Bacteriophages suppress CRISPR–Cas immunity using RNA-based anti-CRISPRs

Bacterial stress and development conditions

The bacterial stress utilized in this research study are noted in Supplementary Table2 Unless otherwise kept in mind, the P. atrosepticum, P. aeruginosa (PAscm, pa14 and pao1) and E. coli stress were consistently grown at 25 ° C, 30 ° C and 37 ° C, respectively, in lysogeny broth (POUND) shaken at 180 rpm or on pound– agar (LBA) plates consisting of 1.5% (w/v) agar. When suitable, supplements and prescription antibiotics were included at the following concentrations: ampicillin, 100 µg ml ^{− 1}; chloramphenicol (Cm), 25 µg ml ^{− 1}; kanamycin, 50 µg ml ^{− 1}; gentamicin, 50 µg ml ^{− 1} for P. aeruginosa or 15 µg ml ^{− 1} for E. coli; tetracycline (Tc), 5 µg ml ^{− 1}; 5-aminolevulinic acid (ALA), 50 µg ml ^{− 1}; isopropyl β-D-1-thiogalactopyranoside (IPTG), 100 µM for P. atrosepticum or 1 mM for PAO1; l– arabinose (Ara), 0.3% (w/v). Bacterial development was determined as the optical density at 600 nm (OD ₆₀₀) utilizing a Jenway 6300 Spectrophotometer.

Phage filtration and titration

The phages utilized in this research study are noted in Supplementary Table2 In short, 2 ml of over night host culture was inoculated into 50 ml pound in a 250 ml flask and bred for 30 minutes. 100 µl of phage lysate was included to the culture and bred over night. A centrifugation action was done (3,220 g for 20 minutes at 4 ° C) to separate the virions from the cell particles. The supernatant was put in a sterilized universal container for storage and a couple of drops of NaCO ₃– filled chloroform were included before completely vortexing the mix to lyse any staying cells. The phage titre was figured out by pipetting 20-μl drops of serial dilutions of the phage stock in phage buffer (10 mM Tris-HCl, pH 7.4, 10 mM MgSO 4, 0.01%( w/v) gelatin) onto an LBA overlay (0.35% w/v) seeded with 100 μl host over night culture. Plaques were counted after incubation over night, with the phage titre represented as PFU per ml. Pseudomonas phages DMS3m and JBD30 were propagated on PA14 ΔCRISPR, wild-type PAO1 or PAsmc Δ cas3 Pectobacterium phage ΦTE was propagated on wild-type P. atrosepticum _Pseudomonas phages were saved at 4 ° C in SM buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 8 mM Mg ₂ SO 4) over chloroform.

Pectobacterium

phage ΦTE was saved at 4 ° C in phage buffer over chloroform.3 DNA seclusion and adjustment The oligonucleotides utilized in this research study are noted in Supplementary Table The polymerases, constraint enzymes, Gibson Assembly mix, USER enzyme and T4 ligase were acquired from New England Biolabs or Thermo Fisher Scientific. DNA from PCRs and agarose gels was cleansed utilizing the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) or QIAEX II Gel Extraction Kit (Qiagen). Limitation digests, ligations and E4 coli changes were done utilizing basic strategies. Plasmid DNA was drawn out from over night cultures utilizing the Zyppy Plasmid Miniprep Kit (Zymo Research) or QIAprep Spin Miniprep Kit (Qiagen) and validated by DNA sequencing. Plasmids and their building and construction information are noted in Supplementary Table Plasmids were presented into P. atrosepticum and P.

aeruginosa

stress by electroporation utilizing basic strategies.9a Selection and cloning of Racr prospects10a–c Candidate Racrs were selected on the basis of their resemblance of series and secondary RNA structure to the appropriate CRISPR repeats in the design system (MAFFT positionings, FastTree roughly maximum-likelihood phylogenetic trees; Extended Data Figs. _and ) and the existence of a promoter series within 250 bp upstream of the Racr prospect (promoter forecast utilizing Bprom and manual curation). The Racr prospects were manufactured as gene pieces consisting of flanking areas (Twist Biosciences) under the control of either the anticipated wild-type promoter or P^{50 BAD51} (Ara-inducible) from the anticipated transcription start website (TSS). RacrIF1 variations were cloned through PCR with mismatched guides and overlap PCR. For alternative 3, a hammerhead ribozyme was presented for Cas6f-independent processing1,

