Bacterial 2′,3′-cGAMP activates a SAVED effector to form membrane-disrupting filaments and restrict phage replication

Uday Tak; Kate Schinkel; Peace Walth; Jian Wei Tay; Erik W Hartwick; Aaron T Whiteley

doi:10.1016/j.chom.2026.03.004

. Author manuscript; available in PMC: 2026 Apr 17.

Published in final edited form as: Cell Host Microbe. 2026 Mar 27;34(4):720–733.e7. doi: 10.1016/j.chom.2026.03.004

Bacterial 2′,3′-cGAMP activates a SAVED effector to form membrane-disrupting filaments and restrict phage replication

Uday Tak ^1,³, Kate Schinkel ¹, Peace Walth ¹, Jian Wei Tay ², Erik W Hartwick ¹, Aaron T Whiteley ^1,^4,^*

PMCID: PMC13085583 NIHMSID: NIHMS2161427 PMID: 41903528

Summary

Mammalian cells initiate antiviral signaling when cGAS detects cytoplasmic DNA and synthesizes 2′,3′-cGAMP, which activates STING. Similarly, bacteria use cyclic oligonucleotide-based antiphage signaling systems (CBASS) to detect phage using ancestral cGAS/DncV-like nucleotidyltransferases (CD-NTases), but are not known to use 2′,3′-cGAMP. Here we discover a bacterial CD-NTase that produces 2′,3′-cGAMP to activate a Saf-2TM-SAVED effector (Cap14) that initiates membrane disruption to restrict phage replication. Cryo-EM reveals that Cap14 binds 2′,3′-cGAMP to form a filament, while electrophysiology suggests cGAMP activates membrane disruption. Swapping the Cap14 transmembrane domain with a nuclease domain yields a functional chimera that exclusively responds to 2′,3′-cGAMP. We hypothesize that other predicted transmembrane effectors in CBASS operons disrupt membranes, and we confirm this by showing that bacterial STING homologues with transmembrane domains restrict phage through membrane disruption. These findings expand our understanding of cGAS-STING-like pathways in bacterial immunity.

Keywords: innate immunity, CBASS, Saf-2TM--SAVED, 2TM-STING, Cap13, Cap14, cGAMP, phage, immunity

Graphical Abstract

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eTOC blurb

Tak et al. discover bacteria use 2′,3′-cGAMP, the same signaling molecule employed by mammalian cGAS-STING, for phage defense. In response to phage, 2′,3′-cGAMP activates filament formation of Cap14, initiating premature cell lysis. Bacterial homologues of STING also disrupt membranes, suggesting membrane perturbation may be an ancient mechanism of antiviral immunity.

Introduction

Cyclic oligonucleotides are signaling molecules that play essential roles in cellular physiology and immune signaling throughout the tree of life. In humans, cyclic GMP–AMP synthase (cGAS) produces the cyclic dinucleotide 2′,3′-cyclic GMP–AMP (2′,3′-cGAMP) in response to binding dsDNA in the cytosol, which is often found during infection, cellular stress, and cancer.¹ 2′,3′-cGAMP binds to the transmembrane receptor stimulator of interferon genes (STING), which activates downstream inflammatory pathways, antiviral signaling, and programmed cell death.^2–4 In this way, cGAS acts as a sensor and STING acts as an adaptor to activate a signaling cascade in response to binding cyclic dinucleotides. STING can also be activated by 2′,3′-cGAMP transported intercellularly^5–10, through 2′,3′-cGAMP packaged in virions¹¹, and by bacterially derived cyclic dinucleotides^12–14, allowing rapid amplification of the innate immune response. Consequently, the cGAS-STING pathway is implicated in several disease pathologies including infection, cancer, and autoimmunity and is a target of host-directed immunotherapy.^1,4

Bacteria encode signaling pathways related to metazoan cGAS-STING called cyclic oligonucleotide-based antiphage signaling systems (CBASS) that restrict bacteriophage replication as a part of the bacterial innate immune system.^15–19 CBASS operons encode structural homologs of cGAS called cGAS/DncV-like nucleotidyltransferases (CD-NTases) that are activated during phage infection to produce cyclic oligonucleotides within infected cells to initiate the immune response.^15,20–23 The cyclic oligonucleotide binds to a cognate intracellular CD-NTase-associated protein (Cap), which activates its effector function to restrict phage replication, often inducing growth arrest or cell death.^{15,18,19,22,24} CBASS pathways signal via a range of cyclic oligonucleotides, including cyclic trinucleotides and isoforms of cGAMP such as 3′,3′-cGAMP and 3′,2′-cGAMP.^{15,21,25–27} However, the 2′,3′-cGAMP isoform has not been identified in bacteria. Motivated by the critical role of 2′,3′-cGAMP in human cGAS-STING signaling, we sought to investigate if bacteria also encode 2′,3′-cGAMP signaling pathways.

