Bacterial cGAS senses a viral RNA to initiate immunity

Abstract

Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host^1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.

Fig. 1. A viral RNA produced during Φ80α-vir infection activates Ssc-CdnE03 in vitro.

a, Detection of phage propagation after spotting tenfold dilutions of the lytic DNA phages Φ80α-vir, ΦNM1γ6, ΦNM4γ4 and Φ12γ3 onto lawns of S. aureus RN4220 harbouring a plasmid expressing an incomplete (Ssc-CdnE03 alone) or intact Ssc-CBASS operon. b, Thin-layer chromatography analysis of Ssc-CdnE03 products in the presence of S. aureus RN4220 crude lysate, whole purified Φ80α-vir particles, host genomic DNA (RN4220 gDNA), phage genomic DNA, and total RNA from S. aureus RN4220 in the presence or absence of Φ80α-vir infection (before the completion of one lytic cycle). An agarose gel stained with ethidium bromide (middle) and SDS–PAGE stained with Coomassie blue (bottom) are shown as loading controls. P_i, free phosphates; int, intermediate cyclase product; CDN, cyclic dinucleotide. Data are representative of three independent experiments. In all main figures, size in nucleotides (nt) is with reference to a single-strand RNA (ssRNA) ladder. c, Agarose gel electrophoresis of the input and output RNA obtained after incubation of Ssc-CdnE03 with no RNA, total RNA extracted from uninfected staphylococci (RN4220) or from cells infected with Φ80α-vir phage. SDS–PAGE stained with Coomassie blue (bottom) is shown as a loading control. Data are representative of three independent experiments. d,As in c, but with input RNA extracted from staphylococci infected with ΦNM1γ6, Φ80α-vir, ΦNM4γ4 or Φ12γ3 phages. Data are representative of three independent experiments. e, Diagram of Φ80α-vir and Φ80α-vir(terS^S74F) genomes with localization of the cabRNA and escaper RNA sequences, respectively. The location of the escaper mutation, C221>T, is shown. f, As in b, but using cabRNA isolated from a pull-down assay. Data are representative of three independent experiments.

Fig. 2. Secondary structures within the cabRNA are required for Ssc-CdnE03 activation.

a, Agarose gel electrophoresis of pulled-down cabRNA treated with RNases T1 or III, or DNase (D). A ssRNA oligonucleotide was digested with RNase T1 as a control. Data are representative of three independent experiments. b, Thin-layer chromatography analysis of Ssc-CdnE03 products in the presence of total RNA extracted from infected cells and treated with RNases III, A, T1 or untreated. Data are representative of three independent experiments. c, Agarose gel electrophoresis of IVT cabRNA, unfolded (left) and folded (right), untreated or treated with RNase T1. Data are representative of three independent experiments. d, As in b, but incubating the cyclase with pulled-down, unfolded or folded IVT cabRNA.

Fig. 3. A positively charged surface within Ssc-CdnE03 binds the cabRNA to initiate immunity.

a, AlphaFold model of Ssc-CdnE03 displayed with surface electrostatics (−77 to +77, red to blue). Inset, positively charged region harbouring the mutated lysine residues 9 and 13. b, Thin-layer chromatography analysis of the products of different Ssc-CdnE03 lysine mutants in the presence of total RNA extracted from infected cells. A representative image of multiple replicates is shown. WT, wild type. c, Electrophoretic mobility shift assay of cabRNA in the presence of increasing concentrations of different Ssc-CdnE03 mutants. d, Growth of staphylococci harbouring the Ssc-CBASS operon with wild-type, K9E or K13E Ssc-CdnE03 measured by optical density at 600 nm (OD₆₀₀) after infection with Φ80α-vir at a MOI of 1. A culture expressing only Ssc-CdnE03 is used as a control. Data are mean ± s.d. of three biological replicates.

