Bacterial strains and growth conditions
Table of Contents
The bacterial strains used in this study are listed in Supplementary Table 1. S. aureus strain RN422025 was grown at 37 °C with shaking (220 RPM) in brain heart infusion (BHI) broth, supplemented with chloramphenicol (10 μg ml−1) or erythromycin (10 μg ml−1) to maintain pC194-based24 or pE194-based plasmids40, respectively. Cultures were supplemented with chloramphenicol (5 μg ml−1) to select for strains with chromosomally integrated Ssc-CBASS or Ssc-CdnE03. Gene expression was induced by the addition of 1 mM isopropyl-d-1-thiogalactopyranoside (IPTG) or 100 ng ml−1 ATC, where appropriate.
The bacteriophages used in this study are listed in Supplementary Table 2. To generate a high-titre phage stock, an overnight culture of S. aureus RN4220 was diluted 1:100 and outgrown to mid-log phase (~90 min) in BHI broth supplemented with 5 mM CaCl2. The culture was diluted to an OD600 of 0.5 (~1 × 108 CFU ml−1). The culture was infected by adding phage at a MOI of 0.1 (~1 × 107 PFU ml−1), or by inoculating with either a single picked plaque or scrape of a frozen stock. The infected culture was grown at 37 °C with shaking and monitored for lysis (full loss of turbidity was typically observed at ~3–4 h). Culture lysates were centrifugated (4,300g for 10 min) to pellet cellular debris. The supernatant was collected, passed through a sterile membrane filter (0.45 μm), and stored at 4 °C. Phage concentrations were determined by serially diluting the obtained stock in tenfold increments and spotting 5 μl of each dilution on BHI soft agar mixed with RN4220 and supplemented with 5 mM CaCl2. After incubation overnight at 37 °C, individual plaques (that is, zones of no bacterial growth) were counted, and the viral titre was calculated.
The plasmids (and details of their construction) and the oligonucleotide primers used in this study are listed in Supplementary Tables 3 and 4, respectively. The coding sequences of Ssc-CBASS and phage gene products were obtained from genomic DNA preparations of S. schleiferi 2142-05 cultures22 or phage stocks41, respectively.
Chromosomal integration of Ssc-CBASS
Ssc-CBASS or Ssc-CdnE03, along with a chloramphenicol resistance (cmR) cassette, was integrated into the hsdR gene (which encodes the defective R-subunit of the restriction-modification system in S. aureus RN4220), an insertion site which was previously shown to not impact growth42. Ssc-CBASS-cmR and Ssc-CdnE03-cmR were amplified from the plasmids pDVB303 and pDVB301 respectively, using primers oDVB565 and oDVB566, which were flanked with loxP sites at both ends followed by 60-bp homology regions to hsdR. Electrocompetent S. aureus RN4220 cells harbouring the recombineering plasmid pPM300 were electroporated with 1−2 μg of PCR product and selected for with chloramphenicol (5 μg ml−1). Potential integrants were screened by colony PCR as well as for functional immunity, and then verified by Sanger sequencing.
Isolation of strictly lytic phage mutants
To construct a virulent mutant of the phage Φ80α26, we used a variation of a method previously described to generate ΦNM1γ627, ΦNM4γ428 and Φ12γ329. Φ80α-vir was isolated as a spontaneous escaper forming a clear plaque following Φ80α infection of a BHI soft-agar lawn of S. aureus RN4220 cells harbouring plasmid pDVB08, which encodes a type III-A CRISPR–Cas system targeting the Φ80α cI-like repressor. PCR of the Φ80α-vir cI gene and Sanger sequencing confirmed an 8-bp deletion.
