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Cyclic tri-adenylate controls a CARF-TM effector in type II Panoptes anti-phage systems [1]

['Sabine Grüschow', 'School Of Biology', 'University Of St Andrews', 'St Andrews', 'United Kingdom', 'Peter Wotherspoon', 'Emma Hilton-Balfe', 'Shirley Graham', 'Malcolm F. White']

Date: 2026-09 

Cyclic nucleotide second messengers are used in all domains of life to amplify viral infection signals and activate cellular defences. In prokaryotes, CBASS (cyclic nucleotide - based antiphage signalling system) and type III CRISPR-Cas systems generate a range of cyclic nucleotides which bind and allosterically activate effector proteins to mount an anti-viral response. Viruses have evolved counter measures to antagonise these signalling pathways in the form of cyclic nucleotide sponges and phosphodiesterases that sequester or degrade these molecules to subvert immunity. Recently, the Panoptes system was shown to function as a guard against these viral tactics. The type I Panoptes polymerase, mCpol, generates cyclic dinucleotides as decoy molecules that, when sequestered by phage proteins, results in the activation of the membrane-permeabilising effector 2TMβ to halt the phage infection cycle. Here, we investigate the type II Panoptes system, demonstrating that it generates cyclic tri-adenylate (cA 3 ) to maintain a CRISPR-associated Rossmann fold-transmembrane (CARF-TM) effector in an inactive, dimeric state. When cA 3 is sequestered or degraded, the CARF protein undergoes conformational changes. In vivo, the absence of cA 3 results in membrane disruption and growth arrest. Type II Panoptes provides defence against phages that express the cA 3 -degrading enzyme Acb1; phage escapers introduce mutations into the acb1 gene to avoid triggering the Panoptes system. These findings expand our understanding of the guard systems that constitute a fascinating component of the bacterial immune system.

Funding: This work was funded by grants awarded to MFW from the Biotechnology and Biological Sciences Research Council ( https://www.ukri.org/councils/bbsrc/ ; Grant REF BB/Z517240/1), which funded the salary of SGr, and the European Research Council ( https://erc.europa.eu/homepage ; Grant REF 101018608), which funded the salary of PW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Although biochemical studies have focussed on Panoptes with a 2TMβ effector, about 40% of systems (hereafter Panoptes type II) are associated with an alternative CARF-family effector with a transmembrane domain (CARF-TM) [ 24 ]. Here, we demonstrate that the type II Panoptes mCpol generates a cyclic trinucleotide (cA 3 ) product which is bound by the associated CARF-TM effector. The system provides immunity against plasmids and phage expressing antidefence proteins that bind or degrade cA 3 . The absence or removal of cA 3 from CARF-TM results in structural changes that ultimately lead to outer membrane (OM) permeabilization and render cells incapable of forming colonies. These data demonstrate another facet of Panoptes-mediated immunity and the potential to guard type III CRISPR-Cas as well as CBASS defence systems.

Recently, two research groups independently demonstrated that one type of mCpol-containing system, which they named “Panoptes” (henceforth Panoptes type I, Fig 1A ), generates cyclic dinucleotides constitutively, maintaining an associated 2TMβ (2 trans-membrane helix, β-strand rich) effector in an inactive state [ 23 , 24 ]. This contrasts with CBASS, and other defence systems relying on cyclic oligonucleotide signalling, which require an active phage infection to trigger cyclic nucleotide synthesis. When a phage expressing an anti-defence sponge protein such as Acb2 infects cells encoding Panoptes type I, the cyclic dinucleotide is depleted, resulting in activation of the 2TMβ-effector, leading to growth arrest and thwarting the phage infection ( Fig 1B ). Panoptes is thus an example of a guard system that may often work in conjunction with CBASS defence to prevent phage from utilising a cyclic nucleotide depletion strategy.

