYMVDDKVHNWQAFENAKRITVNAKLNYQVAYEDNLVYKF hin1523 Hin1523 _ hin1523.cif AlphaFold 3 modifies phage molecules to avoid recognition modifies adenine residues to N6-methyladenine. Diverse set of REases from type I RM systems RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Prophage in Haemophilus influenzae 10.1093/nar/gkr1039 PF02086 hin1523_model.html Hin1523 was cloned from the Haemophilus influenzae Rd strain. Like Hia5, it exhibited DNA methyltransferase activity in vitro. Using [³H]-AdoMet and λ DNA, incorporation of methyl groups was confirmed and was absent in the D194A mutant (ECO_0000315). Thin-layer chromatography (ECO_0000325) identified m6A as the modified base. REase protection assays showed resistance of plasmid DNA from Hin1523-expressing E. coli to multiple methylation-sensitive restriction enzymes, though the protection was less extensive than that seen with Hia5. HPLC revealed that Hin1523 methylated ~30% of adenines in λ DNA. Kinetic analysis showed preference for CA and TA dinucleotides, with GA methylated less efficiently. Poly(A) tracts remained unmethylated, suggesting similar minimal sequence specificity as Hia5 but with lower catalytic efficiency. MSEYLEYQNAIEGKTMANKKTFKQAPLPFIGQKRMFLKHVEIVLNKHIDGEGEGWTIVDVFGGSGLLSHTAKQLKPKATVIYNDFDGYAERLNHIDDINRLRQIIFNCLHGIIPKNGRLSKEIKEEIINKINDFKGYKDLNCLASWLLFSGQQVGSVEALFAKDFWNCVRQSDYPTAEGYLDGIEVISESFHKLIPRYQNQDKVLLLLDPPYLCTRQESYKQATYFDLIDFLRLINLTKPPYIFFSSTKSEFIRYLNYMQESKTDNWRAFENYKRIVVKASASKDGIYEDNMIYKF nma1821 Nma1821 _ nma1821.cif AlphaFold 3 modifies phage molecules to avoid recognition modifies adenine residues to N6-methyladenine. Diverse set of REases from type I RM systems RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Prophage in Haemophilus influenzae 10.1093/nar/gkr1039 PF02086 nma1821_model.html Nma1821 was cloned from Neisseria meningitidis type A strain Z2491. It demonstrated strong DNA methyltransferase activity in vitro, similar to Hia5. Incorporation of [³H]-methyl groups into DNA was confirmed and lost in the D191A mutant (ECO_0000315). Thin-layer chromatography (ECO_0000325) showed the methylated base to be m6A. Plasmid DNA from E. coli expressing Nma1821 was resistant to most of the same methylation-sensitive REases tested for Hia5, indicating extensive adenine methylation. While HPLC data specific to Nma1821 was not shown, its restriction protection profile was highly similar to that of Hia5, implying similarly widespread, non-specific adenine methylation. This positions Nma1821 as a highly active, non-specific DNA adenine methyltransferase MMQKYHSTAPLPFVGQKRYFIKHFTKVLSQIPADGKHWTIVDVFGGSGLLAHVAKRIKPQARVIYNDYDNYSDRLRHIPDYNRLREQIAQIVGGIPKGSRLDPERTRSVQQTITNFQGHIDVRVLSSWLLFSAKQANSLEQLLGFEFYNKVRQSPYSIAADYLDGLEITQQDYNLLMAEHQHNPNTLLVLDPPYVSTAQGAYAADKYFNMVSFLRMIQYMRPPFILFSSTRSEALDYFQFLQECEPDKYRRFSGYNIVSLDAKMGKGIEYQDNMIYKID rad Rad _ rad.cif AlphaFold 3 degrades or sequesters molecules utilised by host defence systems degrades msDNA and ncRNA of the retron. Escherichia coli type 1A retron (Ec83) Retron https://defensefinder.mdmlab.fr/wiki/defense-systems/retron Escherichia coli phage ΦSP15 10.1101/2023.03.15.532788;10.1038/s41467-024-53789-y PF13155 Rad (ORF75 of SP15, named retron anti-defense or Rad) was pinpointed within an 8 kb Anti-Defense Island (ADI) whose deletion impaired phage infectivity against E. coli strains carrying retrons Ec67, Ec78, and Ec83. Cloning of this ORF restored infectivity, confirming Rad’s anti-retron function. Expression (ECO_0000017) of Rad in E. coli carrying retrons Ec67, Ec78, or Ec83 restored infectivity of otherwise sensitive phages (T5n or ΦSP15m). Single-point mutations in conserved residues (R13E, P33T, I88T, D135H, E156H) led to partial loss of Rad activity (ECO_0001113). Double mutations completely abolished anti-retron function (ECO_0001113). Co-expression of Rad with retron systems led to a marked decrease in the production of msDNA and its precursor ncRNA (msr-msd), but not RT or effector protein transcripts. This implies Rad degrades the ncRNA necessary for msDNA synthesis, directly interfering with retron function. Results on Rad are not included in the peer-review version of the paper. MLNSLYNISINLKEEPNMNVDELTQHLLSRGFDTDKYHCWLSPEGWLTVPLYDFSGMLRGYQTYNPSAPKGHGKCPFEAKYFTYSTTQCVWGLETLNGDEKVVLIAESVFKAVALHNAGYPALAMLGSSPGKALLKQLKLLPFKLVAVGDNDPAGEKFARKLNGFVSPVDVDEMSTENLKNFLAMKLNF orf126 ORF126 _ orf126.cif AlphaFold 3 unknown _ _ Broad-spectrum counter-defense _ Enterobacteriophages 10.1101/2023.04.06.535777 orf126 was identified as a broad-spectrum counter-defense gene that enhanced phage infectivity in multiple E. coli strains. Its expression (ECO_0000017) increased plaquing efficiency for several phages, indicating suppression of host defenses. Transposon knockout of capsule and O-antigen biosynthesis genes phenocopied orf126 expression, suggesting that orf126 impairs surface barrier defenses. Affinity purification-mass spectrometry (AP-MS) revealed that orf126 shares host binding partners with known anti-defense protein orf116 (abc1), further supporting its counter-defense role (ECO_0001096). MTIDYRRTYFFDTKRKVNNIISGIASLDIMIYAIQGKAGKEVLAEILSERNIAIPPTLRCLTPEELKALAFVVCKSQLKTTTVGGRMKVVLRVLLLITH orf148 ORF148 _ orf148.cif AlphaFold 3 unknown _ _ Broad-spectrum counter-defense _ Enterobacteriophages 10.1101/2023.04.06.535777 orf148 expression conferred increased phage infectivity in diverse E. coli strains (ECO_0000017), independent of variable defense gene repertoires. Like orf126, transposon inactivation of capsule or O-antigen biosynthesis genes mirrored orf148’s effect, suggesting disruption of conserved envelope barriers. These infection phenotypes across multiple hosts and phages indicate orf148 acts as a general anti-barrier defense protein. MINAKEELLLALKNTNSEVKCIKIEFGYYGDKEVWVLPVGYTEKDIEDFLDNLDFKYDSGFGGQLLYGNVWFTDGTWLERGEYDGSEWWEYKTTPAIPEECRTINGEVDRTLLLN gnarl1 Gnarl1 _ gnarl1.cif AlphaFold 3 unknown _ Escherichia coli O-antigen-based barrier O-antigen-based barrier _ Klebsiella phage vB_KpnM_KpV79 https://www.ncbi.nlm.nih.gov/nuccore/NC_042041.1 10.1101/2023.04.06.535777 gnarl1 (orf48) enhanced infection by phage T5 in multiple E. coli strains where wild-type O-antigen structures normally block phage adsorption. Expression of gnarl1 (ECO_0000017) induced detectable downshifts in O-antigen banding patterns, similar to seroconverting lysogens. These effects were mirrored by host mutants lacking O-antigen biosynthesis genes. Together, these results indicate gnarl1 modifies or downregulates O-antigen to facilitate phage entry. MTEQDQRLKQFDEKLAELEKTIKQVQEQRREYINRKGLNK gnarl2 Gnarl2 _ gnarl2.cif AlphaFold 3 unknown _ Escherichia coli O-antigen-based barrier O-antigen-based barrier _ Escherichia phage Mu 10.1101/2023.04.06.535777 gnarl2 (orf63) expression sensitized E. coli strains to phage T5 and altered O-antigen structures, as revealed by LPS electrophoresis. MAKVIIEIKNTVSGIKGRNLRTSIAVDGSAELDGDEGTLAGMVALLVLNKSQKIINESAHEAIEILKNDGVITSGRVTEMAVEKTCH gnarl3 Gnarl3 _ gnarl3.cif AlphaFold 3 unknown _ Escherichia coli O-antigen-based barrier O-antigen-based barrier _ Escherichia phage Mangalitsa 10.1101/2023.04.06.535777 gnarl3 (orf92) expression led to increased susceptibility to phage T5 and T4 across multiple hosts. AP-MS identified interactions between gnarl3 and host UDP-glucose biosynthesis proteins (ECO_0001096), especially GalU. Overexpression of galU completely suppressed the gnarl3 phenotype, restoring resistance. MNYALYQYINRDGVVAHALVNTKTKDVMLADTVIYFERGNLVWKPAAHPEYVWLAIQNDKHHKIVAMATHPHFIKARG ipii Ipii _ ipii.cif AlphaFold 3 unknown _ Escherichia coli type IV RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Binds and inhibits the gmrS/gmrD complex (glucose-modified hydroxymethylcytosine restriction endonuclease) 10.1101/2023.04.06.535777 IpII (orf143) enhanced T2 and T6 phage infectivity in ECOR21, which normally restricts these phages via a fused Type IV R-M enzyme, GmrSD. In a ∆wecA background lacking O-antigen, transposon disruption of the GmrSD locus mimicked the effect of IpII, confirming it as the target. Cloned expression of GmrSD blocked T2, but this restriction was relieved by co-expression of IpII (ECO_0000017). MKTYQEFIAEARVGAGKLEAAVNKKAHSFHDLPDKDRKKLVSLYIDRERILALPGANEGKQAKPLNAVEKKIDNFASKFGMSMDDLQQAAIEAAKAIKDK orf55 ORF55 _ orf55.cif AlphaFold 3 synthesises and restores essential molecules depleted by bacterial defence (putative) predicted to act as a nick-sensing, ATP-dependent DNA ligase (ipTM = 0.95, pTM = 0.95) that reverses the effect of effector nucleases. Escherichia coli type III Avs Avs https://defensefinder.mdmlab.fr/wiki/defense-systems/avs DruSM1 phage 10.1101/2024.04.14.589459 PF01068;PF14743 orf55_model.html ORF55 was identified via a knockout screen where its deletion led to >100-fold reduction in infectivity against E. coli expressing AVAST type III, accompanied by small plaque formation. Complementation of the ΔORF55 mutant with a plasmid-borne ORF55 restored infectivity and plaque size, confirming its anti-AVAST function. Binding nicked DNA and ATP is supported by computational structure modeling evidence (ECO_0006368). MAFKPHLATDAVEEKIKFPCCILPKIDGVRGLNPDGRIVGRSLKLFKNRHTSAIFSGPQYMGYDGELAAGVETDPDLCRKTTSAVNTIEGEPFLKWHIFDLCAESVAELGYEARYNMMKDFITTQHAKGELLDLQVVPMYVVKSLQELLYWENIWLDMGYEGIIIRDPEKPYKHGRGTVREGGYLRIKRFIQEDAIVLDIIEGETNLNEATVNELGRTTRSSHQENKVPNGMIGTLVCKDVKTGNTINVSPGKLTQEDKIYYWNNPDKIKGRTISYKHFPHGVKDKPRFANFMHFRDESDQALD orf83 ORF83 _ orf83.cif AlphaFold 3 unknown _ Escherichia coli type III Avs Avs https://defensefinder.mdmlab.fr/wiki/defense-systems/avs DruSM1 phage 10.1101/2024.04.14.589459 PF10686 orf83_model.html Deletion of ORF83 significantly reduced infectivity (>100-fold EOP decrease) in AVAST type III-expressing bacteria. Ectopic expression of ORF83 in these strains restored infectivity of the ΔORF83 phage, confirming anti-AVAST activity. MLLVVTGGRDFKHAEYIYAQLDKLHMQRRITTLRHGDADGVDRICAQWAERNGIKTEAFPAPWDNLSLPNTKIRYNAKGAYNAQAGAYRNQLMLNTDPKPDHAIVFPGGAGTMDMYRRIKASGIPYTLAE orf46 ORF46 _ orf46.cif AlphaFold 3 unknown _ Escherichia coli type I BREX BREX https://defensefinder.mdmlab.fr/wiki/defense-systems/brex DruSM1 phage 10.1101/2024.04.14.589459 In Brex type I-bearing E. coli, ΔORF46 phages exhibited strongly reduced infectivity. Complementation with plasmid-encoded ORF46 fully restored infectivity, confirming its anti-Brex activity. MNEETVILMQKIQRLQDELNDAICRAASKKIHSELEIHQRQVTSEAVVEQVVVNLNISLKGF orf72 ORF72 _ orf72.cif AlphaFold 3 unknown _ Escherichia coli type I BREX BREX https://defensefinder.mdmlab.fr/wiki/defense-systems/brex DruSM1 phage 10.1101/2024.04.14.589459 Deletion of ORF72 caused a loss of infectivity against Brex type I, and coexpression of ORF72 with the defense system rescued phage infectivity. MSKVTHREQDKMRYTMKTNARRALKTRWGQVARDAHEQLIHQDEIGFYIVEKEVLQVLRDHKKAQQPEQKVPAPTAAPASSVSVLRPMVNPEIVKSCLEALEQEEEKDMKHAKAATQVSEIKIEEPKAAAPKQDVKTARCDFKNGARKPLKGKTAEVWAMGDKLLKELGRTPELKEMKAAMPSYNGTTVAIQFYAWRKYNGLDA orf71 ORF71 (Druad1) _ orf71.cif AlphaFold 3 unknown _ Escherichia coli type I Druantia Druantia https://defensefinder.mdmlab.fr/wiki/defense-systems/druantia DruSM1 phage 10.1101/2024.04.14.589459 ORF71 deletion impaired infectivity of DruSM1 against E. coli harboring the Druantia type I defense system. Complementation restored infectivity and plaque size. ORF71 (Druad1) expression was linked to m6A DNA methylation, altering phage DNA to evade recognition by Druantia. MFTLEIKYFGSDWEVEDVFNNRYDAEQTGKFMLSQGLITNWRVL orf65 ORF65 _ orf65.cif AlphaFold 3 unknown _ Escherichia coli hhe hhe https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_hhe DruSM1 phage 10.1101/2024.04.14.589459 ΔORF65 mutants showed reduced infectivity (EOP decrease of 10^−3) and small plaques in hhe-expressing bacteria. Complementation with ORF65 restored infectivity. MVMLNKVYSTMGADYKVVRHPRTGKFEAYIRPMGEGWMYYGTYDDAEQAYKEAESAAEASEIMARYDVDDYYLS orf58 ORF58 _ orf58.cif AlphaFold 3 unknown predicted to bind ATP and NAD as a dimer. Triggers retron Ec86. Escherichia coli Sir2+HerA and DUF4297+HerA Sir2+HerA;DUF4297+HerA https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_her;https://defensefinder.mdmlab.fr/wiki/defense-systems/gao_her DruSM1 phage 10.1101/2024.04.14.589459 PF23791 Deletion of ORF58 reduced infectivity in Sir2+HerA- and DUF4297+HerA-expressing strains, indicating anti-defense roles. Complementation with ORF58 restored infectivity in both systems. MDVLLYIHRNTTEDRKMFDADKAVQATEADVIEWYKTKAELQKLQARERALRDKIIKSYFPAPSEGTNKVEITGGVMKMTHKIDRKIDLPSLNGILGDLIKVGVNVDQLVENKPVLKVAAWRKLTAEQAAVFNQCVESKVGSASLEIVPNKA tlaloc Tlaloc _ tlaloc.