Isolation of ΦJ1, ΦJ2, and ΦJ4
S. aureus strains NRS52, NRS102, and NRS110 from the Network on Antimicrobial Resistance in S. aureus (NARSA) repository (BEI/NIAID) were grown overnight at 37 °C with shaking (200 RPM) in Mueller Hinton II (MHII) broth. The next day, cultures were diluted 1:100 into 10 ml fresh MHII and grown for one hour to enter early log phase. Prophages were then induced by adding ciprofloxacin at a final concentration of 0.8 mg ml−1 to each culture. Following a 4 h incubation at 37 °C, each culture was spun down, and the supernatants filtered through a 0.22 μm syringe-driven filter. Singles plaques of these filtrates were obtained via serial dilution onto lawns of S. aureus RN4220, and high-titre phage stocks were produced as described above.
Soft agar phage infection
One-hundred microlitres of an overnight bacterial culture was mixed with 5 ml BHI soft agar supplemented with 5 mM CaCl2 and poured onto BHI agar plates to solidify at room temperature (~15 min). Phage lysates were serially diluted tenfold and 4 μl was spotted onto the soft-agar surface. Once dry, plates were incubated at 37 °C overnight and visualized the next day. Individual plaques (zones of no bacterial growth) were counted manually.
Liquid culture phage infection
Overnight cultures were diluted 1:100 in BHI supplemented with 5 mM CaCl2 and the appropriate antibiotic for selection, outgrown at 37 °C with shaking to mid-log phase (~90 min), and normalized to OD600 0.5. For the desired MOI, a calculated volume of phage stock was added to each culture and 150 μl was seeded into each well of a 96-well plate. OD600 was measured every 10 min in a microplate reader (TECAN Infinite 200 PRO) at 37 °C with shaking.
Total RNA was extracted from S. aureus cells using a Direct-Zol RNA MiniPrep Plus Kit (R2072). Extracted RNA was treated with TURBO DNase (Thermo Fisher Scientific) before cDNA first-strand synthesis with SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) using random hexamers. qPCR was performed using Fast SYBR Green Master Mix (Life Technologies) and 7900HT Fast Real-Time PCR System (Applied Biosystems) with primer pairs for the S. aureus housekeeping gene ptsG (oDVB426/427), cdnE03 (oDVB610/611) or cap15 (oDVB614/615).
Protein expression and purification
Ssc-CdnE03, Sha-CdnE01, and various mutants were expressed and purified using the following approach: transformed BL21 (DE3) E. coli were grown in LB broth at 37 °C with shaking to mid-log phase (OD600 0.6–0.8), at which point the culture was cooled on ice for 10 min and induced with 0.2 mM IPTG for 16 h at 18 °C. Bacteria were harvested, resuspended in lysis buffer (25 mM Tris pH 7.4, 300 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol), and subjected to a single freeze–thaw cycle. The cells were incubated on ice with lysozyme, DNase I, and EDTA-free protease inhibitor cocktail. After incubating on ice for 40 min, the cells were lysed using sonication. Lysates were clarified by centrifugation and applied to cobalt affinity resin. After binding, the resin was washed extensively with lysis buffer prior to elution with lysis buffer containing 300 mM imidazole. Eluted proteins were then proteolysed with TEV protease to remove the affinity tag during overnight 4 °C dialysis to reaction buffer (25 mM HEPES-KOH pH 7.5, 250 mM KCl, 5% glycerol, 2 mM β-mercaptoethanol). The cleaved proteins were then passed over cobalt resin to collect the remaining tag (or uncleaved protein) and concentrated using 10,000 MWCO centrifugal filters (Amicon). Purified proteins were visualized by SDS–PAGE and used for downstream in vitro assays.