Type III CRISPR systems use their catalytic Cas10 subunit, which has two Palm polymerase domains, to generate cOA species ranging from 3 to 6 AMP subunits [ 8 , 9 ]. These activate a diverse range of effector proteins, many of which include a CRISPR-associated Rossmann Fold (CARF) sensor domain, to provide an immune response [ 18 , 19 ]. The recent discovery of variants that make the signalling molecule SAM-AMP by conjugating S-adenosyl methionine and ATP [ 20 ] underscores the flexibility of the polymerase to accept a range of building blocks for signal generation. In 2015, Burroughs and colleagues described a minimal CRISPR-related polymerase (mCpol), distantly related to Cas10, which was sometimes associated with CARF domain proteins characteristic of type III CRISPR effectors [ 21 ]. In some cases, the mCpol cyclase is fused to a Csx1-family ribonuclease effector protein, potentially representing a single-protein antiviral defence system that functions via cyclic nucleotide signalling [ 22 ]. In other cases, 2 gene operons include mCpol next to a gene encoding a predicted membrane-bound effector [ 22 ]. mCpol has been suggested as the ancestral protein that gave rise to Cas10 and the evolution of the class 1 CRISPR systems [ 22 ].

In recent years, a large number of antiviral defence systems have been discovered in prokaryotes, leading to the concept of the bacterial immune system [ 1 , 2 ]. One major approach, used in both prokaryotes and eukaryotes, is the generation of nucleotide second messengers by specialised nucleotide cyclase enzymes activated by viral infection [ 3 , 4 ]. Notable examples include the cGAS/STING pathway in metazoa which signals via cGAMP [ 5 ] and its bacterial equivalent CBASS (cyclic nucleotide-based antiphage signalling system) [ 6 ], Pycsar (Pyrimidine cyclase system for antiphage resistance) which generates cyclic pyrimidines [ 7 ] and the type III CRISPR-Cas system signalling via production of cyclic oligoadenylate (cOA) species [ 8 , 9 ]. This approach has the advantage of achieving amplification of the primary signal of infection—for example, the type III CRISPR system detects 1 viral mRNA molecule and generates over 1,000 cyclic nucleotides in response [ 10 ]. Viruses, in turn, target the cyclic nucleotides to neutralise cellular defences using specialised phosphodiesterases that degrade cGAMP in eukaryotes [ 11 ] and bacteria [ 12 ], ring nucleases that target cOA species to nullify CRISPR-based immunity [ 13 ] and sponge proteins that bind cyclic nucleotide second messengers [ 14 , 15 ]. Sponge proteins are particularly adept at sequestering a range of cyclic nucleotides used by immune defence systems and are widely encoded by phage [ 16 , 17 ].

Results

Type II Panoptes signals via cyclic tri-adenylate (cA 3 ) While the characterised type I Panoptes systems synthesise cyclic dinucleotides and utilise a 2TMβ-family effector, type II systems with a CARF-TM effector remain unstudied. We designed synthetic genes encoding mCpol and CARF-TM from the cyanobacterium Cylindrospermum stagnale PCC7417 (Cst) for expression in E. coli and purified the recombinant proteins. The Cst mCpol protein was tested for nucleotide cyclase activity and shown to synthesise 3′3′3′-cA 3 in the presence of ATP and Mg2+ (Fig 2). The presence of other NTPs did not alter the reaction product. Two further mCpol enzymes associated with a CARF-TM effector, from Sphingomonas (Sph) and Ahniella affigens (Aaf), also synthesised 3′3′3′-cA 3 as shown by HPLC analysis and co-injection with synthetic 3′3′3′-cA 3 (S1 Fig). The turnover number for all three mCpol enzymes was approximately 1 h−1 in vitro (S2 Fig), making mCpol significantly slower than Cas10 from Type III CRISPR systems [25]. Although in vitro reaction rates should be treated with caution, these data suggest mCpol may generate relatively low concentrations of cA 3 in cells. This could be consistent with a function as a decoy molecule for phage sponges and phosphodiesterases, providing sensitivity to changes in cA 3 concentration in vivo. Residues D11 and D63 of Cst mCpol correspond to the canonical metal-binding residues of Cas10 and Palm family polymerases more generally. We mutated D63 and the neighbouring D64 to generate Cst mCpol[D63N/D64N]. This variant was completely inactive, as expected (Fig 2). PNG larger image

TIFF original image Download: Fig 2. Type II mCpols synthesise 3′3′3′-cA 3 . HPLC analysis of Cst, Sph and Aaf mCpol reaction products, showing production of cA 3 in the presence of ATP and Mg2+. The Cst mCpol[D63N, D64N] variant (Cst[DDNN]) was catalytically inactive. The data underlying this Figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003934.g002