RacrIF1 was then cloned downstream of BioBrick constitutive promoters of various strength (BBa_J23110, bba_j23100 and bba_j23112) to examine dosage responsiveness. In-depth info on the prospect Racrs is noted in Supplementary Table

.1d Expression of Racr prospects and associated constructs2b,d For the experiments provided in Fig. 4c,d,f and Extended Data Figs. and , RacrIF1 and its variations, hybrid and canonical crRNAs and the separated RacrIF1 repeat, were revealed in P.NC_018012.1 atrosepticum _{from a plasmid with a p15A origin of duplication (copy variety of around 10) either under the control of its wild-type promoter (}: 4,787,341– 4,787,695 bp) or with the anticipated TSS downstream of the PNC_018012.1 BAD1f promoter (4c,d: 4,787,535– 4,787,695 bp) (Extended Data Fig. NC_018012.1). For the titration showed in Extended Data Fig. _{, RacrIF1 (}: 4,787,535– 4,787,695 bp) was revealed from the P3f,g BAD promoter under various Ara concentrations or the BioBrick constitutive promoters. For the experiments displayed in Fig. , Racr prospects evaluated in PAO1 or PA14 were revealed from the _{Escherichia– Pseudomonas} (ColE1-pRO1600) shuttle bus vector pHERD30T with their anticipated TSS downstream of the P1e BAD3e promoter. RacrIF1 (experiment in Extended Data Fig. 9b) and RacrIC1 (experiment in Fig. and Extended Data Fig. ) were cloned with the anticipated wild-type promoter for the 5 ′ RACE assay. In Pseudomonas stress, pHERD30T duplicates from the P.^{52 aeruginosa53} plasmid pRO1600 oriV and duplication protein (copy variety of around 13)

,

Phage-resistance assay Triplicate cultures of hosts bring either a phage-targeting spacer (+ CRISPR) or a non-targeting control (– CRISPR), and consisting of a plasmid revealing a prospect Racr or an empty-vector control (EV), were grown overnight in 5 ml pound supplemented with the suitable prescription antibiotics and inducers. For P. _atrosepticum, a soft LBA overlay (0.35% w/v) consisting of 100 μl of the over night cultures was put onto an LBA plate supplemented with the matching prescription antibiotics and inducers. For PAO1, pasmc and pa14, a soft LBA overlay (0.5% w/v) consisting of 150 μl of the over night cultures and supplemented with 10 mM MgSO ₄ was put onto an LBA plate supplemented with 10 mM MgSO ⁴ and the matching prescription antibiotics and inducers. Phage titres were figured out by pipetting 2.5 μl (or 5 μl for ΦTE) drops of serial dilutions of phage stock (roughly 10 10 PFU per ml) in phage buffer onto the agar overlay and plates were bred overnight. Plaques were counted after incubation over night, with the phage titre represented as PFU per ml. One plaque was counted in the very first dilution in which no plaques were noticeable when plaques were too little to count. Type I-F Racr prospects were evaluated in P.1d atrosepticum3g PCF610 bring the ΦTE targeting plasmid pPF1423 (for assays in Figs. 2b, 4d and Extended Data Figs. and ) and P.2d atrosepticum4c,f PCF188 (for assays in Extended Data Figs. and ) with the phage ΦTE, and P. aeruginosa PA14 with the phage DMS3m. Type I-E Racr prospects were evaluated in PAsmc, type V-A Racrs in PAO1:: MbCpf1:: crRNA24 (PAO1:: V-A) and type I-C Racrs in PAO1 tagged with a I-C CRISPR– Cas system (PAO1:: I-C), all of which were contaminated with the phage JBD30. The particular non-targeting (– CRISPR) control stress were P. atrosepticum PCF610 with the non-targeting plasmid pPF975 or wild-type P. atrosepticum, PAscm Δ