Nucleotide synthesis assays
Nucleotide synthesis assays were performed using a variation of the method described by Whiteley et al.8. The final reactions (50 mM 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) pH 9.4, 50 mM KCl, 5 mM magnesium acetate, 1 mM DTT, 25 or 250 uM individual NTPs, trace amounts of [α-32P]NTP, 5 uM nucleic acid ligand, and 5 μM enzyme) were started with the addition of enzyme. All reactions except for those with RNA activator (2 h) were incubated overnight at 37 °C. For reactions with total RNA extracts, 500 ng was added to each condition. The sequences of the ssRNA oligonucleotides used as activators are reported in Supplementary Table 5. Reactions were stopped with the addition of 1 U of alkaline phosphatase, which removes triphosphates on the remaining NTPs and enables the visualization of cyclized nucleotide species. After a 1 h incubation, 0.5 μl of the reaction was spotted 1.5 cm from the bottom of a PEI-cellulose thin-layer chromatography (TLC) plate, spaced 0.8 cm apart. TLC plates were developed in 1.5 M KH2PO4 pH 3.8 until the buffer front reached 1 cm from the top (~ 12 cm). The TLC plates were completely dried, covered with plastic wrap and exposed to a phosphor screen before detection by a Typhoon Trio Imager System.
For the putative activator screening in vitro, RN4220 cells were lysed using lysostaphin (5 mg ml−1) treatment at 37 °C for 1 h, clarified lysate was then added to nucleotide synthesis reactions. Phage particles were enriched using polyethylene glycol (PEG8000) precipitation. Resuspended phage was then treated with DNase and RNase to ensure that only phage structural elements remained. Genomic DNA from RN4220 was prepared according to the Wizard Genomic DNA Purification Kit (A1120). Genomic DNA of phage was isolated following phage particles purification, using polyethylene glycol precipitation and CsCl gradient, according to the manufacturer’s protocol. Total RNA was extracted from S. aureus cells with or without infection using a Direct-Zol RNA MiniPrep Plus Kit (R2072).
To purify the Ssc-CdnE03 cyclic nucleotide product for mass spectrometry analysis, nucleotide synthesis reaction conditions were scaled up to 1 ml reactions containing 5 μM Ssc-CdnE03, 250 uM ATP, 250 uM GTP, approximately 5 ng of cabRNA, in 50 mM CAPSO pH 9.4, 50 mM KCl, 5 mM Mg(OAc)2, 1 mM DTT buffer. Reactions were incubated with gentle shaking for 24 h at 37 °C followed by Quick CIP (NEB) treatment for 4 h at 37 °C. Following incubation, reactions were filtered through a 10,000 MWCO centrifugal filter (Amicon) to remove protein.
Nucleotide high-resolution mass spectrometry analysis
All solvents and reagents used for chromatography were liquid chromatography–mass spectrometry grade. Ultrahigh performance liquid chromatography–high-resolution mass spectrometry (UPLC-HRMS) data were acquired on a Sciex ExcionLC UPLC coupled to an X500R mass spectrometer, controlled by SCIEXOS software. Chromatography was carried out on a Waters XBridge BEH Amide Column XP (2.1 × 150 mm, 2.5 µm), under the following conditions: 100% B from 0.0 to 1.0 min, from 100% to 70% B from 1.0 to 8.9 min, 60% B from 9.0 to 13.0 min, 100% B from 13.2 to 20.0 (A: 10 mM ammonium formate + 0.1% formic acid; B: 90% acetonitrile in 10 mM ammonium formate + 0.1% formic acid buffer), with a flow rate of 0.40 ml min−1 and 0.5 µl of injection volume. HRMS analysis were performed in positive and negative electrospray ionization mode in the range m/z 100–1,200 for MS1 and MS2 scans; the maximum candidate ions subjected for Q2-MS2 experiments was 7, declustering potential of 80 V, collision energy of 5 V and temperature of 500 °C. For electrospray ionozation high-resolution mass spectrometry (ESI–HRMS) experiments the spray voltage was set in 5,500 V, the Q2 collision energy at 30 V with a spread of 10 V, whereas the spray voltage for ESI–HRMS was set in 4500 V and the Q2 collision energy at 35 V with a spread of 10 V. The concentration of the standard solutions was 6.25 µM, and all the solutions were centrifuged (13,000 rpm × 3 min) before injection. The molecular ions for ESI modes were analysed for all compounds, but the fragmentation in ESI mode showed a better consistency and was consequently used for the structural analysis. The data analysis was carried out with MestReNova software (14.3.0), data output was converted with MSConvert from Proteowizard, MS2 mirror plot was obtained from GNPS using averaged MS2 spectra from GNPS molecular networking.