Type II Panoptes is active in vivo and responds to cA 3 levels in cells To test the activity of Panoptes type II in vivo, we performed transformation assays, where the effector was introduced to a strain containing the mCpol cyclase. A serial dilution of the transformation mixture was then plated onto selective medium and both cyclase and effector expression were induced. The plates were incubated overnight and inspected for colony growth. If the cyclase and effector combination give rise to a growth defect or kill the host cell, fewer colonies are observed compared to controls. We used Cst mCpol protein as the cyclase with either its cognate CARF-TM effector or NucC, a previously characterised dsDNA-endonuclease associated with CBASS and type III CRISPR defence [26,27]. NucC is activated by cA 3 and was included as a control to detect cA 3 production. In this experiment, mCpol was expressed under control of the T7 promoter (IPTG inducible), while NucC or CARF-TM were expressed under control of the arabinose-inducible pBAD promoter. As expected, expression of NucC with the catalytically inactive mCpol resulted in efficient transformation, as NucC is inactive in the absence of cA 3 (Fig 3A). In contrast, expression of the CARF-TM effector alone resulted in toxicity and therefore no viable colonies in the absence of mCpol. When the NucC plasmid was transformed into cells expressing Cst mCpol, no transformants were observed, consistent with activation of the nuclease by cA 3 . In contrast, the toxicity of the CARF-TM effector was moderated with co-expressed mCpol. Truncation of the CARF-TM effector to remove the predicted transmembrane domain completely abolished activity, demonstrating that the transmembrane (TM) domain is essential. Expression of the inactive D63N, D64N variant of Cst mCpol did not alleviate the toxicity of CARF-TM, confirming the requirement for cA 3 synthesis. PNG larger image

TIFF original image Download: Fig 3. Panoptes type II is functional in vivo. A. Plasmid challenge assay using the Cst Panoptes system in E. coli. Cells expressing the wild-type or inactive variant mCpol were transformed with a plasmid that expresses the CARF-TM, CARF or NucC effectors. The cA 3 -activated nuclease NucC prevented cell growth only in the presence of mCpol, whereas CARF-TM did so in its absence. The transmembrane domain of CARF-TM was required for activity. (w.t.: wild type; mut.: catalytically inactive mCpol[D63N, D64N]). B. Plasmid challenge assay as in (A), where a second gene is introduced on the plasmid encoding the effector. The presence of a cA 3 -degrading ring nuclease (Crn4) or a phage sponge protein (Acb2) activated CARF-TM activity in the presence of mCpol, leading to toxicity. C. Efficiency of plaquing (EOP) assay using the Aaf Panoptes system (mCpol and either CARF-TM (WT) or CARF (ΔTM) in E. coli. Bas31 (T5 family) was insensitive to mCpol. Phages Bas35-37 were sensitive while closely related Bas43, which carries a nonsense mutation in the acb1 gene, escapes defence. D. Close-up of plaques for Bas35, showing reduction in plaque size when Panoptes is active. E. Quantification of EOP for replicates of the data shown in (C). Four biological replicates were analysed and plaques counted. Mean plaque numbers and standard deviations are shown along with individual data points. The data underlying this panel can be found in S1 Data. F. EOP assay of Bas35 and escaper variants (1a, 1b, 2, 3a, 3b) against wild type and inactive (ΔTM) Aaf type II Panoptes. The observed mutations are mapped onto their position in the acb1 gene. 1a,b and 3a,b represent biological replicates purified from a single plaque and sequenced independently. https://doi.org/10.1371/journal.pbio.3003934.g003 We also performed the plasmid challenge assay using the previously characterised Vibrio metoecus (Vme) type III CRISPR-Cas Cmr complex instead of the type II Panoptes mCpol as the cyclase component (S3 Fig). When activated, the Vme Cmr complex produces 3′3′3-cA 3 with a trace of cA 4 , and its cognate effector is the NucC nuclease [26]. The Vme Cmr complex was able to fulfil the same role as Cst mCpol, reducing the growth defect imparted by CARF-TM. This confirmed that 3′3′3′-cA 3 was the relevant signalling molecule for type II Panoptes. Overall, these data establish Panoptes type II as a defence system that signals via cA 3 , using a signalling logic similar to Panoptes type I. To explore this further, we challenged the defence system with two different proteins that are known to bind or degrade cA 3 : the phage sponge protein Acb2 [14] and the cA 3 -degrading ring nuclease Crn4 [28]. Expression of either of these proteins from a plasmid resulted in the activation of Panoptes type II, reflected in cell toxicity when the CARF-TM effector was induced by arabinose (Fig 3B). This mimics the situation where a phage utilising a sponge or ring nuclease to subvert CBASS or type III CRISPR defence would activate Panoptes type II defence. To extend this analysis, we turned to the BASEL collection of E. coli phages [29]. Some phages in this collection encode the anti-CBASS phosphodiesterase Acb1 [30], which degrades a range of cyclic di- and tri-nucleotides including cA 3 . Doherty and colleagues demonstrated that the acb1 gene is disrupted in phage Bas43, but intact in closely related phages such as Bas35-37 of the Tevenvirinae family [23]. They showed that disruption of acb1 correlated with resistance to the type I Panoptes system. We recapitulated this analysis using the type II Panoptes system, observing a clear resistance phenotype for Bas43, where the cA 3 -degrading Acb1 protein is non-functional (Fig 3C). Bas35-37, with functional Acb1, were all sensitive to type II Panoptes defence, resulting in fewer and smaller plaques (Fig 3D and 3E), consistent with the data obtained from plasmid challenge experiments. In the course of these experiments, we isolated a number of escaper mutants for phage Bas35 which generated larger plaques in the presence of active type II Panoptes defence. Sequencing of the PCR-amplified acb1 region in these escapers revealed single-nucleotide mutations predicted to deactivate the acb1 gene (Fig 3F). Although the complete phage genomes were not sequenced, these mutations in acb1 are likely to be responsible for the observed phenotypes. In sum, the in vivo data all point to a key role for cA 3 depletion in the activation of type II Panoptes. Phage challenged by Panoptes defence escape by deactivating the genes encoding cA 3 -depletion proteins. This, of course, could render them sensitive to CBASS and type III CRISPR defence.