cas3

and wild-type PAO1.1e Conjugation-efficiency assay⁵⁴ For the experiment displayed in Fig. , conjugation performance was evaluated in a comparable way to that explained formerly E. coli ST18 was the donor for the conjugation of the untargeted control (– CRISPR, pPF953) and type I-F (+ CRISPR, pPF954) targeted plasmids. Plasmid pPF954 consists of a protospacer targeted by spacer 1 from CRISPR1 (type I-F) and the canonical GG PAM. Receivers were wild-type P. _atrosepticum that have either a plasmid revealing RacrIF1 from the P _BAD promoter (+ RacrIF1, pPF2846) or an empty-vector control (– Racr1F1, pPF781). Pressures were grown overnight in three in 5 ml pound supplemented with Cm and Ara for receivers, or 5 ml pound supplemented with Tc and ALA for donor stress. One ml of over night culture was washed and pelleted two times with pound supplemented with ALA to eliminate the prescription antibiotics. Pellets were resuspended in 0.5 ml pound supplemented with ALA and Ara, and the OD

600

was adapted to 1. Recipients and donors were blended in a 1:1 ratio, and 10 μl was found on LBA supplemented with ALA and Ara, and bred at 25 ° C for 24 h. Next, the breeding areas were scraped with a sterilized loop and resuspended in 0.5 ml PBS, and dilution series were plated either onto pound supplemented with Cm and Ara for recipient counts or with the addition of Tc for choice of transconjugant counts. Conjugation performance was computed as the ratio of transconjugants per recipient cells. _{Co-expression and filtration of Cas6f and RNA} For co-expression and filtration of Cas6f and RNA variations, plasmids pPF2644 (His ₆— Cas6f and type I-F crRNA repeat– spacer– repeat), pPF2868 (His ₆— Cas6f and RacrIF1), pPF2869 (His ⁶— Cas6f and RacrIF1 _GCmut) and pPF2640 (His 6– Cas6f alone) were changed into E. _coli LOBSTR cells. Over night cultures were utilized to inoculate 500 ml pound plus kanamycin in a 2 l baffled flask and bred at 37 ° C and 180 rpm to an OD ₆₀₀ of 0.2– 0.3, followed by incubation at 18 ° C and 180 rpm to an OD 600 of 0.6. Expression was caused with 1 mM IPTG, and proteins were revealed for 20 h at 18 ° C and 180 rpm. Cells were gathered at 10,000 ^g for 10 minutes at 4 ° C, and the pellet was resuspended in 10 ml g ^{− 1} (wet-cell mass) lysis buffer (50 mM HEPES-NaOH, pH 7.5, 300 mM KCl, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT) and 10 mM imidazole) supplemented with 0.02 mg ml ^{− 1} DNase I, one tablet cOmplete EDTA-free protease inhibitor (Roche), 0.67 mg ml − 1 lysozyme and 0.1 mM phenylmethylsulfonyl fluoride. Cells were lysed by ultrasonication and the lysate was clarified by centrifugation at 15,000

g

for 15 minutes at 4 ° C. The cleared lysate was affinity cleansed utilizing a 1 ml HisTrap ™ FF (Cytiva) column equilibrated in lysis buffer and eluted utilizing a gradient versus elution buffer (lysis buffer consisting of 500 mM imidazole). Elution portions were pooled and focused utilizing a 10 kDa Nominal Molecular Weight Limit Amicon Ultra-4 Centrifugal Filter Unit (Amicon) and filled onto a Superdex 75 Increase 10/300 GL (GE Healthcare) column equilibrated in SEC buffer (20 mM HEPES-NaOH, pH 7.5, 100 mM KCl, 5% (v/v) glycerol and 1 mM DTT). Protein concentrations were figured out utilizing a NanoDrop One Spectrophotometer (Thermo Fisher) and a Qubit Protein Assay Kit (Invitrogen). Aliquots of protein were saved at − 80 ° C. Protein samples were separated on an SDS– PAGE gel (Bolt 4 to 12%, Bis-Tris, 1,0 mm (Invitrogen)) and stained with Coomassie blue.2b Expression and filtration of type I-F Cascade _{For expression and filtration of the type I-F Cascade displayed in Fig.} , plasmids pPF1635 (Cas8f– Cas5f– Cas7f) and pPF2644 (His ₆— Cas6f and type I-F crRNA repeat– spacer– repeat) or pPF2868 (His 6— Cas6f and RacrIF1) were co-transformed into E.