Results

Discovery of bacterial CD-NTases that synthesize 2′,3′-cGAMP

To identify bacterial CD-NTases that synthesize 2′,3′-cGAMP, we screened the enzymatic products produced by a panel of CD-NTases that we identified previously.²⁰ Recombinant CD-NTases were incubated with α³²P-radiolabelled NTPs to monitor cyclic dinucleotide production. The reaction was quenched by treatment with calf intestinal phosphatase, and the reaction products were resolved using thin layer chromatography (TLC) (Figure 1A). To identify 2′–5′ bonds in CD-NTase reaction products, the reactions were further digested with nuclease P1 which exclusively hydrolyzes 3′–5′ phosphodiester linkages. Cyclic oligonucleotides composed of only 3′–5′ phosphodiester linkages, such as 3′,3′-cGAMP, are degraded with P1 treatment (Figure 1C and S1C). In contrast, cyclic oligonucleotides composed of one 2′–5′ and one 3′–5′ phosphodiester linkage such as 2′,3′-cGAMP, only the 3′–5′ linkage is hydrolyzed by P1, and the remaining 2′–5′-linked linear oligonucleotide is preserved (Figure 1D and S1C). Using this approach, we identified five CD-NTases (49, 39, 05, 35, and 36) that produced P1-resistant ³²P-radiolabelled products (Figure 1B).

Figure 1. — (A) TLC analysis of enzyme reaction products produced when the indicated CD-NTases were incubated with α³²P-radiolabelled NTPs. Reactions were stopped by addition of calf intestinal phosphatase. *Vibrio cholerae* DncV was used to produce 3′,3′-cGAMP in lane one. Catalytically dead (CD) DncV^DID79AIA; inorganic phosphate (P_i); origin (Ori.). Representative of n = 2 biological replicates.

(B) Analysis as in (A) after treatment with nuclease P1, which cleaves 3′–5′ phosphodiester bonds. Spots remaining after P1 treatment contain non-3′–5′ linked ³²P, such as 2′–5′ linkages. Representative of n = 2 biological replicates.

(C) Chemical structure of 3′,3′-cGAMP and schematic for P1 treatment.

(D) Chemical structure of 2′,3′-cGAMP and schematic for P1 or VacV poxin treatment.

(E) Phylogenetic analysis of CdnB homologues. Abbreviations for bacterial species analyzed in this study are provided in parentheses.

(F) ELISA measurement of synthetic nucleotide standards at 50 nM, used as a control for (G). A one-way ANOVA with Dunnett’s multiple comparison test was used, * P ≤ 0.05.

(G) ELISA quantification of 2′,3′-cGAMP from enzyme reaction products produced when the indicated synthase is incubated with ATP and GTP. Where indicated, reaction products were further treated with VacV poxin. Limit of detection (LOD) was determined as in (F) Data are mean ± standard error of the mean (SEM) from representative of n = 2 biological replicates. (−) indicates buffer + ATP/GTP with no enzyme added. A paired Student’s t-test was used, * P ≤ 0.05.

We selected CD-NTase05 from Desulfofalx alkaliphila for further analysis and found that this enzyme only produced a product when ATP and GTP were provided as substrates and the α³²P-radiolabel was only protected from phosphatase when [α³²P] ATP was used (Figure S1A). These findings suggested that CD-NTase05 produced the linear nucleotide pppG(2′,5′)pA in the reaction conditions used. CD-NTases and cGAS produce cyclic dinucleotides by first catalyzing synthesis of a linear dinucleotide intermediate from two NTPs, followed by catalyzing a second reaction that closes the linear intermediate into a cyclic dinucleotide (Figure S1D).²⁰ Mammalian cGAS produces linear pppG(2′,5′)pA as an intermediate.²⁸ Our results suggested that CD-NTase05 likely catalyzed the same reaction, however, the reaction may be incomplete due to buffer conditions or lack of unknown activation factors.

We searched for CD-NTase05 homologs to identify additional enzymes that may synthesize 2′,3′-cGAMP. Previous analysis of CD-NTases organized these enzymes into sequence-related clades denoted A–H and showed that enzymes within clades produced similar cyclic oligonucleotides.²⁰ CD-NTase05 is a member of Clade B02 (Figure S1E), and we renamed this enzyme DaCdnB (D. alkaphilum cGAS/DncV-like nucleotidyltransferase from clade B). We identified homologues of DaCdnB and selected a subset for characterization (Figure 1E, S1B, Table S2). Homologues from Bacillus thuringiensis (BtCdnB) and Clostridium botulinum (CbCdnB) synthesized oligonucleotide products that migrated similarly to DaCdnB. These products were also resistant to nuclease P1 (Figure S1C). A consistent issue with evaluating CD-NTases is that we do not know their cognate activator provided by phage during infection. In some cases this can be overcome by supplementing the reaction with manganese (Mn²⁺), which activates nucleotidyltransferases such as cGAS and CD-NTases.^20,29,30 Under these conditions, BtCdnB and CbCdnB produced a reaction product with an altered migration pattern, thus we hypothesized that Mn²⁺ enables CdnB to complete the reaction from linear pppG(2′,5′)pA to 2′,3′-cGAMP (Figure S1B).