The CARF-TM effector binds cA 3 Modelling with Alphafold3 [31] strongly supported a dimeric structure for the CARF-TM effector (Fig 4A), consistent with the dimeric organisation of many other CRISPR-associated CARF effectors [22]. cA 3 was modelled bound at the interface between the two subunits. To express and purify recombinant CARF-TM, we had to co-express the protein with mCpol, generating cA 3 to keep the effector in an inactive state. Under these conditions, CARF-TM with a non-cleavable N-terminal polyhistidine tag could be purified to homogeneity via immobilised metal affinity and size exclusion chromatography in the presence of the detergent DDM (S4A and S4B Fig). We also purified a truncated form of the effector (CARF), where the TM domain was replaced with a non-cleavable C-terminal hexahistidine tag. The truncated protein also required the presence of the cyclase for efficient purification. We observed two bands in the final purified sample for full-length CARF-TM and for the truncated CARF protein (S4C and S4D Fig); both were confirmed by mass spectrometry to be the effector. An elevated absorbance at 260 nm for both the full-length and truncated proteins was suggestive of the presence of nucleic acid. We extracted nucleotides from both proteins and analysed them by HPLC, confirming the presence of cA 3 in roughly equimolar amounts to the protein dimer (Fig 4B). PNG larger image

TIFF original image Download: Fig 4. CARF-TM model structure and cA 3 binding. A. Alphafold3 model of CARF-TM in complex with cA 3 , including POPG molecules to simulate the membrane bilayer. The position mutated to generate the truncated CARF protein is shown by black arrows. B. HPLC analysis of the supernatant of heat-denatured CARF-TM and CARF confirmed the presence of cA 3 in the purified proteins. The data underlying this panel can be found in S1 Data. C. Radiolabelled cA 3 (100 nM) was incubated with increasing CARF-TM (125 nM–2 µM dimer) or CARF (200 nM–7 µM dimer) and analysed by EMSA. D. The CARF: [32P]-cA 3 complex was challenged with a 100-fold excess of a range of cyclic nucleotides, as indicated. Only 3′3′3′-cA 3 was able to displace the radiolabelled bound ligand. https://doi.org/10.1371/journal.pbio.3003934.g004 These observations demonstrated that cA 3 remains bound to both the full-length and truncated effector during purification, suggesting a low dissociation constant for cA 3 binding. To explore ligand specificity, we investigated binding of 32P-radiolabelled cA 3 by electrophoretic mobility shift assay (EMSA). At high concentrations of CARF-TM or CARF domain (approximately 1 µM dimer), a single retarded species consistent with cA 3 binding became visible (Fig 4C). This high apparent dissociation constant likely reflects the fact that both proteins were already bound to unlabelled cA 3 , requiring displacement by competition with the radioactive species. The retarded radioactive cA 3 moiety was abolished when a 100-fold molar excess of cold 3′3′3′-cA 3 was added, but binding was unaffected by a range of other cyclic nucleotide species (Fig 4D). Overall, these data demonstrate that both the CARF-TM effector and its truncated, soluble CARF domain bind cA 3 specifically and with a high enough affinity to persist during protein purification. However, binding is not irreversible and is subject to competition by free cA 3 in vitro.