coli

LOBSTR cells. Protein was revealed and cleansed as explained above with the following adjustments: lysis buffer consisted of 15 mM imidazole, elution portions were pooled and focused utilizing a 30 kDa Nominal Molecular Weight Limit Amicon Ultra-4 Centrifugal Filter Unit (Amicon) and focused samples were filled onto a HiLoad 16/600 Superdex 200 pg (GE Healthcare) column equilibrated in SEC buffer.2c RNA seclusion from protein portions _{For the experiment displayed in Fig.} , the various RNA variations were separated from the cleansed His ₆— Cas6f or type I-F complex by phenol– chloroform extraction, ethanol rainfall and dealt with on a denaturing gel consisting of 15% (v/v) 19:1 polyacrylamide, 7 M urea and 0.5 × TBE (45 mM Tris, 45 mM Boric acid, pH 8.3, 1 mM EDTA) (Novex). The gel was stained with SYBR gold (Invitrogen) and RNA was revealed utilizing the Odyssey Fc imaging system (LICOR). For samples with cleansed His

6

— Cas6f just, the quantity of protein was stabilized before RNA seclusion.1b,c Small RNA extraction and sequencing1d,g For the experiments displayed in Fig. and Extended Data Fig. , three cultures of wild-type P. _atrosepticum that have either a plasmid revealing RacrIF1 from its wild-type promoter (+ RacrIF1, pPF2845) or an empty-vector control (– RacrIF1, pPF781) were grown overnight in 5 ml pound supplemented with Cm. The over night cultures were subcultured into 25 ml pound supplemented with Cm in 250-ml flasks from a beginning OD ₆₀₀ of 0.05 and bred for 15 h as much as fixed stage while keeping track of culture development (OD 600). Next, 1 ml (in three) of each culture was centrifuged for 1 minutes at 13,000

g

The supernatant was disposed of and the pellet was resuspended in 1 ml RNAlater Stabilization Solution (Invitrogen) and saved at − 20 ° C. The little RNA portion (less than 200 nt) was drawn out utilizing the mirVana miRNA Isolation Kit according to the producer’s directions. Recurring genomic DNA was eliminated by treatment with TurboDNase (Thermo Fisher) according to the producer’s directions, and the lack of gDNA was validated by PCR. RNA concentration, stability and pureness were figured out utilizing a NanoDrop One Spectrophotometer (Thermo Fisher), a Qubit RNA High Sensitivity (Invitrogen) and an Agilent 2100 Bioanalyzer system with an RNA nano chip. Library preparation and sequencing of little RNA samples were performed by Vertis Biotechnologie (Freising). In short, the little RNA samples were very first treated with T4 polynucleotide kinase. Oligonucleotide adapters were ligated to the 5 ′ and 3 ′ ends of the RNA samples. First-strand cDNA synthesis was done utilizing M-MLV reverse transcriptase with the 3 ′ adapter as guide. The resulting cDNA was enhanced with PCR utilizing a high-fidelity DNA polymerase. The cDNA was cleansed utilizing an Agencourt AMPure XP set (Beckman Coulter Genomics) and was evaluated by capillary electrophoresis. For Illumina NextSeq sequencing, the cDNAs were pooled in roughly equimolar quantities. The cDNA swimming pool was cleansed utilizing the Agencourt AMPure XP set (Beckman Coulter Genomics) and was evaluated by capillary electrophoresis. The guides utilized for PCR amplification were developed for TruSeq sequencing according to the directions of Illumina. The NGS libraries (6 samples) were single-read sequenced on an Illumina NextSeq 500 system utilizing a read length of 75 bp at a depth of 10.2– 11.5 million checks out and were returned as series in FASTQ format.⁵⁵ RNA-seq analysis⁵⁶ Generated checks out in FASTQ format were at first processed by getting rid of adaptors and low-grade checks out utilizing Trimmomatic The quality of the checks out was evaluated utilizing FastQC v. 0.11.9 (ref. ) Processed checks out were lined up to the P.BX950851.1 atrosepticum⁵⁷ (genome accession number ⁵⁸) utilizing Bowtie 2 (ref.