Fig. 4. Phage mutants that evade Ssc-CBASS immunity do not produce cabRNA.

a, Growth of staphylococci harbouring either an incomplete (Ssc-CdnE03 alone) or intact Ssc-CBASS operon measured by optical density at 600 nm after infection with Φ80α-vir or Φ80α-vir(terS^S74F) at a MOI of 1, the latter in the presence or absence of TerS overexpression using plasmid pTerS. Data are mean ± s.d. of three biological replicates. b, Detection of phage propagation after spotting tenfold dilutions of Φ80α-vir or Φ80α-vir(terS^S74F) onto lawns of the S. aureus RN4220 strains described in a. c, Thin-layer chromatography analysis of Ssc-CdnE03 products in the presence of total RNA extracted from uninfected staphylococci or cells infected with Φ80α-vir or Φ80α-vir(terS^S74F), the latter in the presence or absence of TerS overexpression using plasmid pTerS. An agarose gel stained with ethidium bromide (middle) and SDS–PAGE stained with Coomassie blue (bottom) are shown as loading controls. Data are representative of three independent experiments. d, Agarose gel electrophoresis of the input and output RNA obtained after incubation of Ssc-CdnE03 with total RNA extracted from uninfected staphylococci (RN4220) or from cells infected with Φ80α-vir, Φ80α-vir(gp46^E105D) or Φ80α-vir(terS^S74F), the latter in the presence or absence of TerS overexpression using plasmid pTerS. Data are representative of three independent experiments. e, As in c, but in the presence of cabRNA, escaper (esc.) RNA, or escaper RNA pre-treated with RNase III. Data are representative of three independent experiments. f, Enumeration of plaque-forming units (PFU) from cultures harbouring Ssc-CdnE03 alone (−) or Ssc-CBASS (+) and either an empty vector (−) or a plasmid with cabRNA (+) under the control of an ATC-inducible promoter. OE, overexpression. Individual data points are shown with error bars representing the mean ± s.e.m for n = 3 biological replicates.

Fig. 5. Mutations reduce the length of the cabRNA but do not eliminate its activating properties.

a, Growth of staphylococci carrying the full Ssc-CBASS locus, measured by optical density at 600 nm after the addition of Φ80α-vir mutants that contain 122, 100 or 57 mutations within the cabRNA sequence at a MOI of 1. An uninfected culture is shown as control. Data are mean ± s.d. of three biological replicates. b, Enumeration of PFU from cultures shown in a, 0.5, 1.0 and 3.0 h after infection. Data are mean ± s.e.m. of three biological replicates. c, Agarose gel electrophoresis of the RNA obtained after incubation of Ssc-CdnE03 with no RNA (−) or total RNA extracted from staphylococci infected with wild-type or cabRNA mutants of the Φ80α-vir phage. Data are representative of three independent experiments. d, Thin-layer chromatography analysis of Ssc-CdnE03 products in the absence of RNA (−) or in the presence of the IVT cabRNA (wild type), cabRNA⁵⁷ and cabRNA¹⁰⁰. A representative image of multiple replicates is shown.

Fig. 6. Diverse staphylococcal phages produce different cabRNAs to initiate the Ssc-CBASS response.

a, Detection of phage propagation after spotting tenfold dilutions of the lytic DNA phages ΦJ1, ΦJ2 and ΦJ4 onto lawns of S. aureus RN4220 harbouring a plasmid expressing an incomplete (Ssc-CdnE03 alone) or intact Ssc-CBASS operon. b, Agarose gel electrophoresis of the RNA obtained after incubation of Ssc-CdnE03 with total RNA extracted from uninfected staphylococci (−) or from cells infected with ΦJ1, ΦJ2 or ΦJ4. Data are representative of three independent experiments. c, Thin-layer chromatography analysis of Ssc-CdnE03 products in the absence of RNA (−) or in the presence the cabRNAs pulled down in the experiment shown in b. A representative image of multiple replicates is shown. d, As in c, but incubating the cyclase with synthetic oligonucleotides with the sequences of the cabRNAs produced by the ΦJ1, ΦJ2 and ΦJ4 phages.