The soluble CARF variant multimerises when cA 3 is not bound We next investigated whether the CARF effector underwent any conformational changes upon loss of cA 3 , mimicking the effect of a cA 3 -scavenging phage protein. Although we could purify small quantities of the full-length CARF-TM effector bound to cA 3 , plasmid challenge experiments indicated that the presence of N- or C-terminal tags abolished function in vivo (S5 Fig). We therefore focussed on the truncated CARF version of the effector for further in vitro studies, cognisant of the limitations inherent in this approach. To deplete cA 3 from the CARF protein, we incubated the purified protein with increasing concentrations of the ring nuclease Crn4 [32]. HPLC analysis of the nucleotide species present following Crn4 incubation confirmed that a significant proportion of the cA 3 present was converted to smaller products, confirming that the ring nuclease can compete with the CARF-TM effector for cA 3 ligands in vitro (S6 Fig). This is consistent with the competition observed by free cA 3 in the EMSA experiments and with the in vivo data that demonstrate Panoptes activation by Crn4 (Fig 3). To examine the effect of cA 3 removal on the effector, we probed structural changes using the fluorogenic dyes Sypro Orange and BODIPY FL L-cystine (BFC). Both dyes are typically used with thermal shift assays; however, we conducted isothermal assays following changes in fluorescence after addition of Crn4 to the cA 3 :CARF complex. The fluorescence intensity of Sypro Orange increases upon binding to hydrophobic regions, which are normally buried in a folded protein but become exposed when the protein denatures and in turn become hidden when an unfolded protein aggregates (Fig 5A). The fluorescence of BFC in its disulfide dimer form is quenched. Cleavage by free thiols, such as exposed cysteine residues, releases the monomeric, fluorescent dye (Fig 5B). Unfolding and/or increased protein chain mobility can expose buried cysteine residues leading to an increase in fluorescence. We observed a decrease in Sypro Orange fluorescence intensity upon Crn4 treatment, consistent with burial of hydrophobic patches upon ligand removal (Fig 5C). For the BFC dye, isothermal incubation of the CARF protein with Crn4 resulted in a significantly larger increase in fluorescence than in the absence of Crn4 or with the catalytically inactive Crn4 [H15A] variant [28] (Fig 5D). PNG larger image

TIFF original image Download: Fig 5. Removal of cA 3 results in oligomerisation of the CARF protein. A. Cartoon of Sypro Orange binding to exposed apolar patches upon protein unfolding followed by occlusion of hydrophobic binding sites through protein aggregation. B. The BFC dimer becomes fluorescent upon cleavage of its disulfide bridge by cysteine residues. C, D. Difference in fluorescence intensity after 50 min incubation of CARF with Crn4 wild type or catalytically inactive H15A variant. The greatest change in fluorescence was observed for CARF with wild-type Crn4. Removal of cA 3 appears to remove available hydrophobic residues (C, Sypro Orange, mean and range of duplicates shown) but expose Cys residues (D, BFC, mean and standard deviation of triplicates shown). E, F. Chemical cross-linking of CARF in the presence of increasing amounts of Crn4. CARF and Crn4 were incubated for 30 min before separating on SDS-PAGE. CARF oligomers labelled with an asterisk in E were quantified by densitometry relative to the CARF dimer band. Statistical analysis in F was performed with Prism 10 (Graphpad) using the Mann–Whitney test (two-tailed). *: p value 0.0286. G. DLS analysis of CARF after incubation with Crn4. The data underlying Fig 5C, 5D, 5F and 5G can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003934.g005 The effect of cA 3 removal on the quaternary structure of the CARF protein was also tested using chemical cross-linking and SDS-PAGE (Fig 5E). In the absence of Crn4, the CARF protein was observed primarily as monomeric and dimeric after crosslinking with very minor amounts of larger species. With increasing amounts of Crn4, progressively higher amounts of large (>dimeric) cross-linked species were observed (Fig 5F). This assay benefited from the unusual amino acid composition of Crn4, which lacks lysine residues and thus cannot cross-link [32]. We also investigated the removal of cA 3 from the CARF domain by dynamic light scattering (DLS). Notably, a precipitate formed when Crn4 was incubated with the CARF domain. As the samples were centrifuged, the precipitate was removed prior to DLS analysis. An approximately 100-fold increase in size of the remaining soluble CARF protein was detected upon cA 3 -depletion (Fig 5G). Together, these observations suggested that cA 3 removal results in structural perturbations of the CARF dimer, leading to unfolding or aggregation in vitro. The absence of the TM domain in these studies is a significant caveat, however. Given the difficulty in studying the full-length CARF-TM protein in vitro, we turned to in vivo analyses.