) with regional criteria and the positioning was transformed to BAM format utilizing SAMtools v. 1.16.1 (ref.

). The positioning was imagined and last images were produced utilizing Geneious Prime 2022.1.1 (Dotmatics).1a,b RNA structure forecast1b The RNA structures in Fig. 2a,c and Extended Data Figs. 3d, 4e,9a, 10,⁵⁹ and ⁶⁰ were anticipated utilizing the RNAfold web server

v. 2.4.9 and imagined by RNA2Drawer

v. 6.3 and Adobe Illustrator v. 27.1e 5 ′ RACE3a To recognize the 5 ′ end of the mRNA encoding RacrIF1 (experiment displayed in Extended Data Figs. 3e and 9b,c) or RacrIC1 (experiment displayed in Fig. 3 and Extended Data Fig. 1d), 5 ′ RACE was utilized to recognize the 5 ′ end of the RNA records utilizing the template-switching enzyme from NEB. In short, RNA was drawn out from over night cultures in three (for RacrIF1, PCF610 bring an empty-vector control (pPF781) or the RacrIF1-expressing plasmid (pPF2845); for RacrIC1, POA1:: IC bring a plasmid revealing the Acr locus under wild-type promoter expression (pSC144)) utilizing the Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research). Later on, a template-switching reverse-transcription response was utilized to produce cDNAs with a universal series of option (presented by a template-switching oligonucleotide) connected to the 3 ′ end of the cDNA (the 5 ′ end of the records) (NEB). A sequence-specific reverse-transcription guide was put so that it binds in the particular Racr or crRNA series. In the 2nd action, the 5 ′ end of the records was recognized by PCR amplification with guides that bind upstream from the Racr processing website and in the template-switching oligonucleotide, respectively. Oligonucleotides utilized are noted in Supplementary Table3b,c PCR items were imagined on gels and tidied up. For RacrIF1 under wild-type promoter expression, the size of the 5 ′ RACE item was imagined on a gel and evaluated on a piece analyser (experiment displayed in Extended Data Fig. 2c), while the 5 ′ RACE item for RacrIC1 was imagined on a gel and sent out for Sanger sequencing for verification (experiment displayed in Extended Data Fig. 3b,c). 5 ′ RACE was likewise done to verify the identity of the RNA types separated by phenol/chloroform extraction and ethanol rainfall from the cleansed type I-F complex (Fig.

). PCR items were A-tailed with DreamTaq polymerase (Thermo Fisher) and dATP and cloned into pGEM-T Easy Vector (Promega). Plasmids were separated from private nests and Sanger sequenced (Extended Data Fig.

).2e CRISPR-primed adjustment assay6b The CRISPR adjustment assays displayed in Fig. ⁶¹ and Extended Data Fig. were carried out as formerly explained An ignorant plasmid control (no matching protospacer, pPF953) and strong (AG PAM alternative, pPF959) and medium (GT PAM alternative, pPF967) priming-inducing plasmids were conjugated as explained above (without Ara) into wild-type P. _atrosepticum consisting of either a plasmid revealing RacrIF1 from the P BAD promoter (pPF2846) or an empty-vector control (pPF781). The priming-inducing plasmids left targeting from the P.6a atrosepticum

type I-F CRISPR– Cas system (Extended Data Fig.