Extended Data Fig. 1. Type I-B CBASS confers anti-bacteriophage immunity in staphylococci.

(a) Schematic of the type I-B CBASS operon present in Staphylococcus schleiferi (Ssc) 2142-05, 2317-03, and 5909-02 genomes, flanked by a type I restriction-modification system. The CBASS operon consists of two genes encoding a cyclase belonging to the E clade, cluster 3 (Ssc-CdnE03) and a two-transmembrane domain-containing effector, Cap15. (b) Detection of phage propagation after spotting ten-fold dilutions of the lytic DNA phages, Φ80α-vir, ΦNM1γ6, ΦNM4γ4, and Φ12γ3 onto lawns of S. aureus RN4220 harboring either an incomplete (Ssc-CdnE03 alone) or intact Ssc-CBASS operon integrated in its genome. (c) Enumeration of plaque-forming units (PFU) from cultures harboring Ssc-CdnE03 alone or Ssc-CBASS immediately before infection (Pre), after initial absorption of the phage (0 h), after one lytic cycle (1 h), and after complete culture lysis (3 h) by Φ80α-vir at MOI 5. Mean ± SEM of three biological replicates is reported. (d) Same as (c) but using ΦNM4γ4. Mean ± SEM of three biological replicates is reported. (e) Growth of staphylococci harboring a Ssc-CBASS operon measured by optical density at 600 nm after the addition of Φ80α-vir at a multiplicity of infection (MOI) of 0, 0.1, 1, or 10. The mean of three biological replicates ± SD is reported. (f) Same as (e) but following the growth of staphylococci expressing only the Ssc-CdnE03 cyclase. The mean of three biological replicates ± SD is reported. (g) Replicates of the infection shown in panel (e), at MOI 10. The re-growth of two of the curves at ~11 h after infection is presumably due to the selection of non-CBASS resistant bacteria, which are present at a very low frequency in the population. (h) Enumeration of colony-forming units (CFU) from cultures harboring Ssc-CdnE03 alone or Ssc-CBASS immediately before infection (Pre), after initial absorption of the phage (0 h), after one lytic cycle (1 h), and after complete culture lysis (3 h) by Φ80α-vir at MOI 5. Mean ± SEM of three biological replicates is reported. (i) Same as (h) but using ΦNM4γ4. Mean ± SEM of three biological replicates is reported. (j) Genome of the Φ80α-vir^GFP phage, showing the site of insertion of the gfp gene, the early (P_E) and late (P_L) promoters activated during the lytic cycle of the phage (green arrows), the gene inactivated to prevent the lysogenic cycle of Φ80α (cI-like repressor, red star), and the terminase subunit genes (terS/L, in red). (k) Fluorescence microscopy of staphylococci harboring Ssc-CdnE03 alone or the full Ssc-CBASS operon at different times after infection with Φ80α-vir^GFP phage, in the presence of propidium iodide. Green cells indicate a successful infection due to the expression of the viral GFP. Red cells indicate activation of the Cap15 CBASS effector, which causes membrane disruption and enables the internalization of the dye. Scale bar corresponding to 2 μm is displayed. Data are representative of three independent experiments.

Extended Data Fig. 2. Activation of Ssc-CdnE03 in vivo and in vitro.