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with AbiH AbiH https://defensefinder.mdmlab.fr/wiki/defense-systems/abih Vibrio phage 1.056.O. https://www.ncbi.nlm.nih.gov/nuccore/MG592436.1 10.1101/2024.06.14.598830 Ectopic co-expression (ECO_0000017) of Tlaloc with AbiH in the Vibrio superhost restored viral infectivity, which was otherwise reduced by ≥5.7 orders of magnitude. VEVFLYLVHVHQLTYCLLMIYINCFQMYLYFLRRRGAIILSLLTGIDPRLQQFCTKSIF enki Enki _ enki.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with AbiH and Retron type II (Ec86) AbiH;Retron https://defensefinder.mdmlab.fr/wiki/defense-systems/abih;https://defensefinder.mdmlab.fr/wiki/defense-systems/retron Vibrio phage 1.080.O. https://www.ncbi.nlm.nih.gov/nuccore/MG592455.1 10.1101/2024.06.14.598830 Ectopic expression (ECO_0000017) of Enki with AbiH and Retron defense systems restored phage infectivity from severe reductions (5.7 log units for AbiH, 3.2 for Retron), confirming its broad-spectrum anti-defense activity. LNVAPFIRPLFLLVKSFFYHSYIPIVVKFKCLKCDVRAKRRAFSLVRHTLDI surt Surt _ surt.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with AbiU AbiU https://defensefinder.mdmlab.fr/wiki/defense-systems/abiu Vibrio phage 1.217.O. https://www.ncbi.nlm.nih.gov/nuccore/MG592588.1 10.1101/2024.06.14.598830 PF10076 Ectopic co-expression (ECO_0000017) of Surt with AbiU in the superhost restored phage infectivity to levels similar to GFP-expressing controls, indicating complete inhibition of AbiU defense. MGHSVEQWTNSIMAQMPRGILWQRSASLDLYKYAAGYAPRLEAVEVSADSLLLEMRPENTQQLLDEWEEYLGLPECQVQNQTFESRRAAVVEKYHRKGGLQAWNIDKLGADLGFEIEVEEIFPHHCLRGCTYPLYEEKYRHLLRIHVRGITQAYATCLDDCLTPLVSQTAAILECTLNQFKLGGKYYEFIYEESV ads38_ ADS38* _ ads38_.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with type V Avs Avs https://defensefinder.mdmlab.fr/wiki/defense-systems/avs Vibrio phage 10.1101/2024.06.14.598830 Ectopic co-expression (ECO_0000017) of ADS38 with Avs defence neutralized the defense effect, as evidenced by restored viral infectivity in the Vibrio superhost. LKLIAFFSPIDLINPANRPIIKGWKGMVLFI nergal Nergal _ nergal.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with type I CBASS CBASS https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass Vibrio phage 1.209.O. https://www.ncbi.nlm.nih.gov/nuccore/MG592579.1 10.1101/2024.06.14.598830 Ectopic co-expression (ECO_0000017) of Nergal with CBASS operons neutralized the defense effect, as evidenced by restored viral infectivity in the Vibrio superhost. MSKHRGKAKRRATWVQTKRSPKNKHRWETEVNRARRKLAYAEYHKMKNRRGRNYV hades Hades _ hades.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with type I DRT DRT https://defensefinder.mdmlab.fr/wiki/defense-systems/drt Vibrio phage 1.066.O. https://www.ncbi.nlm.nih.gov/nuccore/MG592443.1 10.1101/2024.06.14.598830 Ectopic expression (ECO_0000017) alongside DRT in the superhost abolished defense-induced resistance to phages, confirming its functional role. MTKFLSHLMLATYQSNTHKFMSDGEMGKIYEVTHAHVSYSRKRLIKEGHSFAFNTSVTKSNMPVQTYKYVGQTNLGLVCVDEKKDAMDGDTLLRLRYRMQSPSVPARQFPVEPGHVSLLGISSLV kali Kali _ kali.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with type I DRT DRT https://defensefinder.mdmlab.fr/wiki/defense-systems/drt Vibrio phage 1.139.B. https://www.ncbi.nlm.nih.gov/nuccore/MG592511.1 10.1101/2024.06.14.598830 When co-expressed ectopically (ECO_0000017) with DRT, Kali restored viral infectivity, supporting its designation as a redundant but distinct anti-DRT factor. MGHKVAKCIFYDEASNIVFSAHGRPAKNMFIPVPQRGDWSDGMPKLYGAFSAGAIDRRREGITVHYRCAGFIKSTSVPGLVSRVPWLKL anhur Anhur _ anhur.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with Septu Septu https://defensefinder.mdmlab.fr/wiki/defense-systems/septu Vibrio phage 1.196.O. https://www.ncbi.nlm.nih.gov/nuccore/MG592565.1 10.1101/2024.06.14.598830 Ectopic co-expression (ECO_0000017) of Anhur in the Vibrio superhost eliminated Septu-conferred resistance, restoring virus replication. MAIVWLKLGCRCWCFYVGKRTPPPRSTRGVLMATGSY svarog Svarog _ svarog.cif AlphaFold 3 unknown _ Vibrio cyclitrophicus superhost with Septu Septu https://defensefinder.mdmlab.fr/wiki/defense-systems/septu Vibrio phage 1.066.O. https://www.ncbi.nlm.nih.gov/nuccore/MG592443.1 10.1101/2024.06.14.598830 Ectopic co-expression (ECO_0000017) of Svarog in the Vibrio superhost eliminated Septu-conferred resistance, restoring virus replication. MEAIIKFALLIAWVAGFVIAKGFWPTLFCVIPFWSFYLVVEKVMTVLGWL adfn AdfN (anti-DarT factor NADAR) _ adfn.cif AlphaFold 3 uncategorised removes ADP-ribose modifications from phage DNA. Escherichia coli type IV TA system DarTG TA https://defensefinder.mdmlab.fr/wiki/defense-systems/dartg T4-like phages 10.1101/2024.07.11.602962;10.1038/s41467-025-56887-7 PF08010 AdfN was identified via recombination experiments between DarTG1-sensitive RB69 and resistant T4/T2 phages. Mapping of recombinant genomes pinpointed a 3′ region of gene 30.3 (renamed adfN) that correlates with DarTG1 resistance. Unlike RB69, which encodes a truncated AdfN protein due to a frameshift mutation, resistant phages encode full-length AdfN. Deletion of adfN in T2 or T4 rendered both phages sensitive to DarTG1, while ectopic expression of AdfN in E. coli restored RB69 infectivity in the presence of DarTG1. Point mutations in conserved catalytic residues (E36A, K43A) or truncation at the RB69 stop codon site impaired activity, indicating the requirement for enzymatic function. In vitro dot blot assays confirmed that purified AdfN protein removes ADP-ribose from DNA, directly counteracting DarT1’s toxic modification. Notably, AdfN did not interact with DarT1 in a two-hybrid assay and failed to protect E. coli from DarT1 toxicity in the absence of phage, suggesting that AdfN acts via enzymatic detoxification of modified DNA, not toxin binding, and requires phage context for full activity. MSELEIRSNFRWPSCALSNFAQWPFVMDGIQFGGLEGFLQGCKVKNVEQQRRIFGLSGLAAQQAGRSYARAQDRGTLFWLGVPFSRYSPAWKELYTNAYFEAAIQNKGFRDALLASKGKVLKHSMASGLTKDDTILTEAEFIDVLNLLRDSL t7_pk T7 protein kinase _ t7_pk.cif AlphaFold 3 adds a post-translational modification and deactivates bacterial defence hyperphosphorylates E. coli early proteins, with a preference for DNA/RNA-binding proteins including DNA-targeting defence systems. Escherichia coli type II Retron (Eco9) and type IV TA system DarTG Retron;TA https://defensefinder.mdmlab.fr/wiki/defense-systems/retron;https://defensefinder.mdmlab.fr/wiki/defense-systems/dartg Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1101/2024.12.20.629319 t7_pk_model.html T7 protein kinase (T7K) was identified as a broad-spectrum anti-defense factor through infection assays comparing wild-type T7 and a Δ0.7 mutant lacking the kinase. Deletion of T7K reduced infectivity in E. coli strains expressing DNA-targeting defense systems such as Retron-Eco9 and DarTG1 (ECO_0001175). Mass spectrometry (ECO_0001096) during infection revealed extensive phosphorylation of host and phage proteins, with Retron-Eco9's RcaT toxin and DarTG1’s DarT toxin among the stoichiometrically modified targets. Phosphomimetic mutations at key T7K-targeted sites (e.g., RcaT S155D, S254D; DarT T103D) abolished defense activity, confirming functional inactivation via phosphorylation. T7K’s C-terminal domain was shown to bind DNA, directing its otherwise promiscuous kinase activity to nucleic acid-binding defense components. MNITDIMNAIDAIKALPICELDKRQGMLIDLLVEMVNSETCDGELTELNQALEHQDWWTTLKCLTADAGFKMLGNGHFSAAYSHPLLPNRVIKVGFKKEDSGAAYTAFCRMYQGRPGIPNVYDVQRHAGCYTVVLDALKDCERFNNDAHYKYAEIASDIIDCNSDEHDELTGWDGEFVETCKLIRKFFEGIASFDMHSGNIMFSNGDVPYITDPVSFSQKKDGGAFSIDPEELIKEVEEVARQKEIDRAKARKERHEGRLEARRFKRRNRKARKAHKAKRERMLAAWRWAERQERRNHEVAVDVLGRTNNAMLWVNMFSGDFKALEERIALHWRNADRMAIANGLTLNIDKQLDAMLMG acb4 Acb4 9E4W acb4.pdb PDB entry degrades or sequesters molecules utilised by host defence systems sequesters the CBASS signalling molecule 3′3′-cGAMP. Yersinia aleksiciae type I CBASS and Citrobacter portucalensis/Escherichia coli type II CBASS CBASS https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass Bacillus phage SPO1 https://www.ncbi.nlm.nih.gov/nuccore/NC_011421.1 10.1101/2024.12.30.630793 Acb4 was identified as an anti-CBASS sponge protein through a biochemical screen using phage-infected lysates tested for binding to the CBASS signal 3′3′-cGAMP. EMSA assays revealed that SPO1-infected lysates shifted radiolabeled 3′3′-cGAMP, indicating strong signal sequestration (ECO_0001807). Activity-guided fractionation and mass spectrometry (ECO_0001096) pinpointed SPO1 gp1.3 (Acb4) as the binding protein. In vitro, Acb4 suppressed Cap5 nuclease activation by sequestering 3′3′-cGAMP, blocking downstream DNA degradation. In vivo, engineered phage T4 Δacb1/Δacb2::acb4 replicated efficiently in E. coli expressing Yersinia CBASS (signaling via 3′3′-cUA), rescuing ~10⁴-fold loss in fitness seen in Acb1/Acb2-deficient T4. EMSA with infected lysates confirmed functional expression of Acb4 during infection. Acb4 failed to counteract Citrobacter CBASS (3′2′-cGAMP signaling), suggesting specificity. A 2.1 Å co-crystal structure showed Acb4 forms a tetramer that selectively binds CBASS signals with high affinity (~157 nM K_D) via nucleobase-specific binding pockets. Mutagenesis of key contact residues impaired binding, confirming their role in CBASS inhibition. MKINAENFECLRESKLKRKVYEDLVKEATFVRVSPKSTVCVVTDHNSFEVIGTSSVYKVENFNDEIGRDTALSQALDSFIKFLAYSGELSDVLENI oad1 Oad1 (OLD-antidefense 1) _ oad1.cif AlphaFold 3 binds and inhibits host defence system binds to the OLD exonuclease. Vibrio cholerae Class 1 OLD nucleases Old exonuclease https://defensefinder.mdmlab.fr/wiki/defense-systems/old_exonuclease Vibrio phage ICP1 https://www.ncbi.nlm.nih.gov/nuccore/NC_015157.1 10.1101/2025.01.06.631583 Oad1 (OLD-antidefense 1) was identified as a direct anti-defense factor encoded by the ICP1 phage, capable of inhibiting the activity of the Class 1 OLD family nuclease Vc OLD. Comparative infection assays showed that ICP1⁽²⁰¹⁸⁾, unlike other ICP1 isolates, was able to robustly infect V. cholerae expressing Vc OLD, indicating the presence of a counter-defense mechanism. Plasmid-based expression (ECO_0000017) of candidate genes from ICP1⁽²⁰¹⁸⁾ revealed that only Gp205⁽²⁰¹⁸⁾ (Oad1) enabled ICP1⁽²⁰⁰⁶⁾ to overcome Vc OLD restriction, while the closely related Gp205⁽²⁰⁰⁶⁾ did not. Allelic swaps confirmed that Oad1 was sufficient to restore phage infectivity in a gp205-deletion mutant, and this activity extended to rescuing plaque size of the unrelated ICP3 phage. Mutational analysis pinpointed key residues (S135, K136, N144) in Oad1 important for counter-defense. Finally, co-immunoprecipitation (ECO_0000085) assays demonstrated a specific in vivo physical interaction between Oad1 and Vc OLD, absent with the non-functional Gp205⁽²⁰⁰⁶⁾ allele, confirming direct