). Pressures with plasmids were grown in three for 24 h in 5 ml pound supplemented with Cm and Tc. These ‘day 0’ cultures were then utilized to inoculate (1:500 dilution) 5 ml fresh pound supplemented with Cm, IPTG and Ara (without Tc choice), and bred in the exact same conditions. This procedure was duplicated for 5 days. Aliquots of culture from each day were combined with 50% glycerol in a 1:1 ratio and frozen at − 80 ° C for future usage. CRISPR variety growth (a sign of adjustment) was evaluated by PCR utilizing the cell glycerol stocks as a design template. PCR items were filled on a 2% agarose gel comprised in 1 × salt borate buffer, run for 30 minutes at 180 V and stained with ethidium bromide.2f CRISPR-primed plasmid clearance assay³⁵ Plasmid clearance, imagined in Fig. 6c, was determined as formerly explained

Cells from the CRISPR-primed adjustment assay (glycerol stocks) were watered down in 1 mL of PBS (1:1,000) and evaluated utilizing a BD LSRFortessa Cell Analyzer (BD Biosciences). A limit was looked for FSC and SSC to discover bacterial cells. The mCherry was delighted utilizing a yellow– green laser (561 nm) and found with a 610/20 nm bandpass filter; 20,000 occasions were taped per sample utilizing BD FACSDiva Software v. 8 (BD Biosciences). Subsequent analysis was done utilizing FlowJo Software v. 10.8.1 (BD Biosciences). Cells were gated on SSC-A/SSC-H and SSC-A/FSC-A, then bifurcated (utilizing BifurGate) into mCherry+ and mCherry − populations (Extended Data Fig.

). The ratio of mCherry − cells to overall cells shows the percentage of cells that cleared the plasmid.⁴⁰ SRUFinderhttps://pypi.org/project/srufinder We developed a devoted bioinformatic algorithmhttps://anaconda.org/russel88/srufinder for discovering SRU prospects in DNA series. The algorithm is offered as a python bundle (7) and a conda bundle (https://github.com/Russel88/SRUFinder/blob/master/data/repeats.fa) and is offered at Zenodo. The algorithm is portrayed as schematics in Extended Data Fig.⁶² As questions, the algorithm utilizes a database of 17,823 non-redundant CRISPR repeat series with recognized associated subtypes (⁶³). Repeats were acquired from the CCtyper⁶⁴ web server (v. December 2020) and de-duplicated utilizing cd-hit-env⁶⁵ at 100% identity and protection. Open reading frames (ORFs) were anticipated utilizing prodigal³⁹ in meta mode, and all ORFs with self-confidence ≥ 80% were masked from the input series. Next, repeat series were lined up with BLASTn⁶⁷ versus the masked input series with job= blastn − brief and word size= 6. Matches with identity less than 90% were disposed of. Matches with protection ≥ 90% were thought about to be complete matches, whereas matches with protection in between 50% and 90% were thought about partial matches. Just the match with the greatest bit rating was kept if any positionings overlapped. All complete matches within 100 bp were clustered into selections, and these repeats were neglected as prospective SRUs. If a partial match was within 100 bp of a singular complete match, it was thought about a mini-array if the identity in between the 2 was ≥ 90% (biopython pairwise2.align.global, default match/mismatch charges, − 1 open/extend space charges, no end space charge)

The staying prospective SRUs were lined up versus the flanking 100 bp (biopython pairwise2.align.local, default match/mismatch charges, − 1 open/extend space charges). Due to the fact that BLAST was observed to miss out on recognizing repeats with numerous inequalities to the inquiry, prospect SRUs revealing partial matches (identity higher than 70%) to any of the 2 flanking areas (100 bp) were disposed of to make sure that the SRUs were really singular. The staying SRUs were then filtered by a bit rating limit of 41.1. This cut-off was set by running the algorithm on both intergenic (as explained above) and intragenic (as above, however with ORF masking reversed) on the IMG/VR3 database⁶⁸, and utilizing recursive partitioning trees (rpart 4.1– 15; ref. ³⁷) to figure out the very best cut-off for identifying prospective SRUs in intergenic areas (real prospects) from prospective SRUs in intragenic areas (most likely false-positive matches). We discovered that 84.0% of the matches with a bit rating ≥ 41.1 were from intergenic areas, compared to 23.9% of matches with any bit rating being from intergenic areas; 84.6% of matches with bit rating of less than 41.1 were from intragenic areas.⁴⁰ Bioinformatic look for SRUs in databases³⁸ Prophages were drawn out utilizing VIBRANT 1.0.1 (ref. ³⁹) from the 104,858 premium genomes from GTDB (Version r95, 2020/10/06; ref. 8a), which yielded 437,636 prophages from 69,688 of the genomes. SRUfinder⁴⁰ was then run versus these GTDB prophages, the PLSDB plasmid database (27,939 plasmid genomes⁶⁹) and the IMG/VR3 database (2,332,702 infection genomes⁴⁰). An infection dendrogram was produced from the taxonomic info supplied in the IMG/VR3 metadata and provided in Extended Data Fig.1 SRUFinder