(a) Regulation of Ssc-CBASS operon. Expression of the Ssc-CdnE03 and Ssc-Cap15 effector genes during log-phase growth of S. aureus RN4220::Ssc-CBASS in the presence or absence of infection by Φ80α-vir measured by RT-qPCR. For each condition, expression ratios were determined by normalizing Cq values for Ssc-CBASS genes to Cq values for the housekeeping gene glcC. The mean of three biological replicates ± SEM is reported. No significant differences were observed between values obtained in the presence or absence of phage, suggesting that there is not transcriptional regulation of the Ssc-CBASS operon upon infection. (b) Growth of staphylococci harboring an overexpression plasmid containing either the Ssc-CdnE03 alone, Ssc-Cap15 alone, or the intact Ssc-CBASS operon under the transcriptional control of a P-spac promoter, measured by optical density at 600 nm after the addition of IPTG. The mean of three biological replicates ± SD is reported. (c) Growth of staphylococci harboring either an incomplete (Ssc-CdnE03 alone) or intact Ssc-CBASS operon, with either wild-type or D86A,D88A mutant Ssc-CdnE03, measured by optical density at 600 nm after the addition of Φ80α-vir at a MOI of 1. The mean of three biological replicates ± SD is reported. (d) Thin-layer chromatography analysis of Ssc-CdnE03 products in the presence of total RNA from S. aureus RN4220 after Φ80α-vir infection, using different radiolabeled nucleotides to investigate the nucleotide composition of the enzymatic product. Pi, free phosphates; int, intermediate cyclase product; cdn, cyclic dinucleotide. Data are representative of three independent experiments. (e) TIC analysis of the reaction products of wild-type Ssc-CdnE03. The retention time of the peak corresponding to the cyclase products coincides with the retention time of the 3’,2’-cGAMP. This peak is not present in the reaction products of the active site mutant cyclase, D86A-D88A. Peaks for other cyclic dinucleotides that contain adenine and guanosine, which do not overlap with 3’,2’ cGAMP, are shown. (f) Comparison of averaged MS/MS spectra of the reaction products of wild-type Ssc-CdnE03 (purple spectrum) and 3’,2’-cGAMP (green spectrum). The most abundant ions are present in both samples (see Supplementary Text for a complete MS analysis). (g) Treatment of the Ssc-CdnE03 reaction products with P1 nuclease. As shown in (d), after treatment of the Ssc-CdnE03 reaction products with calf intestinal phosphatase (CIP), an intermediate species is observed when ATP, but not when GTP, is radiolabeled (“-”). Given that the Ssc-CdnE03 product is 3’,2’-cGAMP, this intermediate should result from the formation of either the canonical (pppG[3’−5’]pA) or the non-canonical (pppA[2’-5’]pG) phosphodiester bond during the first step of the cyclase reaction. To distinguish between these two possibilities, the reaction products were treated with CIP and P1 nuclease (“+”), which cleave 5’ and 3’ phosphodiester bonds, respectively. This treatment eliminated both the ³²P-labeled intermediate and cyclic nucleotide when ATP was radiolabeled. In contrast, when GTP was radiolabeled, treatment with both enzymes eliminated the radioactive cyclic nucleotide to generate the linear intermediate. This result is consistent with the formation of a (pppG[3’-5’]pA) intermediate, as shown in the Supplementary Text File. Data are representative of three independent experiments. (h) Diagram of the ΦNM1γ6 genome showing the localization of the cabRNA sequence. (i) Northern blot analysis of RNA extracted from S. aureus/pSsc-CBASS cells before (-) and 30 min after (+) infection with Φ80α-vir, using a cabRNA probe. A 400-nt RNA that is present only during phage infection, presumably the cabRNA, is marked by the arrowhead. Data are representative of three independent experiments.

Extended Data Fig. 3. Analysis of cabRNA activation of Ssc-CdnE03.