inhibition of Vc OLD by Oad1. MCTHIITISGEKLDELGKLIDHKHNINFSTKFSIYEKVPSTGADISESNDSKDKIRQQAKDILDKIQSHQEEEESTPEWMEGFEDLIRSDYVLRKGDYLLLEDLSLEQKVFLQNSLKTTTEFTFLEEDYNYAYFNSKGSFVGLNYGDPFVVKKITFNDLFIPKQ anti_rerI1 Anti-TerI1 _ anti_rerI1.cif AlphaFold 3 binds and inhibits host defence system prophage encoded anti-defence protein counteracting self-encoded defence system, TerI. TerI targets the terminase complex of invading phages, and anti-TerI1 and anti-Terl2 counteract Terl through direct interaction during prophage induction to allow virion production. Prophage ϕ10403S TerI defence system (active in Listeria monocytogenes) TerI _ Prophage ϕ10403S 10.1101/2025.02.27.640495 anti_rerI2 Anti-TerI1 (encoded by LMRG_01518) was identified as a self-immunity factor that neutralizes the anti-phage defense protein TerI. Its function was revealed through a suppressor screen using terI-expressing bacteria repeatedly infected with phage ϕ10403S, which selected for a phage mutant with enhanced early gene expression and resistance to TerI inhibition. Overexpression of LMRG_01518 in terI-expressing cells restored virion production to wild-type levels, confirming its counter-defensive role. Deletion of LMRG_01518 (ECO_0001038) impaired virion production upon prophage induction, resulting in capsids devoid of DNA, a phenotype rescued by inactivating terI expression. This demonstrates that Anti-TerI1 counteracts TerI activity in vivo. When overexpressed alone, Anti-TerI1 also rescued virion production during exogenous phage infection, confirming its ability to neutralize TerI's anti-phage activity. MNNIKQAIIKLETILENGNAIESGSFVKYSVIKNILNLLEKDQELKIIEMEVELNGVEDSIENAALLEKRLSEAKSLVEDLASTINSLEIKVK anti_rerI2 Anti-TerI2 _ anti_rerI2.cif AlphaFold 3 binds and inhibits host defence system prophage encoded anti-defence protein counteracting self-encoded defence system, TerI. TerI targets the terminase complex of invading phages, and anti-TerI1 and anti-Terl2 counteract Terl through direct interaction during prophage induction to allow virion production. Prophage ϕ10403S TerI defence system (active in Listeria monocytogenes) TerI _ Prophage ϕ10403S 10.1101/2025.02.27.640495 anti_rerI1 Anti-TerI2 (encoded by LMRG_02984) was independently identified as a second self-immunity protein opposing TerI activity. Its anti-defense function was initially obscured due to potential antisense transcriptional silencing of terI; however, co-expression experiments with a codon-modified synthetic terI (to eliminate antisense effects) confirmed that LMRG_02984 could restore virion production in TerI-expressing cells. Like Anti-TerI1, deletion of Anti-TerI2 (ECO_0001038) led to severely reduced virion production and DNA-less capsids, dependent on active terI. Bacterial adenylate cyclasebased two-hybrid assays demonstrated direct interaction between Anti-TerI2 and TerI, supporting a physical neutralization mechanism. Furthermore, overexpression of Anti-TerI2 restored phage infectivity during exogenous infection in TerI-expressing bacteria, underscoring its role as a functional anti-defense factor. MIFTVNSFPQSGHEFTPGLTVNTCEHSGQVPSVPFLIISSLAIFRLCSLICLSNFFESNLILTPILPTSLHKNYSTVKGRTERRTKCQIYK dap2 Dap2 _ dap2.cif AlphaFold 3 binds and inhibits host defence system directly binds to the Lon protease to prevent the degradation of the phage-encoded HNH endonuclease. Pseudomonas aeruginosa Lon-mediated antiviral defence Lon-mediated antiviral defence _ Pseudomonas phage PaoP5 https://www.ncbi.nlm.nih.gov/nuccore/NC_029083.1 10.1101/2025.03.13.642734 dap1 Dap2 was identified as an anti-defense factor through infection assays using a Δdap2 mutant of phage PaoP5. Deletion of dap2 (ECO_0001038) reduced phage burst size and resulted in smaller plaques in Pseudomonas aeruginosa PAO1, independent of the T3SS system, indicating an additional anti-defense function. Pull-down assays (ECO_0006249) and mass spectrometry identified Lon protease as a Dap2 binding partner (ECO_0001096). MYDKAQVLLWVGDNLLDYTRKSDGSPMTFLWYEEELKEAIGYSKSTYDFRRVDFHFVTEKKVTAYTLHECDTGPTTELNGTYTLQALKEIIAEMEEENEN dap1 Dap1 _ dap1.cif AlphaFold 3 uncategorised binds and shields the Lon-protease target, phage HNH endonuclease. Pseudomonas aeruginosa Lon-mediated antiviral defence Lon-mediated antiviral defence _ Pseudomonas phage PaoP5 https://www.ncbi.nlm.nih.gov/nuccore/NC_029083.1 10.1101/2025.03.13.642734;10.1038/s41564-024-01719-5 dap2 Dap1 was previously characterized as a partial anti-defense factor that binds directly to the phage HNH endonuclease, shielding it from Lon-mediated degradation. In this study, a PaoP5Δdap1 mutant showed reduced plaque size and phage productivity (ECO_0001038). When combined with a dap2 deletion (PaoP5Δdap1Δdap2), the phenotype was exacerbated, producing tiny plaques and ~90% empty capsids as observed by electron microscopy. One-step growth assays confirmed a severe drop in burst size (~6.68% of wild-type), and in vitro degradation assays revealed that Dap1 alone only partially protected HNH from Lon degradation. However, full protection was restored only when Dap1 and Dap2 were co-expressed, confirming that Dap1 complements Dap2’s anti-defense activity through a distinct, synergistic mechanism of physical shielding of HNH, in contrast to Dap2’s inhibition of Lon itself. MKTKEIDVSNFTAEQFDAFLEYCQYAQLKVGEGVVDKIREWMEHPISVDGPDDSPRCLYIRDHPEDRFISYGTGLRSKWQPDMYALYRPTFQTKCVMTLGEADKYVIVGGKPLSLDELQATLAKQGVIVTFGRV sequestin Sequestin _ sequestin.cif AlphaFold 3 degrades or sequesters molecules utilised by host defence systems bind and sequester the TIR-produced signaling molecules 3′cADPR and His-ADPR Bacillus cereus MSX-D12 type I Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Metagenomics data (IMG_VR ID: IMGVR_UViG_3300045988_056527) 10.1101/2025.07.12.664507 Sequestin proteins were identified as anti-Thoeris defense factors through infection assays where co-expression of Sequestin with the type I Thoeris system in Bacillus subtilis restored phage SBSphiJ infectivity, leading to culture collapse and plaque formation. When Sequestin genes were engineered into the SBSphiJ phage genome, infectivity was similarly restored in Thoeris-expressing cells, confirming functional inactivation. NADase assays showed that lysates from Sequestin-expressing infected cells failed to activate ThsA, indicating depletion of the 3′cADPR immune signal. Biochemical binding was demonstrated by size-exclusion chromatography (ECO_0000325) and HPLC (ECO_0001272), where Sequestin bound and removed 3′cADPR from solution, with chloroform denaturation releasing the intact molecule. AlphaFold3 modeling placed 3′cADPR in conserved inter-protomer binding pockets, and alanine substitutions in key residues (E12, Q47, R61) abolished anti-defense function, confirming their role in sequestration (ECO_0006341). MEKSVFDRLLTEYKELETKTTKLRDFLINKIDKTSIDNLNKDLLIAQLKAMEAYLTILSIRIGLNQPTQEEKQLDEAKALAKSTINE lockina LockinA _ lockina.cif AlphaFold 3 degrades or sequesters molecules utilised by host defence systems bind and sequester the TIR-produced signaling molecules 3′cADPR and His-ADPR Bacillus cereus MSX-D12 type I Thoeris and Bacillus amyloliquefaciens Y2 type II Thoeris Thoeris https://defensefinder.mdmlab.fr/wiki/defense-systems/thoeris Metagenomics data (IMG_VR ID: IMGVR_UViG_3300014204_000177) 10.1101/2025.07.12.664507 Lockin was identified as a broad-spectrum anti-Thoeris sponge family by infectivity assays showing that multiple Lockin proteins restored phage SBSphiJ propagation in cells expressing type I and/or type II Thoeris systems. Infected cells co-expressing Lockin and ThsB failed to accumulate 3′cADPR, as shown by NADase-based assays. Biochemical validation included size-exclusion chromatography (ECO_0000325) and HPLC (ECO_0001272) confirming direct binding and reversible sequestration of 3′cADPR by Lockin. Crystallography of LockinA with 3′cADPR revealed a homo-hexameric complex (ECO_0001171) with six deep inter-protomer pockets sequestering the signaling molecule. Mutations in pocket-coordinating residues K45 and K48 impaired anti-defense activity, confirming their role in nucleotide binding. MKEKDLGITEVRGAKANITDLVVYGNGDTFALLCKASSQEQGWMKSTKVCNVYGGCIVQVTTQQRNPDGSYALAEALTFVPNNHIDTSGNTRFIGKI acb5a Acb5a _ acb5a.cif AlphaFold 3 degrades or sequesters molecules utilised by host defence systems cleave and inactivate 3′3′-cGAMP and related molecules Escherichia albertii MOD1-EC1698 type I CBASS CBASS https://defensefinder.mdmlab.fr/wiki/defense-systems/cbass Metagenomics data (IMG_VR ID: IMGVR_UViG_3300024284_000007) 10.1101/2025.07.12.664507 Acb5 was identified as an anti-CBASS enzyme through growth and plaque assays showing that co-expression of Acb5 with a CBASS system from E. albertii restored infectivity of phages T2 and Bas60. In phage-infected cells expressing the CD-NTase enzyme, co-expression of Acb5 led to complete loss of detectable 3′3′-cGAMP, measured by ELISA (ECO_0000267). Thin-layer chromatography (ECO_0000140) and HPLC ( ECO_0001272) analyses demonstrated enzymatic cleavage of 3′3′-cGAMP by Acb5 into 2′3′-cAMP and 2′3′-cGMP, with similar partial activity against 3′3′-cUA. AlphaFold3 models predicted conserved interfacial binding pockets for both substrate and cleavage products, stabilized by conserved residues (e.g., R10, H61, W38), whose alanine mutations abolished anti-defense function. MRRFKVYRPNPPEGYLESGTANPPEEVQFEGVVFSDGTVCVRWLTEFRSHSLWSSLADLVKVHGHSEYGTLWEWLDE psib Psib 3NCT_A psib.pdb PDB entry binds and inhibits host defence system binds to RecA protein. Escherichia coli SOS response SOS stress response _ Escherichia coli plasmid 10.1111/j.1365-2958.1992.tb01539.x;10.1016/j.molcel.2009.07.026 PF06290 In a recA441 strain, which constitutively activates the SOS response, overexpression of PsiB from plasmid R6-5 or from an engineered F plasmid with a Tn10 promoter prevented LexA cleavage and SOS induction (as measured by sfiA expression). This demonstrated that PsiB directly inhibits RecA’s coprotease activity even without exogenous DNA damage. Immunoquantification showed that ~1500 PsiB tetramers per cell were sufficient to suppress RecA coprotease activity, even in cells with ~100,000 RecA monomers. However, suppression of UV-induced SOS required higher PsiB levels (~40,000 tetramers), indicating that PsiB targets a subset of RecA molecules—likely those bound to transient ssDNA during replication. MKTELTLNVLQTMNAQEYEDIRAAGSDERRELTHAVMRELDAPDNWTMNGEYGSEFGGFFPVQVRFTPAHERFHLALCSPGDVSQVWVLVLVNAGGEPFAVVQVQRRFASEAVSHSLALAASLDTQGYSVNDIIHILMAEGGQV lar Lar _ lar.cif AlphaFold 3 unknown _ Escherichia coli K-12 type 1 RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage lambda https://www.ncbi.nlm.nih.gov/nuccore/NC_001416.1 10.1111/j.1365-2958.1995.tb02438.x PF14354 Lar anti-restriction protein (Lar) was identified as a functional homolog of Ral through restriction alleviation and modification enhancement assays in E. coli strains expressing the Type IA R-M system EcoKI. Lar activity was confirmed by expression of lar from the Rac prophage in sbcA mutants (E.colu) and the hybrid phage λreverse. Cloning of lar into plasmids (e.g., pGK10, pGK15) enabled restriction alleviation as measured by increased phage titre of unmodified λ on restricting strains (ECO_0000017). Enhancement of modification was demonstrated by infecting cells expressing lar with unmodified phage and showing increased protection of progeny (ECO_0000017). MRYEKVKPCPFCGCPSVTVKAISGYYRAKCNGCESRTGYGGSEKEALERWNKRTTGNNNGGVHV dmd Dmd 5HY3_B dmd.pdb PDB entry binds and inhibits host defence system binds toxins (LsoA and RnlA) and neutralises them. Escherichia coli type II TA systems RnlA-RnlB and LsoA-LsoB TA