Nuclease P1 cleavage analysis
Nuclease P1 cleavage analysis was performed using Ssc-CdnE03 reactions labelled with α-32P-ATP or α-32P-GTP. Radiolabelled nucleotide products were incubated with nuclease P1 (80mU; N8630, Sigma) in buffer (30 mM sodium acetate pH 5.3, 5 mM ZnSO4, and 50 mM NaCl) for 30 min at 37 °C in the presence of Quick CIP (NEB). Reactions were terminated by heat inactivation at 95 °C for 2 min before PEI-cellulose TLC analysis as described above.
RNA extraction from phage infection
Ten millilitres of a mid-log phase S. aureus RN4220 culture normalized to OD600 0.5 was infected with phage at MOI 10. Infection was allowed to proceed for 30 min, just before the completion of the first burst. Cells were pelleted at 4,300g for 5 min and flash-frozen with liquid nitrogen. The pellet was resuspended in 150 μl PBS and 50 μl lysostaphin (5 mg ml−1) and incubated at 37 °C for 30 min. Total RNA was extracted from S. aureus cells using a Direct-Zol RNA MiniPrep Plus Kit (R2072). In brief, 450 μl Trizol was added to the lysate, vigorously vortexed and centrifuged at 16,000g for 30 s. In total, 650 μl of 100% ethanol was added to the supernatant and the samples were thoroughly vortexed. The entire volume was passed through a Zymo-Spin IIICG Column followed by in-column treatment with DNase I for 15 min at room temperature. The column was washed according to the manufacturer’s protocol and RNA was eluted in 100 μl nuclease-free water.
RNA pull-down assay
His6–MBP-tagged or His6-tagged Ssc-CdnE03 as well as Sha-CdnE01 were expressed and purified as described above. Purified His6–MBP tag alone was prepared alongside as a negative control. After immobilizing ~0.2 mg of protein on cobalt resin, the column was washed extensively with lysis buffer prior to the addition of 5 ml of lysis buffer containing 1 mM MgCl2, 5 units of RNaseOUT (ThermoFisher, 10777019), and 100 μg of total RNA extracted from cultures with or without phage infection. The RNA was incubated with the tagged Ssc-CdnE03 on the column for 40 min before washing the column with 5 volumes of lysis buffer. The column was treated with His6-tagged TEV protease to release the Ssc-CdnE03 and bound RNA. Eluted protein was collected for each sample and combined with TRI Reagent (Zymo Research, R2050-1-200). RNA was then extracted according to the Direct-Zol RNA MiniPrep Plus Kit (R2072) manufacturer’s protocol. The final RNA product was run on a 2% agarose 1× TAE gel and stained with SyBr Gold or ethidium bromide. Eluted protein samples were collected as controls for visualization by SDS–PAGE.