(a) Quantification of the cyclase reaction products of triplicates of the experiment shown in Fig. 3d. p values determined using a t-test (unpaired, two-tailed), individual data points are shown with error bars representing the mean +/− s.e.m of n = 3 technical replicates representative of three independent experiments. (b) Thin-layer chromatography analysis of Ssc-CdnE03 reaction products in the presence of the cabRNA isolated from a pull-down assay, or the sense or antisense strands of in vitro transcribed (IVT) cabRNA. IVT RNA was subjected to heat refolding (see Methods). An agarose gel stained with ethidium bromide (middle) and SDS-PAGE stained with Coomassie blue (bottom) are shown as loading controls. Pi, free phosphates. Data are representative of three independent experiments. (c) Same as in (b) but incubating the cyclase with pulled-down RNA or different synthetic RNA oligonucleotides described in detail in the Supplementary Sequences File and Supplementary Methods Table 5. (d) Quantification of the cyclase reaction products of triplicates of the experiment shown in (c), induced by the pull-down, hairpin−1 and random dsRNA oligonucleotide. P values determined using a t-test (unpaired, two-tailed), individual data points are shown with error bars representing the mean +/− s.e.m of n = 3 technical replicates representative of three independent experiments. (e) Growth of staphylococci harboring either an incomplete (Ssc-CdnE03 alone, “-”) or intact Ssc-CBASS (“+”) operon and an empty vector (“-”) or a plasmid encoding cabRNA (“+”) under the control of an aTc inducible promoter measured by optical density at 600 nm. The mean of three biological replicates ± SD is reported. (f) Agarose gel electrophoresis of the input and output RNA obtained after incubation of Ssc-CdnE03 with total RNA extracted from cells infected with Φ80α-vir or Φ80α-vir(terS^S74F) phage, in the presence or absence of cabRNA plasmid overexpression. Data are representative of three independent experiments.

Extended Data Fig. 4. Structural analysis of Ssc-CdnE03.

(a) Structures of porcine OAS1 bound to dsRNA (PDB: 4RWO), Ssc-CdnE03 and Sha-CdnE01 (AlphaFold rank #1 models). Black lines define a conserved primary dsRNA-binding surface present in OAS1 that seems conserved in the CBASS cyclases. (b) Surface electrostatics of the structures shown in (a); blue and red, positive and negative charge, respectively. Black lines define the dsRNA-binding surface. (c) Structural alignment of Ssc-CdnE03 (red) and crystal structure of porcine OAS1:dsRNA (PDB: 4RWO) (blue) with zoomed-in cutaways highlighting conservation of the active site (top inset) and positively charged residues within the ligand binding surface (bottom inset). (d) Quantification of the cyclase reaction products of triplicates of the experiment shown in Fig. 4d. p values determined using a t-test (unpaired, two-tailed), individual data points are shown with error bars representing the mean +/− s.e.m of n = 3 technical replicates representative of three independent experiments. (e) Quantification of propidium iodide (PI) fluorescence at 615 nm of S. aureus cultures expressing the Ssc-CdnE03 alone, or the full CBASS system harboring a wild-type, K9E or K13E cyclase, 15 min after infection with Φ80α-vir. p values determined using a t-test (unpaired, two-tailed). Individual data points are shown with error bars representing the mean +/− s.e.m for n = 6 biological replicates. (f) Quantification of the fraction of the cyclase bound to cabRNA in the experiment shown in Fig. 4c. p values determined using a t-test (unpaired, two-tailed), Individual data points are shown with error bars representing the mean +/− s.e.m n = 3 technical replicates representative of three independent experiments. (g) Agarose gel electrophoresis of the input and output RNA obtained after incubation of Ssc-CdnE03, wild-type or K9E/K13E mutant, with total RNA extracted from cells infected with Φ80α-vir. Data are representative of three independent experiments. (h) Replicates of the infection growth curve shown in Fig. 4d, for the cultures expressing Ssc-CdnE03^K13E.

Extended Data Fig. 5. The Sha-CdnE01 cyclase also binds the cabRNA during Φ80α-vir infection.