https://defensefinder.mdmlab.fr/wiki/defense-systems/rnlab Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1111/j.1365-2958.2012.07975.x PF17587 Dmd of bacteriophage T4 was identified as a broad-spectrum antitoxin through phage infection experiments in E. coli. T4 Δdmd mutants exhibited severely impaired growth on E. coli strains carrying either the chromosomal rnlA-rnlB or the plasmid-encoded lsoA-lsoB toxin-antitoxin (TA) systems (ECO_0001175), implicating Dmd in suppression of both toxins (RnlA and LsoA). In contrast, wild-type T4 grew normally under the same conditions, indicating that Dmd inactivates these host toxins. Co-expression of Dmd rescued the growth defects caused by RnlA and LsoA overexpression in ΔrnlAB strains. Immunoprecipitation (ECO_0005644) and pull-down (ECO_0006249) assays confirmed direct physical interaction between Dmd and the toxins LsoA and RnlA. Upon T4 infection, host antitoxins RnlB and LsoB rapidly degraded, while Dmd accumulated and associated with the toxins, replacing native antitoxins and blocking toxin activity. These results demonstrate that Dmd acts as a protein antitoxin by directly binding and neutralizing multiple non-cognate toxins to promote T4 phage propagation. MELVKVVFMGWFKNESMFTKEITMMKDDVQWATTQYAEVNKALVKAFIDDKKVCEVDCRG alt Alt _ alt.cif AlphaFold 3 adds a post-translational modification and deactivates bacterial defence performs ADP-ribosylation of MazF, abolishing its toxicity. Escherichia coli type II TA system MazEF TA https://defensefinder.mdmlab.fr/wiki/defense-systems/mazef Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1111/mmi.13225 PF03496 Infection assays showed that deletion of mazEF increased T4 phage burst size, implicating MazF as a defense factor. Mass spectrometry (ECO_0001096) revealed that MazF was mono-ADP-ribosylated with a 542 Da modification only during T4 infection, and this modification was absent when Alt was deleted (ECO_0001038), confirming Alt dependency. Site-directed mutagenesis identified Arg4 as the modified residue, and in vitro assays demonstrated that Alt catalyzes this modification using β-NAD⁺. Functional assays showed that ADP-ribosylation of MazF by Alt reduced its RNA cleavage activity by ~70%, supporting Alt’s role in neutralizing MazF toxicity during infection. MELITELFDEDTTLPITNLYPKKKIPQIFSVHVDDAIEQPGFRLCTYTSGGDTNRDLKMGDKMMHIVPFTLTAKGSIAKLKGLGPSPINYINSVFTVAMQTMRQYKIDACMLRILKSKTAGQARQIQVIADRLIRSRSGGRYVLLKELWDYDKKYAYILIHRKNVSLEDIPGVPEISTELFTKVESKVGDVYINKDTGAQVTKNEAIAASIAQENDKRSDQAVIVKVKISRRAIAQSQSLESSRFETPMFQKFEASAAELNKPADAPLISDSNELTVISTSGFALENALSSVTAGMAFREASIIPEDKESIINAEIKNKALERLRKESITSIKTLETIASIVDDTLEKYKGAWFERNINKHSHLNQDAANELVQNSWNAIKTKIIRRELRGYALTAGWSLHPIVENKDSSKYTPAQKRGIREYVGSGYVDINNALLGLYNPDERTSILTASDIEKAIDNLDSAFKNGERLPKGITLYRSQRMLPSIYEAMVKNRVFYFRNFVSTSLYPNIFGTWMTDSSIGVLPDEKRLSVSIDKTDEGLVNSSDNLVGIGWVITGADKVNVVLPGGSLAPSNEMEVILPRGLMVKVNKITDASYNDGTVKTNNKLIQAEVMTTEELTESVIYDGDHLMETGELVTMTGDIEDRVDFASFVSSNVKQKVESSLGIIASCIDIANMPYKFVQG acrva4 AcrVA4 6NM9_A acrva4.pdb PDB entry binds and inhibits host defence system induces dimerisation of Cas12-crRNA, blocking dsDNA binding by the complex. Moraxella bovoculi and Lachnospiraceae bacterium type V-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Moraxella bovoculi 22581 10.1126/science.aau5138 AcrVA4 was identified from Moraxella bovoculi strain 58069 using a cell-free TXTL assay. The TXTL system includes plasmids that encode fluorescent proteins (GFP and RFP). Guide RNAs (gRNAs) are also included that direct Cas12a to specifically target and cleave the reporter plasmids. Genomic fragment GF59 increased fluorescent reporter expression, indicating inhibition of Cas12a-mediated DNA cleavage. Subsequent testing of individual open reading frames revealed GF59 candidate 2 as AcrVA4. In vitro assays confirmed that AcrVA4 inhibits dsDNA cleavage by both MbCas12a and LbCas12a, but not AsCas12a. Biochemical data further showed stronger inhibition of MbCas12a from strain 58069 than from strain 22581. In HEK293T genome editing reporter cells, AcrVA4 selectively blocked LbCas12a-induced editing, matching its in vitro activity. It did not affect SpyCas9, indicating target specificity. MYEIKLNDTLIHQTDDRVNAFVAYRYLLRRGDLPKCENIARMYYDGKVIKTDVIDHDSVHSDEQAKVSNNDIIKMAISELGVNNFKSLIKKQGYPFSNGHINSWFTDDPVKSKTMHNDEMYLVVQALIRACIIKEIDLYTEQLYNIIKSLPYDKRPNVVYSDQPLDPNNLDLSEPELWAEQVGECMRYAHNDQPCFYIGSTKRELRVNYIVPVIGVRDEIERVMTLEEVRNLHK acrva5 AcrVA5 6IUF_A acrva5.pdb PDB entry adds a post-translational modification and deactivates bacterial defence functions as an acetyltransferase and modifies Cas12a. Moraxella bovoculi and Lachnospiraceae bacterium type V-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Moraxella bovoculi 58069 10.1126/science.aau5138 AcrVA5 was discovered within the same GF59 genomic fragment as AcrVA4, identified as GF59 candidate 3. The TXTL system includes plasmids that encode fluorescent proteins (GFP and RFP). Guide RNAs (gRNAs) are also included that direct Cas12a to specifically target and cleave the reporter plasmids. TXTL screening showed elevated fluorescence of both reporters, suggesting CRISPR inhibition. In purified protein assays, AcrVA5 inhibited dsDNA cleavage by MbCas12a and LbCas12a, but not AsCas12a, and showed no inhibition of MbCas12a from strain 58069. In mammalian cells, AcrVA5 inhibited LbCas12a-mediated genome editing but did not impact AsCas12a or SpyCas9. These findings support AcrVA5 as a selective inhibitor of certain Cas12a orthologs. MKIELSGGYICYSIEEDEVTIDMVEVTTKRQGIGSQLIDMVKDVAREVGLPIGLYAYPQDDSISQEDLIEFYFSNDFEYDPDDVDGRLMRWS aca4 Aca4 _ aca4.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1126/science.aau5174 PF16509 _ MTEEQFSALAELMRLRGGPGEDAARLVLVNGLKPTDAARKTGITPQAVNKTLSSCRRGIELAKRVFT aca5 Aca5 _ aca5.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1126/science.aau5174 PF15943 _ MSLTEYIDKNFAGNKAAFARHMGVDAQAVNKWIKSEWFVSTTDDNKIYLSSVRREIPPVA aca6 Aca6 _ aca6.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1126/science.aau5174 PF13384;PF13560;PF01381 _ MTAMKEWRARMGWSQRRAAQELGVTLPTYQSWEKGIRLSDGSPIDPPLTALLAAAAREKGLPPIS aca7 Aca7 _ aca7.cif AlphaFold 3 unknown _ _ CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/avs _ 10.1126/science.aau5174 _ MIDARKHYDPNLAPELVRRALAVTGTQKELAERLDVSRTYLQLLGKGQKSMSYAVQVMLEQVIQDGET acric1 AcrIC1 _ acric1.cif AlphaFold 3 unknown _ Moraxella bovoculi type I-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Moraxella bovoculi 10.1126/science.aau5174;10.1093/nar/gkab006 DMS3m phage was genetically engineered to carry the acrIC1 gene. This phage was then used to infect PAO1IC, a strain expressing the PaLML1 Type I-C system and a phage-targeting crRNA. The engineered phage produced plaques on PAO1IC at levels comparable to a control strain lacking CRISPR targeting. In CRISPRi assays, it did not relieve transcriptional repression, indicating that Cascade still binds DNA. MNNLKKTAITHDGVFAYKNTETVIGSVGRNDIVMAIDATHGEFNDKNFIIYADTNGNPIYLGYAYLDDNNDAHIDLAVGACNEDDDFDEKEIHEMIAEQMELAKRYQELGDTVHGTTRLAFDDDGYMTVRLDQQAYPDYRPENDDKHIMWRALALTATGKELEVFWLVEDYEDEEVNSWDFDIADDWREL acrie4_if7 AcrIE4-IF7 7VZM acrie4_if7.pdb PDB entry binds and inhibits host defence system its N-terminal domain targets the PAM interaction site of the Cas8e subunit, and the C-terminal domain disables target DNA recognition at the PAM interaction site in the Cas8f subunit. Pseudomonas aeruginosa SMC4386 type I-E and I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa SMC4386 10.1126/science.aau5174;10.1093/nar/gkac096 AcrIE4-IF7 is a chimeric protein that inhibits both type I-E and I-F CRISPR-Cas systems. Upon ectopic expression in P. aeruginosa (ECO_0000017), it restored replication of phages JBD8 (I-E) and DMS3m (I-F), confirming dual activity. MSTQYTYQQIAEDFRLWSEYVDTAGEMSKDEFNSLSTEDKVRLQVEAFGEEKSPKFSTKVTTKPDFDGFQFYIEAGRDFDGDAYTEAYGVAVPTNIAARIQAQAAELNAGEWLLVEHEA acrie5 AcrIE5 _ acrie5.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts high-confidence interactions between Cas5e (https://www.ncbi.nlm.nih.gov/protein/QZE32570.1) and AcrIE5 (ipTM = 0.81, pTM = 0.86). Pseudomonas aeruginosa SMC4386 type I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa SMC4386 10.1126/science.aau5174 In Pseudomonas aeruginosa strains ectopically expressing AcrIE5 (ECO_0000017) and harboring active type I-E CRISPR-Cas targeting phage JBD8, phage replication was restored, demonstrating CRISPR inhibition. Binding it supported by computational structure modeling evidence (ECO_0006368). MSNDRNGIINQIIDYTGTDRDHAERIYEELRADDRIYFDDSVGLDRQGLLIREDVDLMAVAAEIE acrie6 AcrIE6 _ acrie6.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa SMC4386 type I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa SMC4386 10.1126/science.aau5174 AcrIE6 was validated via ectopic expression (ECO_0000017) in P. aeruginosa with type I-E CRISPR-Cas targeting phage JBD8. Phage plaque formation indicated that AcrIE6 effectively inhibited CRISPR immunity. MNNDTEVLEQQIKAFELLADELKDRLPTLEILSPMYTAVMVTYDLIGKQLASRRAELIEILEEQYPGHAADLSIKNLCP acrie7 AcrIE7 _ acrie7.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa SMC4386 type I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa SMC4386 10.1126/science.aau5174 AcrIE7 showed activity against type I-E CRISPR-Cas when ectopically expressed in P. aeruginosa (ECO_0000017). Phage replication assays using JBD8 confirmed successful immune evasion. MIGSEKQVNWAKSIIEKEVEAWEAIGVDVREVAAFLRSISDARVIIDNRNLIHFQSSGISYSLESSPLNSPIFLRRFSACSVGFEEIPTALQRIRSVYTAKLLEDE acrif11 AcrIF11 6KYF acrif11.pdb PDB entry adds a post-translational modification and deactivates bacterial defence ADP-ribosylates N250 of the Cas8f subunit, a residue required to recognise the PAM, within the crRNA-guided surveillance (Csy) complex. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa 10.1126/science.aau5174;10.1016/j.molcel.2020.09.015 AcrIF11 was discovered as a widespread type I-F inhibitor. When ectopically expressed in P. aeruginosa strains with type I-F CRISPR targeting DMS3m (ECO_0000017), it allowed phage replication, indicating potent inhibition. Biochemical assays demonstrated that AcrIF11 catalyzes ADP-ribosylation of the Csy complex in the presence of NAD, specifically modifying residue N250 of the Cas8f subunit, a critical site for PAM recognition. This modification abolished the DNA binding ability of the Csy complex, confirmed through electrophoretic mobility shift assays (EMSA, ECO_0001807), mass spectrometry (ECO_0001096), and site-directed mutagenesis (e.g., Cas8f N250A). AcrIF11’s enzymatic activity required the presence of the Cas7.6f subunit for binding, and its NAD-binding and catalytic residues were essential for inhibition, as shown by in vitro cleavage and binding assays using catalytic mutants (ECO_0000015). MSMELFHGSYEEISEIRDSGVFGGLFGAHEKETALSHGETLHRIISPLPLTDYALNYEIESAWEVALDVAGGDENVAEAIMAKACESDSNDGWELQRLRGVLAVRLGYTSVEMEDEHGTTWLCLPGCTVEKI acrif12 AcrIF12 _ acrif12.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Pseudomonas aeruginosa 10.1126/science.aau5174 AcrIF12 was identified using aca4-associated loci and confirmed via ectopic expression in type I-F CRISPR-active P. aeruginosa (ECO_0000017). Phage plaque assays showed restored replication of DMS3m. MAYEKTWHRDYAAESLKRAETSRWTQDANLEWTQLALECAQVVHLARQVGEELGNEKIIGIADTVLSTIEAHSQATYRRPCYKRITTAQTHLLAVTLLERFGSARRVANAVWQLTDDEIDQAKA acrif13 AcrIF13 7FI4 acrif13.pdb PDB entry binds and inhibits host defence system binds to the Cas5f-8f tail and Cas7.6f subunits of the Csy complex to block target DNA recognition. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Moraxella phage Mcat5 https://www.ncbi.nlm.nih.gov/nuccore/823079777 10.1126/science.aau5174;10.1016/j.jbc.2022.101636 AcrIF13 from Moraxella catarrhalis was ectopically expressed in P. aeruginosa (ECO_0000017), completely inhibiting type I-F CRISPR-Cas immunity and restoring phage replication. Competitive binding experiments demonstrated that AcrIF13 targets the Cas5f-Cas8f tail of the complex, similarly to AcrIF2, and also requires Cas7.6f for stable binding. Native gel electrophoresis confirmed that AcrIF13 does not bind the Cas5f-8f heterodimer alone, unlike other Acrs, indicating multivalent interactions. Electrophoretic mobility shift assays (EMSA, ECO_0000096) showed that AcrIF13 potently blocked DNA binding to the Csy complex at a 1:1 molar ratio, confirming its role as a DNA mimic. Site-directed mutagenesis of acidic residues on AcrIF13 and basic residues on Cas8f and Cas7f reduced binding affinities by up to 40-fold (e.g., D113K mutant KD = 67.65 nM), further confirming critical electrostatic interactions mediating inhibition. MKLLNIKINEFAVTANTEAGDELYLQLPHTPDSQHSINHEPLDDDDFVKEVQEICDEYFGKGDRTLARLSYAGGQAYDSYTEEDGVYTTNTGDQFVEHSYADYYNVEVYCKADLV acrif14 AcrIF14 7DU0 acrif14.pdb PDB entry binds and inhibits host defence system binds to the Cas7.4/7.6f subunits of the Csy complex, inducing strong non-specific DNA binding activity. Pseudomonas aeruginosa type I-F CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Moraxella phage Mcat5 https://www.ncbi.nlm.nih.gov/nuccore/823079777 10.1126/science.aau5174 AcrIF14, encoded in phage Mcat5, was validated through ectopic expression in type I-F CRISPR-active strains (ECO_0000017). It restored DMS3m replication, confirming inhibition. Cryo-EM structures of the Csy–AcrIF14 complex (ECO_0006181) showed two AcrIF14 molecules engaging the Cas7.4f and Cas7.6f subunits, sterically blocking crRNA–DNA hybridization. Key interface residues (e.g., R84, Y89, E91, F104, Y105) were identified via mutational analysis; pull-down (ECO_0006249) assays and cleavage assays confirmed their importance for inhibition. Additionally, AcrIF14 induced strong non-sequence-specific dsDNA binding when complexed with Csy, evidenced by electrophoretic mobility shift assays (EMSA, ECO_0000096) using dsDNA (non-specific). This DNA-binding activity was abolished in AcrIF14 mutants targeting its positively charged N-terminal patches, and in Csy complexes bearing mutations in the Cas8f PAM-recognition loop (K247E, N250D) or the R-loop Binding Channel (R207E/R219E/R258E). MKKIEMIEISQNRQNLTAFLHISEIKAINAKLADGVDVDKKSFDEICSIVLEQYQAKQISNKQASEIFETLAKANKSFKIEKFRCSHGYNEIYKYSPDHEAYLFYCKGGQGQLNKLIAENGRFM acrva1 AcrVA1 6NMD_B acrva1.pdb PDB entry binds and inhibits host defence system binds to Cas12a by mimicking the PAM and triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to inactivate the Cas12a complex (blocks DNA binding). Moraxella bovoculi, Acidaminococcus sp. and Lachnospiraceae bacterium type V-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Moraxella bovoculi 58069 10.1126/science.aau5174;10.1016/j.chom.2019.05.004 AcrVA1 was ectopically expressed in both bacterial and human cells (ECO_0000017). In P. aeruginosa expressing MbCas12a and targeting phage JBD30, AcrVA1 restored phage growth. In human U2-OS cells, AcrVA1 coexpression with Cas12a and EGFP-targeting crRNA abolished gene editing, confirming strong inhibition across multiple Cas12a orthologs. Size exclusion chromatography (ECO_0000325) demonstrated that AcrVA1 binds directly to both LbCas12a alone and LbCas12a-crRNA complexes, indicating crRNA-independent binding. Functional inhibition was confirmed by in vitro DNA cleavage assays, where addition of AcrVA1 completely blocked Cas12a endonuclease activity. Cryo-EM analysis (ECO_0006181) revealed AcrVA1 inserts into the PAM-binding cleft of Cas12a, mimicking the DNA PAM duplex and inducing structural rearrangements that displace the REC1 domain. AcrVA1 also exhibited Cas12a-dependent RNase activity, cleaving the crRNA guide. Cleavage resulted in two crRNA fragments, and was abolished by mutating key active site residues (R41A, H42A, H45A -- ECO_0001113) in AcrVA1’s α2 helix. Alanine substitutions at interaction interfaces (e.g., D95A/S96A) impaired both binding and RNase function (ECO_0001113). The crRNA cleavage was independent of Cas12a’s own nuclease activity, confirming AcrVA1 contains an intrinsic RNase active site. These findings support AcrVA1-mediated inhibition of Cas12a via dual mechanisms: PAM mimicry and destruction of crRNA guidance. MSKAMYEAKERYAKKKMQENTKIDTLTDEQHDALAQLCAFRHKFHSNKDSLFLSESAFSGEFSFEMQSDENSKLREVGLPTIEWSFYDNSHIPDDSFREWFNFANYSELSETIQEQGLELDLDDDETYELVYDELYTEAMGEYEELNQDIEKYLRRIDEEHGTQYCPTGFARLR acrva2 AcrVA2 7CI1_A acrva2.pdb PDB entry unknown _ Moraxella bovoculi type V-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Moraxella bovoculi 58069 10.1126/science.aau5174 AcrVA2 and its ortholog AcrVA2.1 inhibited Cas12a in P. aeruginosa strains via ectopic expression (ECO_0000017), restoring phage titer in plaque assays targeting JBD30. However, they showed limited or no activity in human cell assays. MHHTIARMNAFNKAFANAKDCYKKMQAWHLLNKPKHAFFPMQNTPALDNGLAALYELRGGKEDAHILSILSRLYLYGAWRNTLGIYQLDEEIIKDCKELPDDTPTSIFLNLPDWCVYVDISSAQIATFDDGVAKHIKGFWAIYDIVEMNGINHDVLDFVVDTDTDDNVYVPQPFILSSGQSVAEVLDYGASLFDDDTSNTLIKGLLPYLLWLCVAEPDITYKGLPVSREELTRPKHSINKKTGAFVTPSEPFIYQIGERLGSEVRRYQSIIDGEQKRNRPHTKRPHIRRGHWHGYWQGTGQAKEFRVRWQPAVFVNSGRVSS acrva3 AcrVA3 _ acrva3.cif AlphaFold 3 unknown _ Moraxella bovoculi type V-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Moraxella bovoculi 58069 10.1126/science.aau5174 AcrVA3 displayed weak inhibition and toxicity in bacteria. Its ortholog AcrVA3.1 was ectopically expressed in P. aeruginosa (ECO_0000017), showing stronger inhibition of Cas12a and partial activity against type I-C, suggesting possible dual specificity without toxicity. MVGKSKIDWQSIDWTKTNAQIAQECGRAYNTVCKMRGKLGKSHQGAKSPRKDKGISRPQPHLNRLEYQALATAKAKASPKAGRFETNTKAKTWTLKSPDNKTYTFTNLMHFVRTNPHLFDPDDVVWRTKSNGVEWCRASSGLALLAKRKKAPLSWKGWRLISLTKDNK acrvia1 AcrVIA1 6VRB_C acrvia1.pdb PDB entry binds and inhibits host defence system binds to the crRNA-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Listeria seeligeri type VI-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Listeriaphage (fLS46) 10.1126/science.abb6151 Phage fLS46 carrying the acrVIA1 gene overcame Cas13a-mediated immunity, while deletion of acrVIA1 (ΔacrVIA1) rendered the phage susceptible to CRISPR targeting. Infection assays with L. seeligeri strains harboring Cas13a-targeting spacers demonstrated that ΔacrVIA1 mutants were cleared, whereas wild-type fLS46 replicated efficiently even under conditions of multiple spacer targeting and extremely low multiplicity of infection. Conjugation assays revealed that plasmid transfer was inhibited by Cas13a unless AcrVIA1 was expressed, confirming its ability to suppress defense. In vitro assays showed that AcrVIA1 binds to the Cas13a–crRNA complex, blocking both cis- and trans-RNase activities. Electrophoretic mobility shift assays (EMSA, ECO_0001807) confirmed that AcrVIA1 prevents target RNA binding. Co-immunoprecipitation (ECO_0000085) and cryo-EM (ECO_0006181) revealed that AcrVIA1 interacts with both crRNA and multiple Cas13a domains, stabilizing crRNA in a conformation incompatible with activation. MIYYIKDLKVKGKIFENLMNKEAVEGLITFLKKAEFEIYSRENYSKYNKWFEMWKSPTSSLVFWKNYSFRCHLLFVIEKDGECLGIPASVFESVLQIYLADPFAPDTKELFVEVCNLYECLADVTVVEHFEAEESAWHKLTHNETEVSKRVYSKDDDELLKYIPEFLDTIATNKKSQKYNQIQGKIQEINKEIATLYESSEDYIFTEYVSNLYRESAKLEQHSKQILKEELN orba OrbA _ orba.cif AlphaFold 3 binds and inhibits host defence system directly binds the ATPase BrxC, disrupting its dimerisation Vibrio cholerae type I BREX BREX https://defensefinder.mdmlab.fr/wiki/defense-systems/brex Vibrio phage ICP1 https://www.ncbi.nlm.nih.gov/nuccore/?term=Vibrio+phage+ICP1 10.1126/science.abg2166;10.1128/jb.00206-24 Deletion (ECO_0001038) of orbA abolished plaque formation and phage genome replication in Vibrio cholerae strains expressing the BREX system from ICEVchInd5. This restriction was relieved either by restoring orbA or deleting the brxC component of BREX, confirming OrbA’s role in counter-defense. Co-immunoprecipitation (ECO_0005644) experiments revealed that OrbA binds directly to BrxC, the ATPase component of BREX. A bacterial two-hybrid assay (ECO_0005805) confirmed this interaction occurs specifically with the Ind5 BrxC variant, which OrbA inhibits, but not with BrxC from BREX systems that resist OrbA. Furthermore, OrbA expression disrupted BrxC multimerization in vivo, a process dependent on intact Walker A and B ATPase motifs, suggesting OrbA inhibits BREX by interfering with BrxC oligomerization and function. MKIANNYTFKQNKDGSVTVYNHGMFCAIIDEGMSRAKRIFLETV forsur_7 Forsur-7 _ forsur_7.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts a direct interaction between Avs3B (https://www.ncbi.nlm.nih.gov/protein/AZY97719.1) and Forsur-7 (ipTM = 0.72, pTM = 0.37). Salmonella enterica type III Avs Avs https://defensefinder.mdmlab.fr/wiki/defense-systems/avs Escherichia phage forsur https://www.ncbi.nlm.nih.gov/nuccore/1802642416 10.1126/science.abm4096 Forsur-7 was identified as a phage-encoded anti-defense factor through co-expression assays with SeAvs3 and its cognate trigger (the PhiV-1 large terminase subunit). In a genetic screen for phage early genes that rescue Avs-mediated toxicity, Forsur-7 partially restored E. coli growth when co-expressed with SeAvs3 and its activator. In vitro assays showed that Forsur-7 weakly inhibited SeAvs3 nuclease activity on double-stranded DNA, indicating it interferes with Avs3 effector function. Additionally, plaque assays demonstrated that Forsur-7 enhances phage propagation on Avs-containing E. coli, supporting its role as a functional Avs inhibitor. Binding it supported by computational structure modeling evidence (ECO_0006368). MATINVYKTITGSLVFVVGELREDAPIVGVSLREGATEADAIAEIRQNIENLGGLFFLLQGGSLNLAKLQEHEPVLVTSFEAEDVDASDFEASRADEEEAGE lidtsur_17 Lidtsur-17 _ lidtsur_17.cif AlphaFold 3 unknown _ Salmonella enterica type III Avs Avs https://defensefinder.mdmlab.fr/wiki/defense-systems/avs Escherichia phage Lidtsur https://www.ncbi.nlm.nih.gov/nuccore/NC_048177.1 10.1126/science.abm4096 Lidtsur-17 was shown to be a potent inhibitor of the Avs3 system. In the same pooled genetic screen with Lidtsur-6 and Forsur-7, Lidtsur-17 was among the most enriched genes, strongly rescuing SeAvs3-induced toxicity in E. coli. In vitro biochemical reconstitution demonstrated that Lidtsur-17 robustly blocked SeAvs3's DNA endonuclease activity in the presence of its trigger protein. In phage plaque assays, Lidtsur-17 restored phage infectivity on strains expressing Avs3, confirming its role in disabling Avs defense in vivo. MAINFEKFNKQQLAVVCQLGNEIDATPDVHDIIEFRGGFIPKVVYVAAVRNNRVDSIGIIQPTRTGAVLARNVTLIASLSAKQMELEGDREGVVAMQMAASLAVRFACEETVHDSYHEWFLACEHVPSDLTETQLSLLFAGEVLDQMLHQLQGGLSELMEAARGVKRATLH lidtsur_6 Lidtsur-6 _ lidtsur_6.cif AlphaFold 3 unknown _ Salmonella enterica type III Avs Avs https://defensefinder.mdmlab.fr/wiki/defense-systems/avs Escherichia phage Lidtsur https://www.ncbi.nlm.nih.gov/nuccore/NC_048177.1 10.1126/science.abm4096 Lidtsur-6 also functioned as an Avs inhibitor, albeit with moderate activity. Identified in the genetic suppression screen, it was found to confer partial protection against SeAvs3-mediated toxicity. Its expression in plaque assays restored infectivity of phages on E. coli harboring SeAvs3, suggesting that it inhibits the defense system in vivo. It does not inhibit the defence system in vitro, suggesting a possible indirect or context-dependent mode of action, possibly affecting Avs assembly or activation. MTKVTVLLTSDKVTLIALLGDAVEDTPSLALDIKPGSDLSAAIAELETQLKKPTAPIMFIVNGGGYSPEAAAQYSPEKVAEFTVDNPALDEFVAAVA