The CARF-TM effector causes growth arrest in the absence of cA 3 We set out to monitor changes in cell viability upon expression of CARF-TM using microscopy. The full-length, untagged CARF-TM was expressed from the T7 promoter in pEHisV5TEV [25], allowing induction of expression using IPTG. We omitted the mcpol gene from these strains, so that the effector would be in the active apo form when expressed. E. coli harbouring CARF-TM, truncated CARF or the empty vector were grown to mid-log phase before induction with 0.1 mM IPTG. Cells collected 1–2 h after induction were stained with Syto9 and propidium iodide (PI) [33]. Both are nucleic acid staining fluorescent dyes; Syto9 is a membrane-permeable dye that will stain live and dead cells, whereas PI can only enter cells with significantly damaged membranes. PI has a higher affinity for nucleic acids than Syto9 and tends to displace Syto9 from the nucleic acid given a high enough concentration. We determined the percentage of PI-dyed cells using fluorescent confocal microscopy (Figs 6A and S7). We observed minor differences between CARF-TM-expressing (7.2% PI-stained) or non-expressing cells (3.5% PI-stained for empty vector, 2.3% for CARF). This suggested that CARF-TM is not a fast-acting bactericidal agent when expressed in E. coli, but rather induces a growth arrest phenotype. PNG larger image

TIFF original image Download: Fig 6. The CARF-TM effector causes growth arrest in the absence of cA 3 . A. Live/dead staining of cells expressing Cst CARF-TM, CARF or empty vector. Samples from 1 h (open circles) or 2 h (closed circles) after induction of the effector protein were analysed by confocal fluorescence microscopy. There was no significant difference in the percentage of PI-stained cells between strains expressing CARF-TM or those lacking the effector (p value 0.11, two-tailed Mann–Whitney test); however, cells expressing the truncated effector CARF had a lower percentage of PI-staining than those expressing full length CARF-TM (p value 0.019). Data show the mean and standard deviation from three independent experiments. More than 1,000 cells were counted in total for each strain. B. Optical density of cultures after induction of protein expression. C, D. Cells expressing CARF-TM lost their ability to proliferate as determined by microdilution assay. Data for panels B, C show the individual values of four biological replicates as symbols with a line representing the mean. One representative example of the microdilution assay is shown in D. eV: empty vector. The data underlying Fig 6A, 6B and 6C can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003934.g006 To follow on from the live/dead staining, we determined the cell density and culturability of the three strains for the first 3 h after IPTG induction. The optical density initially increased for all three strains, levelling off for the CARF-TM-expressing strain after 2 h, consistent with growth arrest upon effector expression (Fig 6B). The growth of cells with the empty vector control slowed significantly on IPTG addition—a phenomenon that was consistently observed and which may relate to the burden IPTG places on cells in a context-dependent manner [34]. We therefore investigated the equivalent growth curves using the Aaf Panoptes system under the control of an arabinose promoter, confirming that Aaf CARF-TM, but not truncated CARF, induced growth arrest in the absence of mCpol, with no growth defect in the presence of mCpol (S8 Fig). Microdilution and colony counting assays for the Cst effector demonstrated that whilst the strains with empty vector or expressing the CARF domain yielded a similar number of colony-forming units (cfu), those for the CARF-TM-expressing strain dropped by 2–3 orders of magnitude (Fig 6C and 6D). Combined with the data from PI staining, this suggests cells experience growth arrest when the CARF-TM effector is activated. While the cells don’t appear to lyse, as observed for type I Panoptes [23], they may still be functionally “dead” to all intents and purposes.

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