cDNA library preparation was performed using the Illumina TruSeq Stranded mRNA (for >100 nt) or Small RNA (for <100 nt) library preparation kits. In brief, reverse transcription of the RNA isolated from the CD-NTase pull-down assays was performed using the Illumina manufacturer’s protocol, or alternatively as follows: RNA was treated with TURBO DNase (Thermo Fisher Scientific) before cDNA first-strand synthesis with SuperScript IV Reverse Transcriptase using random hexamers. Second-strand synthesis of the cDNA was performed with Q5 DNA polymerase at 15 °C for 2 h, followed by 75 °C for 10 min in the presence of RNase H and DMSO. Chemical fragmentation was then performed using the Illumina manufacturer’s protocol, or alternatively as follows: cDNA was sheared to 150-bp fragments using an S220 Covaris Focused-Ultrasonicator (peak incident power: 175 W, duty factor: 10%, cycles per burst: 200, treatment time: 430 s, temperature 4 °C) in S-Series Holder microTUBEs (PN 500114). Quantification and quality check of cDNA libraries were performed by Qubit 4.0 Fluorometer and Agilent Bioanalyzer/Tapestation, respectively. 12 pM of indexed cDNA libraries was loaded on an Illumina MiSeq instrument for either single-read (150 cycle) or paired-end sequencing (2 ×75 cycle). Bowtie2 via the Galaxy open-source interface43 was used to align sequencing reads to phage and host genomes and then visualized using Geneious Prime. A custom Python script was used to convert the output SAM alignments into CSV files containing the number of aligned reads at each nucleotide location along a given reference genome.
RNA structure prediction
RNA secondary structures were analysed using the ViennaRNA 2.0 package31 and visualized via the SnapGene interface.
In vitro transcription of cabRNA
IVT was performed according to the Thermo Scientific TranscriptAid T7 High Yield Transcription Kit protocol (K0441). Linear dsDNA for the cabRNA, and terSS74F phage escaper RNA sequences were PCR-amplified using oCR190/193 (sense cabRNA), oCR191/192 (antisense cabRNA), and oDVB691/oCR193 (terSS74F phage escaper RNA). The target sequence was placed downstream of a T7 promoter, which was inverted for antisense transcription reactions. For high yield in vitro transcription reactions, 1 μg of PCR product was combined with TranscriptAid Enzyme mix and NTPs. Following a 4 h incubation period at 37 °C, transcripts were purified according to the Direct-Zol RNA MiniPrep Plus Kit (R2072) manufacturer’s protocol. To stimulate the refolding and formation of a structured RNA product, the purified IVT samples were heated at 95 °C for 5 min in a heat block, which was slowly cooled down to room temperature over 1 h. Where indicated, IVT products were either heat-treated (folded) or untreated.
Electrophoretic mobility shift assay
Analysis of in vitro protein-nucleic acid complex formation was performed as previously described18. 1 μM cabRNA or escaper RNA was incubated with Ssc-CdnE03 at a concentration of 0, 1, or 10 μM. Complex formation was performed in the reaction buffer: 50 mM CAPSO pH 9.4, 50 mM KCl, 5 mM magnesium acetate, 1 mM DTT. Reactions (20 μl) were incubated at 4 °C for 25 min before separation on a 2% agarose gel using 1× TB buffer as running buffer. The agarose gel was stained with ethidium bromide and complex formation was visualized using an Amersham ImageQuant 800 (Cytiva). Fraction of RNA bound in each sample was calculated by dividing the mean intensity of the shifted band over the sum of the mean intensity for the shifted and unshift bands. Mean intensity of signal was generated using Fiji (measure tool).
In vivo Ssc-Cap15 activation assay
Overnight cultures of S. aureus RN4220 harbouring either Ssc-CdnE03 alone or the full Ssc-CBASS operon were diluted 1:100 in BHI supplemented with 5 mM CaCl2, 10 μM propidium iodide (PI), and the appropriate antibiotic for selection, outgrown at 37 °C with shaking to mid-log phase (~90 min), and then normalized to OD600 0.5. Cells were then infected with phage at MOI 10 and allowed for grow at 37 °C for another 10 min before collection (4,000 rpm, 5 min), and resuspension of 2 × 108 cells in 200 μl 1× PBS. Upon binding to DNA or RNA in cells propidium iodide fluorescence is enhanced 20 to 30-fold Excitationmax = 540 nm/Emissionmax = 617 nm, a process which requires disruption of the bacterial membrane. Thus, cGAMP-induced membrane disruption by Ssc-Cap15 was measured by adding cells to a 96-well plate and using a multi-well fluorescence scanner to report emission in each well at 615 nm. Experiments were performed with six independent clones (biological replicates), each with three technical replicates.