(a) Alignment of porcine OAS1, D. erecta cGLR, and bacterial CdnE03s. Conserved residues are highlighted in blue. (b) Alignment of the N-terminal helix, PβCD and C-terminal domains CD-NTases from staphylococcal species, belonging to different families (in different colors). The conserved basic residues involved in cabRNA binding by Ssc-CdnE03 are highlighted in red. Accession numbers are: S. nepalensis WP_096808822.1; S. hominis WP_0494417910.1; S. epidermidis WP_020363757.1 (CdnB03); S. pseudintermedius WP_101458501.1; S. saprophyticus WP_041080732.1; S. argenteus WP_076688059.1; S. hominis WP_071561002.1; S. sp. EGD-HP3 WP_021459889.1; S. haemolyticus WP_037559004.1; S. equorum WP_071664859.1; S. vitulinus WP_016911376.1; S. epidermidis W23144 (Cd–E03) - EES35648.1; S. sp. HMSC077B09 WP_070485035.1; S. schleiferi WP_050329628.1; S. chromogenes WP_037576868.1. (c) Detection of phage propagation after spotting ten-fold dilutions of the lytic DNA phages, Φ80α-vir, ΦNM1γ6, ΦNM4γ4, and Φ12γ3 onto lawns of S. aureus RN4220 harboring a plasmid expressing an incomplete (Sha-CdnE01 alone) or intact Sha-CBASS operon. (d) Agarose gel electrophoresis of the input and output RNA obtained after incubation of Sha-CdnE01 with total RNA extracted from uninfected staphylococci (RN4220) or from cells infected with Φ80α-vir phage. Data are representative of three independent experiments.

Extended Data Fig. 6. Isolation and characterization of CBASS escapers.

(a) An overnight culture of S. aureus RN4220 was diluted and outgrown to early log-phase, at which time Φ80α-vir at an MOI of 1 was added. Just before the first burst (~30 min), 1% ethyl methanesulfonate (EMS) was added to generate mutations. Infections in the presence of EMS were allowed to proceed at 37 °C for 4 h to allow phage to propagate and lyse the culture. Culture supernatants were collected and used to infect staphylococci expressing Ssc-CBASS to enrich for phage escapers. Supernatants of these cultures were serially diluted and spotted on a lawn of S. aureus RN4220::Ssc-CBASS or Ssc-CdnE03. A control experiment without the addition of the EMS mutagen is shown. (b) Diagram of Φ80α-vir genome with localization of four unique escaper mutations identified in terS or gp46. (c) Growth of staphylococci harboring either an incomplete (Ssc-CdnE03 alone) or intact Ssc-CBASS operon measured by optical density at 600 nm after the addition of Φ80α-vir or Φ80α-vir(gp46^E105D) at MOI 1. The mean of three biological replicates ± SD is reported. (d) Detection of phage propagation after spotting ten-fold dilutions of Φ80α-vir or Φ80α-vir(gp46^E105D) onto lawns of S. aureus RN4220 harboring either an incomplete (Ssc-CdnE03 alone) or intact Ssc-CBASS operon, the latter in the presence or absence of Gp46 overexpression using plasmid pGp46. (e) Thin-layer chromatography analysis of Ssc-CdnE03 reaction products in the presence of total RNA extracted from uninfected staphylococci or cells infected with wild-type or gp46^E105D Φ80α-vir. Pi, free phosphates. (f) Quantification of propidium iodide (PI) fluorescence at 615 nm of S. aureus cultures expressing the Ssc-CdnE03 alone or the full CBASS system, 15 min after infection with wild-type or different mutant versions of Φ80α-vir. p values determined using a t-test (unpaired, two-tailed). Individual data points are shown with error bars representing the mean +/− s.e.m n = 9 biological replicates.

Extended Data Fig. 7. Mechanism of escape mediated by the terS mutation.