ardu ArdU _ ardu.cif AlphaFold 3 binds and inhibits host defence system (putative) shares high similarity to ArdA (PDB ID: 2w82), which binds to the MTase of the type I RM complex and blocks it. AlphaFold 3 predicts a moderate-confidence complex between MTase (https://www.ncbi.nlm.nih.gov/protein/WP_010883983.1) and ArdU (ipTM = 0.76, pTM = 0.76). Deinococcus radiodurans RM systems RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Deinococcus radiopugnans plasmid pUE30 10.1128/AEM.66.9.3856-3867.2000 PF07275 ArdU was identified as a putative antirestriction protein based on sequence similarity to known antirestriction proteins such as ArdA from Yersinia pestis plasmid pMT-1, and others from enterobacterial plasmids like pKM101 and ColIb-P9. It was predicted to be highly acidic (pI 4.25), a characteristic feature of known Ard proteins. To test its functional role, the ardU gene was removed from plasmid pI3, resulting in derivative pI5. Transformation assays in Deinococcus radiodurans R1 showed that deletion of ardU led to a 50- to 100-fold decrease in transformation frequency compared to wild-type pI3, indicating that ArdU enhances transformation efficiency. However, ArdU was not required for replication or stable maintenance of the plasmid. These results support a role for ArdU in overcoming host restriction barriers during plasmid uptake, consistent with antirestriction function. Binding it supported by computational structure modeling evidence (ECO_0006368). MTYTHPLIIERHPDAPALWIGCLAAYNAGKLHGAWMQASSDTAEMFGAIEEILKASPEPHAEEWDIMDTDNMPTEAGRTLDSAATYVAALDALSRADAAEIVAAWVEWRGAEEMDADKITDAYLGRFDSVEDYAAQYLDDSGALQEVPEWLRPYINTAALGRDMEINGDVYEGKNGHFFNGHA acria1 AcrIA1 _ acria1.cif AlphaFold 3 unknown abolishes spacer acquisition by the type I-A CRISPR/Cas system. Sulfolobus islandicus type I-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Sulfolobus spindle-shaped virus Ragged Hills https://www.ncbi.nlm.nih.gov/nuccore/AY388628.1 10.1128/jb.00747-18;10.1016/j.jmb.2023.167996 PF01930;PF12705 acria1_model.html Viral Cas4 protein (AcrIA1) was identified as an anti-adaptation factor through plasmid challenge and transformation assays in Sulfolobus islandicus overexpressing both the CRISPR activator Csa3a and a virus-encoded Cas4 from SSV Ragged Hills (SSVRH). Overexpression of AcrIA1 resulted in a near-complete loss of new spacer acquisition, as confirmed by the absence of expanded PCR bands in CRISPR arrays and <1% acquisition efficiency based on high-throughput sequencing. Despite this loss, the remaining spacers retained normal length distribution, indicating that AcrIA1 did not affect spacer size but specifically interfered with acquisition activity. Protospacers from the few acquired spacers lacked the conserved 3′ A/G motif, suggesting interference with motif recognition. These findings indicate that AcrIA1 inhibits the CRISPR-Cas subtype I-A system by suppressing PAM- and motif-dependent spacer acquisition, enabling potential escape of viral DNA from host immunity. MRSKMIKKEEKDNKIYITVKDEETGIEWTAVVEKVEFEWCVKQKEELEVEDAEKSVMLDYALFGNCAIPKVTAEEYKNSLTKYTGEKMSRLLHILYNYEIVSQNDTKNIWVTELSRCLRRSYLMRKEGKTKVGLNEAMKMHIGSGLHMRLQSLLRKHGFETEVRVQRKTALGFQIVGRIDVYDKEENVIYELKYTHNDKLDSVRLNNYLRQLNYYIEMANAMKGYLVIVHADGSVEEIKRDWAETDLEKRANAFGIYVEENTLPPKKSRPDAECIECPFYNFCWGKL arda ArdA 2W82_A arda.pdb PDB entry binds and inhibits host defence system binds to the MTase of the type I RM complex and blocks it. Triggers Ronin defence. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Plasmid pKM101 10.1128/jb.174.15.5079-5085.1992 ardr;ardc;ardk PF07275 Infection assays using bacteriophage λ demonstrated that ArdA inhibits restriction and modification by multiple E. coli type I systems (EcoA, EcoB, EcoD, EcoK, and EcoR124), with efficiency of plating (EOP) restored by factors of up to 10,000 (Table 1). ArdA also showed partial activity against the type II enzyme EcoRI, but not against type III (EcoPI) or McrA/McrBC systems, indicating specificity. Genetic evidence from Tn5 mutagenesis localized ArdA activity to a defined region of pKM101, where insertions abolished antirestriction function. A neighboring regulatory gene, ardR, was shown to suppress ArdA expression, while its disruption derepressed activity by 200-fold. Another locus, ardK, prevented ArdA-mediated lethality when cloned in trans, confirming ArdA's kil-type potential if unregulated. MTDITTPSVYVGTYHKYNCGSIAGAWLDLTDFDSSEEFYERCRELHANEADPEFMFQDWEGIPSDMASECHINWDFINGFKQAREEGNEAAFVAFVDLFNSTDFDLFRDAYMGEAKDEETFAEEYLNDSGLLNEIPESVARYFDIVAYARDLFIGDFSLHDGHVFNMTC ardk ArdK 7BBQ ardk.pdb PDB entry unknown the mechanism is not clear, apart from its role in regulating the expression of ardA and ardB. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Plasmid pKM101 10.1128/jb.175.15.4843-4850.1993 ardr;arda;ardc PF16509 ArdK was identified as a transcriptional regulator of the antirestriction genes ardA and ardB through promoter activity assays and stability experiments using plasmid derivatives with and without upstream regulatory sequences. Expression of ardB from constructs containing the upstream conserved CUP region (e.g., pCAT66-4) resulted in plasmid instability unless ArdK was provided in trans, suggesting repression of a strong promoter (P2) by ArdK. Promoter probe assays showed that ArdK specifically repressed P2 activity but not P1. Maxicell experiments confirmed expression of a 15-kDa ArdK protein from the ardK gene. Combined with ArdR, ArdK completely abolished promoter activity from CUP sequences upstream of ardB and ardA, indicating coordinated repression at the transcriptional level. Sequence homology in the regulatory regions of ardA and ardB (94% identity) further supports ArdK’s dual role in their regulation. MAQKNRISETEWKQLLPQMASFAHITTDIGYSVLVKGEKSSDVATRVGRSKQNISSTVKRIWDLYQNTTLKAENGEPLKLVQVWIPASLAETVLKEAAKYSINNITTSEMEKK ardr ArdR _ ardr.cif AlphaFold 3 unknown the mechanism is not clear, apart from the role in regulating the expression of ardA and ardB. Escherichia coli type I RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Plasmid pKM101 10.1128/jb.175.15.4843-4850.1993 arda;ardc;ardk ArdR was shown to repress expression of both ardA and ardB, particularly the downstream P1 promoters, through deletion mapping and promoter activity assays using CUP-region constructs. Promoter probe plasmids (e.g., pCAT66-6) showed significant chloramphenicol acetyltransferase (CAT) activity in the absence of ArdR, which was abolished upon its expression, indicating ArdR-dependent repression. Maxicell analysis confirmed expression of a ~20-kDa protein from ardR. The gene is located adjacent to ardB, and deletion of ardR led to derepression of ardB-mediated antirestriction activity. These results demonstrate that ArdR acts as a transcriptional repressor of both ard genes and can function synergistically with ArdK to silence both P1 and P2 promoters. MLATLQHPTAWQLPDRLMLLELLMFDYRNSDQERYGQQIYHHYRKQGNHRWDTSVHQDSGGQYAIIFRHSFSKKQADGVKRTMIRDETVIRAGTAQELTEATFPDFQDSDILKASDFFKSLIQRKAADVTQTDI sam_lyase S-adenosyl-methionine_lyase 6ZNB sam_lyase.cif PDB entry degrades or sequesters molecules utilised by host defence systems degrades S-adenosyl-methionine (SAM) and inhibits SAM synthase. SAM is believed to be a co-factor in BREX-mediated and RM-mediated exclusion. Escherichia coli type I RM;Escherichia coli type I BREX RM;BREX https://defensefinder.mdmlab.fr/wiki/defense-systems/brex;https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T3 https://www.ncbi.nlm.nih.gov/nuccore/NC_047864.1 10.1128/JVI.19.1.136-145.1976;10.1016/j.celrep.2023.112972 PF23780 Deletion mutants lacking gene 0.3 encoding S-adenosyl-methionine lyase failed to overcome host restriction and exhibited no detectable SAMase activity in infected E. coli cells (ECO_0001038). Assays of cell extracts infected with various T3 strains showed that wild-type T3 induced strong SAMase activity, while T3 mutants such as R1, R4, R7, R12, and R13 showed both restriction sensitivity and complete loss of SAMase function. An amber mutant (HR2) produced SAMase only in suppressor hosts, confirming gene-linked inactivation. However, strain 3356 retained the ability to overcome restriction despite loss of SAMase activity, indicating that SAMase production is not the sole mechanism for bypassing restriction systems. Electrophoresis of phage-infected protein samples revealed the absence of the 0.3 protein in deletion and amber mutants, directly linking gene 0.3 to SAMase function. The enzyme cleaved radiolabeled S-adenosylmethionine in vitro, forming detectable products by thin-layer chromatography (ECO_0000325), establishing enzymatic activity of the SAMase gene product. MIFTKEPAHVFYVLVSAFRSNLCDEVNMSRHRHMVSTLRAAPGLYGSVESTDLTGCYREAISSAPTEEKTVRVRCKDKAQALNVARLACNEWEQDCVLVYKSQTHTAGLVYAKGIDGYKAERLPGSFQEVPKGAPLQGCFTIDEFGRRWQVQ riia RIIA _ riia.cif AlphaFold 3 unknown _ Escherichia coli RexAB RexAB https://defensefinder.mdmlab.fr/wiki/defense-systems/rexab Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1128/jvi.61.12.3790-3794.1987 PF13589 riia_model.html Plasmid-expressed rexA and rexB were sufficient to restrict T4 rII mutants, even in the absence of lambda cI repressor, establishing a direct functional interaction between RIIA and rex products. Growth restriction of wild-type T4 (rIIA⁺, rIIB⁺) by rex was demonstrated in engineered E. coli strains expressing rex genes under strong promoters (e.g., prex20) and was more stringent at higher temperatures. This indicates that RIIA is involved in mitigating rex-induced abortive infection under normal conditions. MIITTEKETILGNGSKSKAFSITASPKVFKILSSDLYTNKIRAVVRELITNMIDAHALNGNPEKFIIQVPGRLDPRFVCRDFGPGMSDFDIQGDDNSPGLYNSYFSSSKAESNDFIGGFGLGSKSPFSYTDTFSITSYHKGEIRGYVAYMDGDGPQIKPTFVKEMGPDDKTGIEIVVPVEEKDFRNFAYEVSYIMRPFKDLAIINGLDREIDYFPDFDDYYGVNPERYWPDRGGLYAIYGGIVYPIDGVIRDRNWLSIRNEVNYIKFPMGSLDIAPSREALSLDDRTRKNIIERVKELSEKAFNEDVKRFKESTSPRHTYRELMKMGYSARDYMISNSVKFTTKNLSYKKMQSMFEPDSKLCNAGVVYEVNLDPRLKRIKQSHETSAVASSYRLFGINTTKINIVIDNIKNRVNIVRGLARALDDSEFNNTLNIHHNERLLFINPEVESQIDLLPDIMAMFESDEVNIHYLSEIEALVKSYIPKVVKSKAPRPKAATAFKFEIKDGRWEKRNYLRLTSEADEITGYVAYMHRSDIFSMDGTTSLCHPSMNILIRMANLIGINEFYVIRPLLQKKVKELGQCQCIFEALRDLYVDAFDDVDYDKYVGYSSSAKRYIDKIIKYPELDFMMKYFSIDEVSEEYTRLANMVSSLQGVYFNGGKDTIGHDIWTVTNLFDVLSNNASKNSDKMVAEFTKKFRIVSDFIGYRNSLSDDEVSQIAKTMKALAA riib RIIB _ riib.cif AlphaFold 3 unknown Triggers Gasdermin defence Escherichia coli RexAB RexAB https://defensefinder.mdmlab.fr/wiki/defense-systems/rexab Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.1128/jvi.61.12.3790-3794.1987 PF13518;PF13551;PF02796 The experimental evidence supporting RIIB activity includes: (1) RIIB translation mutants with reduced protein levels showed graded susceptibility to rex-mediated restriction, correlating with the amount of RIIB produced. For example: (a) rII mutants expressing 46% and 39% of wild-type RIIB had partial restriction. (b) Mutants expressing 7%, 3%, or 0% (deletion mutant r638, ECO_0001038) were fully restricted. (2) The restriction level correlated quantitatively with RIIB levels and rex expression levels (modulated via IPTG induction or promoter strength). (3) Complementation data showed that wild-type RIIB activity is required to overcome rex restriction, and that loss of RIIB leads to arrest of phage development in rex⁺ hosts. (4) Temperature-dependent effects further confirmed RIIB’s role: restriction by rex was stronger at 42°C and weaker at 30°C, matching expected temperature sensitivity of translation or protein stability. MYNIKCLTKNEQAEIVKLYSSGNYTQQELADWQGVSVDTIRRVLKNAEEAKRPKVTISGDITVKVNSDAVIAPVAKSDIIWNASKKFISITVDGVTYNATPNTHSNFQEILNLLVADKLEEAAQKINVRRAVEKYISGDVRIEGGSLFYQNIELRSGLVDRILDSMEKGENFEFYFPFLENLLENPSQKAVSRLFDFLVANDIEITEDGYFYAWKVVRSNYFDCHSNTFDNSPGKVVKMPRTRVNDDDTQTCSRGLHVCSKSYIRHFGSSTSRVVKVKVHPRDVVSIPIDYNDAKMRTCQYEVVEDVTEQFK