Structural prediction and analysis of Ssc-CdnE03
The amino acid sequence of Ssc-CdnE03 sequence was used to seed a position-specific iterative BLAST (PSI-BLAST) search of the NCBI non-redundant protein and conserved domain databases (composition-based adjustment, E-value threshold 0.01). Putative domains identified from this search include a C-terminal nucleotidyltransferase (NT) domain of 2′,5′-oligoadenylate (2–5 A) synthetase (NT_2-5OAS) domain (residues 61–204; E-value 2.63 × 10−15) and an N-terminal tRNA nucleotidyltransferase (CCA-adding enzyme) domain (residues 5–158; E-value 8.01 × 10−4). A structure of the Ssc-CdnE03 was predicted using AlphaFold (ColabFold). Following structure determination, pairwise structural comparison of the rank 1 model to the full PDB database was performed using DALI. The ConSurf database was used to visualize conserved structural features of the Ssc-CdnE03. Structural alignments and generation of surface electrostatics with apo-OAS1 (PDB:4RWQ) and OAS1:dsRNA (PDB:4RWO) were performed using PyMOL.
Generation of GFP-tagged Φ80α-vir
Wild-type Φ80α-vir was passaged on a liquid culture of S. aureus RN4220 harbouring a plasmid (pDVB434) encoding the gfp gene flanked by 500-nucleotide upstream and downstream homology arms corresponding to Φ80α gp18 and gp19, respectively. To isolate individual plaques, the lysed culture supernatant spotted onto a lawn of RN4220 harbouring a type II-A Sau CRISPR–Cas targeting plasmid (pDVB435) in BHI soft agar for counter-selection against wild-type phage and enrichment of Φ80α-vir::GFP. The gp18 and gp19 genes were amplified by PCR and the gfp insertion was confirmed by Sanger sequencing.
Time-lapse fluorescence microscopy
S. aureus cells harbouring Ssc-CBASS or lacking Cap15 were loaded onto microfluidic chambers using the CellASIC ONIX2 microfluidic system. After cells became trapped in the chamber, they were supplied with BHI medium with 5 mM CaCl2 and 10 μM propidium iodide under a constant flow of 5 μl h−1. After 1 h, GFP-tagged Φ80α-vir was flowed through the chambers for 1 h, before switching back to growth medium. Phase contrast images were captured at 1,000× magnification every 2 min using a Nikon Ti2e inverted microscope equipped with a Hamamatsu Orca-Fusion SCMOS camera and the temperature-controlled enclosure set to 37 °C. GFP was imaged using a GFP filter set and propidium iodide stain with DSRed filter set, both using an Excelitas Xylis LED Illuminator set to 2% power, with an exposure time of 300 ms. Images were aligned and processed using the NIS Elements software. Further downstream analysis of images was performed with Fiji v.2.3.0.
Generation and isolation of escaper bacteriophages
Overnight cultures of S. aureus RN4220 were diluted 1:100 and outgrown at 37 °C with shaking for 1 h, infected with Φ80α-vir (MOI 1) for 20 min, and then treated with 1% EMS, a chemical mutagen. Cultures were allowed to lyse for 3 h before pelleting debris and sterile-filtering the supernatant to obtain an EMS-treated mutant phage library. One-hundred microlitres of RN4220 overnight cultures harbouring Ssc-CBASS were infected with a high-titre mutant phage library in BHI soft agar and then plated. After incubating at 37 °C overnight, individual phage plaques were picked from the top agar and resuspended in 50 μl of BHI liquid medium. Phage lysates were further purified over two rounds of passaging on RN4220 harbouring Ssc-CBASS.