(a) Agarose gel electrophoresis of the escaper RNA generated during infection with Φ80α-vir(terS^S74F) isolated from a pull-down assay, treated with RNase T1 or III. Data are representative of three independent experiments. (b) Electrophoretic mobility shift assay of cabRNA and the escaper (long) cabRNA pulled down during infection with the Φ80α-vir(terS^S74F) phage, in the presence of increasing concentrations of Ssc-CdnE03. (c) Quantification of the fraction of the cyclase bound to cabRNA in the experiment shown in (b). p values determined using a t-test (unpaired, two-tailed), individual data points are shown with error bars representing the mean +/− s.e.m n = 3 technical replicates representative of three independent experiments. (d) Thin-layer chromatography analysis of Ssc-CdnE03 reaction products in the presence of cabRNA or in vitro transcribed escaper RNA. Pi, free phosphates. (e) Wild-type ΦNM1γ6 was propagated in liquid cultures of S. aureus RN4220::Ssc-CBASS harboring a plasmid-borne terS gene, wild-type or carrying the S74F mutation. Culture supernatants were collected and serial dilutions were spotted onto lawns of S. aureus RN4220::Ssc-CdnE03 or RN4220::Ssc-CBASS. (f) Growth of staphylococci harboring pTerS, providing IPTG-inducible expression of the Φ80α-vir TerS protein, measured by optical density at 600 nm after the addition of the inducer. The mean of three biological replicates ± SD is reported. (g) Same as (f) but using pGp46 plasmid, providing IPTG-inducible expression of the Φ80α-vir Gp46 protein. (h) Same as (f) but using pGp40-47 plasmid, providing IPTG-inducible expression of the complete Φ80α-vir viral capsid.

Extended Data Fig. 8. Characterization of Φ80α-vir phages carrying mutations in the cabRNA.

(a) Growth of staphylococci expressing the Ssc-CdnE03 cyclase alone, measured by optical density at 600 nm after the addition of Φ80α-vir mutants that contain 122, 100 or 57 mutations within the cabRNA sequence, at MOI 1. An uninfected culture is shown as control. The mean of three biological replicates ± SD is reported. (b) Enumeration of PFU from cultures shown in (a), 0.5, 1.0 and 3.0 h after infection. Mean ± SEM of three biological replicates is reported. (c) competition assays between Φ80α-vir and Φ80α-vir(terS^S74F) or Φ80α-vir(cabRNA¹²²). A 1:1 ratio mix of both phages was used to infect S. aureus RN4220 and supernatants were collected every three hours to enumerate total and Φ80α-vir(terS^S74F) or Φ80α-vir(cabRNA¹²²) PFUs (mutant phages were enumerated through plating on lawns of staphylococci expressing the Ssc-CBASS system, on which wild-type phages cannot form plaques), and diluted into a fresh bacterial culture. Each dilution constitutes one passage. p values determined using a t test (unpaired, two-tailed), individual data points are shown with error bars representing the mean +/− s.e.m of n = 3 biological replicates. (d) Detection of phage propagation after spotting ten-fold dilutions of Φ80α-vir or Φ80α-vir(cabRNA¹²²) onto lawns of S. aureus RN4220 harboring either an incomplete (Ssc-CdnE03 alone) or intact Ssc-CBASS operon. (e) Thin-layer chromatography analysis of Ssc-CdnE03 reaction products in the presence of cabRNA⁵⁷ or cabRNA¹⁰⁰ isolated from pull-down assays. Pi, free phosphates. (f) Quantification of the cyclase reaction products of triplicates of the experiment shown in Fig. 5e. p values determined using a t-test (unpaired, two-tailed), individual data points are shown with error bars representing the mean +/− s.e.m of n = 3 technical replicates representative of three independent experiments.

Extended Data Fig. 9. Characterization of Ssc-CBASS immunity against ΦJ1, ΦJ2 and ΦJ4 phages.

(a) Phylogenetic tree of the phages used in this study, generated using Jalview. The scale bar indicates the genetic distance calculated by this software. (b) Diagram of the ΦJ1/2 genomes showing the localization of the cabRNA sequence within the terS/L region. (c) Same as (b) but for the ΦJ4 genome.