adfb AdfB (anti-DarT factor B) _ adfb.cif AlphaFold 3 binds and inhibits host defence system binds the toxin, DarT. Vibrio cholerae type IV TA system DarTG TA https://defensefinder.mdmlab.fr/wiki/defense-systems/dartg Vibrio phage ICP1 https://www.ncbi.nlm.nih.gov/nuccore/NC_015157.1 10.1128/mbio.00111-24 AdfB (anti-DarT factor B) was identified as a phage-encoded anti-defense factor through selection and sequencing of ICP1 phage mutants that escaped restriction by the DarTG toxin-antitoxin system in clinical V. cholerae isolates. A conserved G62D mutation in the ICP1 core genome gene gp145 conferred escape from DarTG-mediated restriction. Deletion of gp145 abolished this escape (ECO_0007379), while complementation with the G62D allele restored it, confirming functional necessity. Genome replication assays revealed that only phage encoding Gp145D62 (AdfB) restored ICP1 DNA replication in the presence of DarTG, directly linking AdfB function to anti-DarT activity. Inducible expression of Gp145D62 in V. cholerae abrogated DarT-mediated cell death, similar to the native antitoxin DarG, while wild-type Gp145G62 did not. Bacterial two-hybrid assays showed that Gp145D62, but not Gp145G62, physically interacted with DarT, and this interaction was disrupted by a Y82A mutation in DarT’s DNA-binding domain. Structural modeling suggested that Gp145D62 mimics the C-terminal DNA-binding inhibitory domain of DarG. Collectively, these data demonstrate that Gp145D62 (AdfB) directly binds and neutralizes DarT toxin activity, allowing phage escape from host defense. MKIINFVNPNNTMDGYKLLVHCVFSGFDYDNTKFYMNREGFYLPEDQFNAFKSCCDIMMDLGIGDRKKEVSLVQGG acrie1 AcrIE1 6AS4 acrie1.pdb PDB entry binds and inhibits host defence system binds as a dimer to Cas3 (blocks DNA cleavage). Pseudomonas aeruginosa SMC4386 type I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage JBD5 https://www.ncbi.nlm.nih.gov/nuccore/NC_020202.1 10.1128/mBio.00896-14 AcrIE1 was identified via plaque assays in Pseudomonas aeruginosa strain SMC4386 expressing individual anti-CRISPR genes from plasmids. Expression (ECO_0000017) of ACR88a-32 from phage JBD88a allowed robust infection by CRISPR-sensitive phage JBD8, which otherwise fails to infect due to targeting by the type I-E CRISPR-Cas system. Transformation efficiency assays in lysogenized strains confirmed loss of CRISPR interference, supporting ACR88a-32’s inhibitory role. MEKKLSDAQVALVAAWRKYPDLRESLEEAASILSLIVFQAETLSDQANELANYIRRQGLEEAEGACRNIDIMRAKWVEVCGEVNQHGIRVYGDAIDRDVD acrie2 AcrIE2 _ acrie2.cif AlphaFold 3 unknown _ Pseudomonas aeruginosa SMC4386 type I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage JBD88a https://www.ncbi.nlm.nih.gov/nuccore/NC_020200.1 10.1128/mBio.00896-14 AcrIE2 was functionally validated using a phage knockout strategy. Wild-type phage DMS3m carrying the acrIE2 gene (gene 30) infected strain SMC4386 efficiently, while a mutant version lacking acrIE2 (replaced by a type I-F anti-CRISPR) was unable to form plaques due to CRISPR interference (ECO_0001038). Complementary assays using JBD8 plaque formation in plasmid-expressing strains further demonstrated ACR3-30's ability to inhibit the type I-E system in vivo. Expression of ACR3-30 had no effect on E. coli’s type I-E system or on type I-F systems MNTYLIDPRKNNDNSGERFTVDAVDITAAAKSAAQQILGEEFEGLVYRETGESNGSGMFQAYHHLHGTNRTETTVGYPFHVMEL acrie4 AcrIE4 _ acrie4.cif AlphaFold 3 binds and inhibits host defence system (putative) AlphaFold 3 predicts high-confidence interactions between CasA (Cas8e, https://www.ncbi.nlm.nih.gov/protein/QZE32573.1) and AcrIE4 (ipTM = 0.92, pTM = 0.89) and Cas6e (https://www.ncbi.nlm.nih.gov/protein/QZE32569.1) and AcrIE4 (ipTM = 0.8, pTM = 0.85). Pseudomonas aeruginosa SMC4386 type I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage D3112 https://www.ncbi.nlm.nih.gov/nuccore/AY394005.1 10.1128/mBio.00896-14 AcrIE4 was identified in phage JBD5 and shown to inhibit the type I-E system using both transformation efficiency assays and plaque assays. Expression (ECO_0000017) of ACR5-34 from a plasmid in SMC4386 enabled efficient plaque formation by CRISPR-targeted phage JBD8. Additionally, a homolog of ACR5-34 found in a P. aeruginosa mobile genetic element (non-phage) was also shown to have anti-CRISPR activity in plaque assays, suggesting this gene may contribute to horizontal gene transfer by evading CRISPR immunity. Binding it supported by computational structure modeling evidence (ECO_0006368). MSTQYTYEQIAEDFRLWGEYMDPNAEMTEEEFQALSTEEKVAMQVEAFGAEA acrie3 AcrIE3 _ acrie3.cif AlphaFold 3 binds and inhibits host defence system primarily binds to Cas8e, but may interact with Cas5e and Cas7e. Pseudomonas aeruginosa SMC4386 type I-E CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Pseudomonas phage DMS3 https://www.ncbi.nlm.nih.gov/nuccore/NC_008717.1 10.1128/mBio.00896-14;10.1016/j.str.2024.10.024 AcrIE3 activity was demonstrated by expressing ACR3112-31 from phage D3112 in P. aeruginosa SMC4386. The expression (ECO_0000017) enabled phage JBD8, which is normally blocked by the CRISPR system, to form plaques at a 10³–10⁵-fold increased efficiency compared to controls. Transformation assays in lysogenized strains supported functional suppression of type I-E activity. MKITNDTTTYEVAELMGSEADELDGRIMMGLLSRECVVDTDDLSEDQWLALIDESQKVRREQFESDEA acriic4 AcrIIC4 7F7P_A acriic4.pdb PDB entry binds and inhibits host defence system binds to Cas9, blocking DNA binding. Neisseria meningitidis type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Haemophilus parainfluenza 10.1128/mBio.02321-18 AcrIIC4Hpa was identified as a Cas9 inhibitor through in vitro cleavage assays using recombinant NmeCas9 and sgRNA-loaded complexes. Addition of AcrIIC4Hpa blocked target DNA cleavage in a concentration-dependent manner, with full inhibition observed at approximately 20-fold molar excess. In E. coli phage Mu immunity assays, AcrIIC4Hpa expression rescued phage infectivity in strains expressing HpaCas9 (its cognate Cas9) and significantly reduced NmeCas9-mediated phage targeting, confirming in vivo inhibition. Co-purification assays demonstrated direct binding between AcrIIC4Hpa and both HpaCas9 and NmeCas9. In HEK293T cells, transient coexpression of AcrIIC4Hpa abolished NmeCas9 genome editing activity without affecting SpyCas9, showing subtype specificity. Electrophoretic mobility shift assays (ECO_0000096) revealed that AcrIIC4Hpa does not interfere with sgRNA loading but blocks target DNA binding, consistent with a mechanism that prevents stable Cas9-DNA association. MKITSSNFATIATSENFAKLSVLPKNHREPIKGLFKSAVEQFSSARDFFKNENYSKELAEKFNKEAVNEAVEKLQKAIDLAEKQGIQF acriic5 AcrIIC5 8JB9 acriic5.pdb PDB entry binds and inhibits host defence system binds to Cas9, blocking DNA binding Neisseria meningitidis type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas MGE in Simonsiella muelleri 10.1128/mBio.02321-18 AcrIIC5Smu was identified as a highly potent NmeCas9 inhibitor through in vitro cleavage assays where it blocked DNA cleavage at lower concentrations than AcrIIC4Hpa, achieving full inhibition at ~7-fold molar excess. In phage immunity assays, AcrIIC5Smu expression fully restored infectivity of phage Mu in E. coli strains expressing NmeCas9 and HpaCas9, indicating strong cross-species inhibition. Although no direct interaction with Cas9 was observed in bacterial co-purification, co-immunoprecipitation (ECO_0005644) from mammalian cell lysates confirmed physical binding between AcrIIC5Smu and NmeCas9. In HEK293T cells, AcrIIC5Smu expression abolished genome editing by NmeCas9 at multiple loci, while editing by SpyCas9 remained unaffected, demonstrating high specificity. Biochemical assays showed that AcrIIC5Smu does not disrupt sgRNA loading but prevents target DNA binding, reducing NmeCas9 DNA affinity by ~6-fold. In live-cell imaging, AcrIIC5Smu eliminated telomeric focus formation by dNmeCas9 while sparing dSpyCas9, verifying functional inhibition of DNA binding in human cells and supporting its utility as a precise anti-CRISPR off-switch. MNNSIKFHVSYDGTARALFNTKEQAEKYCLVEEINDEMNGYKRKSWEEKLREENCASVQDWVEKNYTSSYSDLFNICEIEVSSAGQLVKIDNTEVDDFVENCYGFTLEDDLEEFNKAKQYLQKFYAECEN acrib2 AcrIB2 _ acrib2.cif AlphaFold 3 unknown hypothesised to act as a DNA mimic, with some tentative evidence that it binds Cas3. Clostridioides difficile type I-B CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Clostridium difficile phage φCD38-2 https://www.ncbi.nlm.nih.gov/nuccore/NC_015568.1 10.1128/msphere.00401-23 AcrIB2 was identified as an inhibitor of the type I-B CRISPR-Cas system through conjugation assays in Clostridioides difficile. A plasmid containing a protospacer targeted by the CRISPR system failed to establish in recipient cells unless AcrIB2 was expressed, indicating interference inhibition. In a self-targeting assay, induction of a CRISPR array targeting the chromosomal hfq gene was lethal unless AcrIB2 was co-expressed, resulting in restored cell viability and growth. Genomic sequencing after CRISPR induction showed loss of DNA coverage near the target site in the absence of AcrIB2, while co-expression of AcrIB2 preserved genomic integrity, confirming interference suppression. Deletion analysis of CRISPR-Cas operons demonstrated that only the partial cas operon is responsible for interference and is the target of AcrIB2. Expression of AcrIB2 in a strain lysogenized with φCD38-2 partially suppressed CRISPR-mediated toxicity, indicating its activity in a prophage context. Mass spectrometry following co-purification with tagged AcrIB2 revealed enrichment of Cas3, suggesting a direct interaction. Structural predictions and conserved sequence features supported a DNA mimicry mechanism of action. MNKQKARRFLRVIDMNIDKIEEEAIKAFKESCLIKETNNIKIYIDIQGKVEAIAVQTWAKLLGDDKEINIFTLNQAPTHLNDMLGEICYVNDYEEFENWCENEWENLDWDSYKKFNKENFEEIAERNIDDSTSVFLEELQKGIESCKQELQNVIEN acriia22 AcrIIA22 7JTA_A acriia22.pdb PDB entry modifies phage molecules to avoid recognition modifies MGE DNA topology. Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Metagenome 10.1371/journal.pbio.3001428 AcrIIA22 was identified as a novel anti-CRISPR through a functional metagenomic screen using a two-plasmid system in E. coli, where its expression allowed retention of a kanamycin-resistant target plasmid otherwise eliminated by SpyCas9. Deletion (ECO_0001038) or mutation of acrIIA22 (orf_1) abolished this protective effect, confirming its necessity and sufficiency for CRISPR inhibition. AcrIIA22 did not bind or inhibit SpyCas9 in vitro directly, suggesting an alternative mechanism. Structural analysis via X-ray crystallography (PDB: 7JTA) revealed homology to PC4-like nucleic acid–binding proteins, predicting a role in DNA interaction. Biochemical assays demonstrated that AcrIIA22 functions as a DNA nickase, converting supercoiled plasmids into relaxed forms in vivo and in vitro. Mutations that impaired this nicking activity (e.g., D14A or natural variant AcrIIA22a) also reduced anti-CRISPR activity in bacterial plasmid protection assays, confirming that DNA nicking underlies its function. Pre-nicked plasmids became resistant to SpyCas9 cleavage in vitro, linking AcrIIA22’s activity to altered DNA topology and impaired Cas9 R-loop formation. MVVEETRDLAETADCVVIEAILVDDGLRYRQLSVGIKDENGDIIRIVPISTVLI ardc ArdC 6SNA_A ardc.pdb PDB entry unknown ssDNA-binding protein with a metalloprotease domain; the mechanism is not clear. Pseudomonas putida type I RM (hsdRMS) RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Plasmid R388 10.1371/journal.pgen.1008750 ardr;arda;ardk PF08401 ArdC was identified as an anti-restriction factor that enhances plasmid conjugation across species barriers by counteracting Type I restriction-modification systems. Deletion of ardC (ECO_0007379) from plasmid R388 drastically reduced conjugation efficiency from E. coli to Pseudomonas putida, but not within E. coli, implicating a specific defense mechanism in P. putida. This effect was rescued by expressing ArdC (ECO_0000017) in the recipient cell, but not in the donor, confirming recipient-side activity. Conjugation into P. putida ΔhsdRMS mutants restored high transfer rates even without ArdC, directly implicating the HsdRMS restriction system as the target. A catalytically inactive ArdC mutant (E229A) retained full antirestriction function in conjugation assays, indicating that metalloprotease activity is dispensable and suggesting that DNA binding is the functional mechanism. RNA-seq analyses during ArdC-mediated conjugation revealed SOS response activation in recipient P. putida, likely due to increased ssDNA exposure. Together, these data establish ArdC as a recipient-expressed, ssDNA-binding antirestriction protein that enables plasmid transfer by protecting incoming DNA from HsdRMS degradation. MTMNLHTQTSVPNPAPATSASPLEQSGSSKTKFSKGKAKPDVYQVVTDSIIEALETGVKPWVCPWKRNGAVSGIPSNFTTGTSYSGINIMMLWYSAAAQGFTDSRWLTYKQAQELGAQVRKGEKGTTAIFYKMLEKETEAGEGEKIPMLKSFTVFNAEQIEGLTLEEKTAPQPVAEFDPLPQVEALFQRTGAKITERGQQAFFRPSTDEIWMPERHLFTDAANFYATGLHELVHWSGAKNRLNREKGGKFGSAGYAFEELIAELGSAFLMADLSIYGEVQHENYIASWLEALKGDKRFIFKAASAASKAHRYLMDF nip Nip _ nip.cif AlphaFold 3 binds and inhibits host defence system binds directly to the TIR (NADase) domain of the anti-phage defense protein SpbK, inhibiting its NADase activity and thereby preventing abortive infection. Bacillus subtilis SpbK SpbK https://defensefinder.mdmlab.fr/wiki/defense-systems/spbk Phage Φ3T 10.1371/journal.pgen.1011551 Nip (NADase inhibitor from phage) was identified as a phage-encoded counter-defense protein that inhibits the SpbK NADase defense system. Its activity was demonstrated through infection assays with wild-type Φ3T and a Δnip mutant. Deletion of nip from Φ3T (Φ3T Δnip) drastically reduced infectivity on B. subtilis strains expressing SpbK (ECO_0001038), whereas reintroduction of nip restored infectivity, confirming its necessity and sufficiency for counter-defense. Co-expression of nip in strains harboring both spbK and yonE abolished SpbK-dependent growth arrest and NAD⁺ depletion, demonstrating functional inhibition of NADase activity. Infection of spbK⁺ strains with Φ3T Δnip, but not with wild-type Φ3T, led to significant NAD⁺ depletion, further supporting this effect during infection. Co-immunoprecipitation experiments revealed that Nip binds directly to the TIR (NADase) domain of SpbK but not to its N-terminal domain. Additional immunoprecipitation assays showed formation of a tripartite complex among Nip, SpbK, and YonE, with Nip-SpbK interaction occurring independently of YonE. These data collectively demonstrate that Nip counteracts SpbK-mediated abortive infection by directly binding and inhibiting its enzymatic activity. MTETKANANVKIHVLADETLSGIKREYVEVDRKAEVGEKIVIVDKNDPGDVYENGAIFTVDRDFPGKKHVESDAARCGGNLHGFILREEYRVLEPTDIVHIDGGRYELTNRKAKVGEKVITITKCDIYSKGEIGTVGYQSPPRYIYVRFETRATGWRVPHEDYRVLVPLDKCEKTFETKNSGYKEIKNLIHNDLGITKQDIQEMISVAVSNEVQKMSESGKLDSIAGVKIESLIEEGFRDGGRLLYGFRERVSQTVSDEVGKRIANVLNINVELKEERN acriia11 AcrIIA11 _ acriia11.cif AlphaFold 3 binds and inhibits host defence system binds SpyCas9 to inhibit dsDNA cleavage. Streptococcus pyogenes type II-A CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Metagenome 10.7554/eLife.46540 AcrIIA11 was identified as a potent anti-CRISPR protein through a two-step functional metagenomic selection that screened for elements protecting a plasmid with a kanamycin resistance gene from cleavage by SpyCas9 in E. coli. A contig derived from a Lachnospiraceae phage conferred the highest level of resistance and was significantly enriched after selection. Targeted mutagenesis of all five ORFs in the contig revealed that orf_3, named acrIIA11, was solely responsible for SpyCas9 inhibition; introducing a stop codon in this ORF eliminated the protective effect. When cloned alone, AcrIIA11 protected plasmids as effectively as known inhibitors such as AcrIIA4. In phage infection assays, AcrIIA11 expression (ECO_0000017) restored infectivity of Mu phage in E. coli expressing a SpyCas9–targeting crRNA, demonstrating functional inactivation of the CRISPR defense. Further support came from in vitro assays: purified AcrIIA11 inhibited SpyCas9-mediated DNA cleavage in a dose-dependent manner, with size exclusion chromatography (ECO_0000325) suggesting that the active form is a dimer. Pull-down assays (ECO_0006249) showed AcrIIA11 binds both apo- and sgRNA-bound forms of SpyCas9, with moderate preference for the latter. Electrophoretic mobility shift assays (ECO_0000096) demonstrated that unlike AcrIIA4, AcrIIA11 does not block SpyCas9–DNA binding but instead forms a supershifted ternary complex, indicating a novel mechanism of inhibition involving both SpyCas9 and dsDNA interaction. MADMTLRQFCERYRKGDFLAKDRETQIEAGWYDWFCDDKALAGRLAKIWGILKGITSDYILDNYRVWFKNNCPMVGPLYDDVRFEPLDEEQRDELYFGVAIDDKRREKKYVIFTARNDYENECGFNNVREVRQFINGWEDELKNEEFYKAREKKRQEMEEANNKFAEIMQRADEILWNLKED tifa Tifa _ tifa.cif AlphaFold 3 binds and inhibits host defence system (putative) may directly interact with the toxin, toxN. AlphaFold 3 predicts a direct interaction between ToxN (https://www.ncbi.nlm.nih.gov/protein/WP_012609144.1) and Tifa (ipTM = 0.72, pTM = 0.75). Escherichia coli type III TA toxIN TA _ Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.7554/eLife.79549 PF23945 TifA was identified as a phage-encoded inhibitor of the bacterial toxin ToxN through experimental evolution of T4 phage on E. coli expressing the toxIN toxin-antitoxin system. Segmental amplification of the dmd-tifA locus was repeatedly selected in independently evolved T4 populations, enhancing infectivity on toxIN-containing hosts. Cloning and overexpression of tifA alone (but not dmd) in E. coli restored T4 infection in toxIN-expressing cells, confirming tifA as the active anti-defense factor. Functional assays showed that co-expression of TifA neutralized ToxN toxicity, rescuing cell growth. Deletion of the tifA start codon abolished this rescue, while a codon-recoded tifA retained activity, indicating that TifA acts as a protein antitoxin. Co-immunoprecipitation (ECO_0000085) experiments demonstrated a physical interaction between TifA and ToxN in vivo. Finally, T4 clones with tifA deletions lost the ability to infect cells with chromosomally encoded toxIN, confirming the necessity of TifA for defense evasion. These findings establish TifA as a direct, proteinaceous anti-toxin that inhibits ToxN-mediated phage defense. Binding it supported by computational structure modeling evidence (ECO_0006368). MHIVLFKPTPYNVRKNTQFKALIADTWELVLDIPAEESPPFGRVEFIKFAVRPTKRQIRQCKRYFRKIVKLEKQFVTCDYAEILK gp5_9 Gp5.9 8B1R_P gp5_9.pdb PDB entry binds and inhibits host defence system mimics DNA and binds to the RecB subunit of RecBCD, competing sterically with DNA. Triggers retron Ec48 defence Escherichia coli RecBCD RecBCD _ Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 10.7554/eLife.83409 Gp5.9 was identified as a RecBCD inhibitor through in vitro helicase assays using purified protein. Addition of Gp5.9 to RecBCD prevented DNA unwinding in a double-stranded break resection assay, indicating potent inhibition of RecBCD activity. The specificity of Gp5.9 for RecBCD was demonstrated by its lack of effect on the AddAB complex homologous to RecBCD. A quantitative fluorescence-based helicase assay showed Gp5.9 inhibits RecBCD with an IC50 of 1.3 nM. Electrophoretic mobility shift assays (ECO_0000096) confirmed that Gp5.9 prevents DNA binding by RecBCD, even in complexes already bound by Abc2, indicating a direct competitive mechanism. CryoEM structural analysis of the RecBCD-Gp5.9 complex revealed that Gp5.9 acts as a DNA mimic (ECO_0006181), occupying the DNA binding site on the RecB arm domain and sterically preventing substrate access. This binding mode explains the observed loss of helicase activity and validates Gp5.9’s function as an anti-RecBCD factor that protects phage DNA from degradation. MSRDLVTIPRDVWNDIQGYIDSLERENDSLKNQLMEADEYVAELEEKLNGTS acriic6 AcrIIC6 _ acriic6.cif AlphaFold 3 binds and inhibits host defence system binds to sgRNA loaded Cas9 preventing the complex from binding target DNA Neisseria meningitidis type II-C CRISPR-Cas CRISPR-Cas https://defensefinder.mdmlab.fr/wiki/defense-systems/cas Prophages in Neisseria meningitidis and Pasteurella multocida No DOI, master thesis (Khan, A.N., 2021. Characterizing a Novel Type II-C Anti-CRISPR, AcrIIC6) AcrIIC6Nme was identified using a guilt-by-association bioinformatic approach and tested for anti-CRISPR activity via in vivo phage targeting assays. Expression of AcrIIC6Nme in E. coli co-expressing Nme1Cas9 and an sgRNA targeting phage Mu restored phage infectivity, indicating inhibition of CRISPR interference. This inhibition was selective, effective only against closely related type II-C Cas9s (Nme1Cas9, Nme2Cas9, HpaCas9, BoeCas9), but not distantly related variants (GeoCas9, CjeCas9, CdiCas9). In vivo pulldown (ECO_0006249) assays demonstrated that AcrIIC6Nme selectively co-purifies with the Nme1Cas9:sgRNA complex but not apo-Cas9, indicating specific binding to the Cas9:sgRNA binary complex. These results support the activity of AcrIIC6Nme as a selective type II-C anti-CRISPR that binds the loaded Cas9 complex and inhibits DNA targeting. MEIIKTGSYRLEKLSGNTYKVYRHQYAIGTLTEYGEVELTDEIIKGYEKKPHSGYWVSEIEKIIKNR dnmp Deoxynucleoside monophosphate kinase 1DEK_A dnmp.pdb PDB entry modifies phage molecules to avoid recognition synthesises hydroxymethyl-deoxycytidine triphosphate (Hm-dCTP) from Hm-dCMP. Escherichia coli RM RM https://defensefinder.mdmlab.fr/wiki/defense-systems/rm Enterobacteria phage T4 https://www.ncbi.nlm.nih.gov/nuccore/NC_000866.4 PMID: 8670851 (there is no DOI) dcmp_hm;bgt PF21448 dnmp_model.html Deoxynucleoside monophosphate kinase (DNK) was functionally validated as an active kinase through structural, biochemical, and comparative analyses. DNK was overexpressed, purified, and crystallized in both binary (DNK–dGMP) and ternary (DNK–dGMP–ATP) complexes, enabling direct observation of substrate binding (ECO_0001823). Crystallographic data revealed well-defined interactions between DNK and dGMP, including specific hydrogen bonds at the base, sugar, and phosphate groups, confirming nucleotide recognition. ATP binding was also structurally resolved, and although β- and γ-phosphates were disordered, modeling based on homologous structures indicated a catalytically competent P-loop conformation. The arrangement of active site residues and the requirement for domain closure to bring phosphates into proximity supported a mechanism of phosphoryl transfer. Furthermore, DNK specificity for dGMP, dTMP, and hmdCMP was supported by kinetic studies and structure-based rationalization of hydrogen bond patterns that exclude non-substrate nucleotides. Mutation data cited (e.g., His206Q inactivation) corroborated the functional importance of residues involved in catalysis. Involved in modification of 5-hydroxymethyl-dCMP. MKLIFLSGVKRSGKDTTADFIMSNYSAVKYQLAGPIKDALAYAWGVFAANTDYPCLTRKEFEGIDYDRETNLNLTKLEVITIMEQAFCYLNGKSPIKGVFVFDDEGKESVNFVAFNKITDVINNIEDQWSVRRLMQALGTDLIVNNFDRMYWVKLFALDYLDKFNSGYDYYIVPDTRQDHEMDAARAMGATVIHVVRPGQKSNDTHITEAGLPIRDGDLVITNDGSLEELFSKIKNTLKVL