Whole-genome sequencing and analysis
Genomic DNA from high-titre phage stocks was extracted using a previously described method41. DNA was sheared to 300-bp fragments using an S220 Covaris Focused-Ultrasonicator (peak incident power: 140 W, duty factor: 10%, cycles per burst: 200, treatment time: 80 s, temperature 4 °C) in S-Series Holder microTUBEs (PN 500114). Library preparation was performed using the Illumina Nextera XT DNA Library Preparation Kit protocol (FC-131-1096). 12 pM of the library was loaded on an Illumina MiSeq instrument for paired-end sequencing (2 × 150 cycles). Bowtie2 via the Galaxy open-source interface43 was used to align sequencing reads to phage and host genomes. A custom Python script was used to convert the output SAM alignments into CSV files.
Generation of recombinant ΦNM1γ6 terS
Wild-type ΦNM1γ6 was passaged on S. aureus RN4220 harbouring Ssc-CBASS and pTerS or pTerSS74F to enable recombination. The infected culture supernatant was spotted onto a lawn of RN4220 with Ssc-CBASS in BHI soft agar to isolate individual escaper plaques. The terS gene was amplified by PCR and the S74F mutation was confirmed by Sanger sequencing.
Generation of recoded Φ80α-vir cabRNA mutants
Wild-type Φ80α-vir was passaged on a liquid culture of S. aureus RN4220 harbouring a plasmid (pDVB442, pDVB443, or pDVB460) encoding a mutant cabRNA sequence with silent transition mutations at the wobble positions of each codon and flanking homology arms. To isolate individual plaques, the lysed culture supernatant spotted onto a lawn of RN4220 harbouring a type II-A Sau CRISPR–Cas targeting plasmid (pDVB444) in BHI soft agar for counter-selection against wild-type phage and enrichment of the recoded mutants. The terS and terL genes were amplified by PCR and the recombined phage mutants was confirmed by Sanger sequencing.
Northern blot analysis
RNA was extracted from S. aureus RN4220 cells, with or without infection by Φ80α-vir, according to the Direct-Zol RNA MiniPrep Plus Kit (R2072) manufacturer’s protocol. RNA samples were diluted in an equal volume of sample buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.3, 8 M Urea, 10% sucrose, 0.05% bromophenol blue, and 0.05% xylene cyanol) and denatured by heating at 65 °C for 15 min, followed by chilling on ice. Denatured samples were separated by 12% polyacrylamide-8 M urea denaturing gel electrophoresis at 250 V in 0.5× TBE buffer (45 mM Tris, 45 mM borate, and 1 mM EDTA, pH 8.0). For blotting, separated RNA was transferred onto BrightStar-Plus positively charged nylon membrane (ThermoFisher, AM10100) by semidry electroblotting in molecular grade water at 300 mA for 60 min. After EDC crosslinking, the membrane was blocked in 6× SSC and 7% SDS in a 65 °C oven for 1 h. The membrane was then incubated overnight at 42 °C with PCR-generated double-stranded DNA probes against the cabRNA labelled with fluorescein. After washing with 0.1% SDS in 3× SSC 3 times for 10 min at 42 °C the blots were imaged using a Typhoon Trio Imager System for detection of fluorescein signal.
Phylogenetic analysis of CD-NTase sequences
The CD-NTases from S. schleiferi are most similar to the CdnE subtype 03 (CdnE03) described by Whiteley et al.8. All CD-NTase enzymes were aligned using TCoffee Multiple Sequence Alignment tool (default parameters) and used to construct a phylogenetic tree with Geneious Prime using the neighbour-joining method and Jukes–Cantor genetic distance model with no outgroup.
All statistical analyses were performed using GraphPad Prism v9.5.1. Error bars and number of replicates for each experiment are defined in the figure legends. Comparisons between groups for viral titre, gene expression, colony-forming units, and signalling intensity were analysed by unpaired parametric t-test, two-tailed with no corrections. Comparisons of signal intensities from phosphor screen images were quantified using Fiji v2.3.
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