was likewise run versus the PHASTER database (65,668 prophage and infection genomes

), however these SRUs were utilized just for discovering prospects for speculative recognition. The filtered output of SRUFinder⁶⁴ can be accessed in Supplementary Data ⁷⁰https://github.com/motu-tool/fetchMGs GTDB prophage analysis⁷¹ A phylogenetic tree of the GTDB-derived prophages consisting of SRUs was made by calling genes with prodigal⁷² and drawing out 40 single-copy marker genes⁷³ utilizing fetchMGs 1.2 (⁷⁴). Each marker gene was then lined up individually with mafft 7.310 (ref. ), the positionings were concatenated and a tree was presumed utilizing FastTree 2.1.10 (ref. ). Clades were collapsed with the collapse_tree_at_resolution function from R-package castor variation 1.7.2 (ref. ⁷⁵) at resolution 0.01 with rename_collapsed_nodes= TRUE. The tree was imagined with iTOL v. 5 (ref. ). Recognition and subtyping of cas operons in the chromosomes was finished with CCtyper 1.2.1 (ref. ). To figure out whether there was a non-random association in between the subtype of the SRU and the subtype of any cas operon in the chromosome, we to start with limited the analysis to SRUs where the host had any 8b cas2 operon (that is, IMG/VR3 hits were left out provided the absence of recognized host associations). For this subset (170 SRUs of 188 overall in GTDB prophages), 82.9% of the SRUs had a

cas operon of matching subtype in the host. When the subtype of the SRU was permuted, there was a mean association of 32.3% with a basic variance of 2.7% throughout 1,000 permutations. The information are shown in Extended Data Fig. and the positionings of SRU series from stress with CRISPR– Cas, together with their matching agreement CRISPR repeats, are offered in Supplementary Data

Association with acr genes To develop whether racr prospects were co-located with ⁷⁶ acr⁶⁵ genes, we utilized acr genes anticipated by artificial intelligence from ref. Just anticipated Acrs with a rating higher than 0.5 were thought about. Acr protein series were lined up versus all infection and plasmid genomes consisting of SRUs with tblastn v. 2.11.0+( ref. ). Just matches with E-values ≤ 0.01 were kept. Just the match with the greatest bit rating was kept if matches were overlapping. To figure out whether SRUs and 3c acr genes are genetically co-located more frequently than random, the variety of acr

and a one-tailed

P worth was computed as: $$ P= frac { acr, {{rm {within}} 1 {rm {kb}}} _ {{rm {random}}} > > acr, {{rm {within}} 1 {rm {kb}}} _ {{rm {observed}}} +1} {mathrm {1,000} +1} $$5 Statistics and reproducibility

The particular test utilized for evaluating analytical significance is shown in the figure legends. The precise

P1 worths of the analytical analyses are specified in Supplementary Table

Protein filtrations, RNA seclusions and the phage infection assay on PAO1:: I-C were separately duplicated two times. Little RNA-seq, 5 ′ RACE, conjugation performance, primed adjustment and the phage infection assay with induction of Racr expression at various ALA concentrations were carried out as soon as with 3 independent biological duplicates. All the other phage infection assays were separately duplicated a minimum of 3 times.

Data visualizationNature Portfolio Reporting Summary Unless specified otherwise, information processing and visualization were carried out in Microsoft Excel v. 16, Prism v. 9.2.0 (GraphPad), SnapGene v. 7.0.2 and Geneious Prime v. 2022.1.1, and consequently modified in Adobe Illustrator v. 27. For gel source information, see Supplementary Fig.