To confirm that the final reaction product of BtCdnB and CbCdnB was indeed 2′,3′-cGAMP, we subjected the enzyme reaction products to a commercial ELISA assay. Both reaction products, along with our human cGAS control, produced a significant signal (Figure 1G). The signal was highly specific for 2′,3′-cGAMP as we did not detect cross-reactivity for other isoforms of cGAMP, linear pppG(2′,5′)pA, cyclic di-AMP, or cyclic di-GMP when using synthetic standards as controls (Figure 1F and 1G). We further validated that the products were 2′,3′-cGAMP by treating the reaction products with VacV Poxin, a 2′,3′-cGAMP-specific hydrolase^31,32, which significantly reduced the detectable signal (Figure 1G). Collectively these data indicate that the BtCdnB and CbCdnB enzymes synthesize 2′,3′-cGAMP.

2′,3′-cGAMP signaling restricts phage replication by initiating bacterial cell lysis

BtCdnB is encoded in a type I CBASS system (Figure 2A), a two-gene system from B. thuringiensis Bt407 (hereafter referred to as BtCBASS) which consists of the CD-NTase and a predicted transmembrane effector named CD-NTase-associated protein 14 (Cap14).^16,18,33 We expressed the BtCBASS system on the chromosome of Bacillus subtilis PY79 under control of its native promoter (Figure 2B). Next, this strain and associated negative controls were challenged with phages and plaque formation was measured using a modified double-agar overlay assay. BtCBASS protected B. subtilis by over 10⁴-fold against phages SPP1 and phiB002 as compared to empty vector or BtCBASS expressing a catalytically inactive BtCdnB (BtCBASS^CD) but did not protect against phages SPβ or phi29 (Figure 2C–F).

We then focused on the kinetics of SPP1 restriction by BtCBASS during liquid culture infection. Infection with SPP1 at a multiplicity of infection (MOI) of 0.04 or 0.4 did not impact growth of strains expressing BtCBASS (Figure 2G) and SPP1 was unable to replicate in these conditions as measured by plaque forming units (PFU, Figure 2H). In contrast, strains expressing an empty vector or BtCBASS^CD decreased in OD₆₀₀ and the PFU of SPP1 increased indicating successful production of infectious virions (Figure 2G, H). At an MOI of 4, which ensured that nearly every bacterium was infected, strains expressing BtCBASS decreased in OD₆₀₀ at 60 minutes post-infection and did not produce infectious progeny whereas strains harboring an empty vector or BtCBASS^CD decreased at 90 minutes post-infection and displayed an increase in SPP1 PFUs indicating successful replication (Figure 2G, H). These results suggested that BtCBASS activates premature cell lysis that prevents completion of the SPP1 replication cycle.

We used fluorescence microscopy to visualize B. subtilis during SPP1 infection at an MOI of 4. At 30 minutes post-infection, bacteria expressing empty vector, BtCBASS, and BtCBASS^CD appeared similar. However, at 60 minutes post-infection, bacteria expressing BtCBASS took up propidium iodide and displayed clearly observable signs of membrane destruction as compared to strains expressing empty vector or BtCBASS^CD (Figure 2I–L). By 90 minutes post-infection, all strains, including empty vector and BtCBASS^CD displayed signs of gross cell lysis (Figure 2I–J). These data suggest that at 60 minutes BtCBASS initiates a premature cell lysis program that is distinct from the phage-mediated cell lysis that occurs at 90 minutes in susceptible strains. These results are consistent with an abortive infection mechanism, in which the antiphage system provides population-level immunity by preventing successful viral replication, in this case by inducing lysis of the infected host.^34,35

We next wanted to confirm that 2′,3′-cGAMP was produced in vivo in response to phage infection. Signal for 2′,3′-cGAMP was only detectable in cell lysates of strains expressing BtCBASS infected with SPP1 or phiB002 as compared to strains expressing empty vector or BtCBASS^CD (Figure 2M). These data confirm that the BtCBASS system produces 2′,3′-cGAMP in vivo, during phage infection and executes host cell lysis to restrict viral replication.

Architecture of Cap14 bound to 2′,3′-cGAMP

To determine the mechanism for 2′,3′-cGAMP-induced cell lysis, we focused on the Cap14 effector encoded in the BtCBASS operon (BtCap14). BtCap14 encodes an N-terminal SAVED-fused 2TM (Saf-2TM) domain that is predicted to have at least 2 transmembrane helices.¹⁸ The C-terminus is a globular SMODS-Associated Fused to Various Effector Domains (SAVED) domain. Both predictions were supported by structure prediction and analysis of the amino acid sequence with TMHMM (Figure 3A, B, S3A).^18,36,37 The Saf-2TM domain has no homology with any well-characterized proteins, whereas SAVED domains are known to function as cyclic oligonucleotide binding domains in CBASS and Type III CRISPR system effectors where they regulate activation of a fused enzymatic domain with a defined function.^{25,27,38–40} Saf-2TM-SAVED effectors are widespread in bacteria, but their mechanism of defense is unclear.¹⁸

We purified full-length BtCap14 in detergent micelles and determined that it bound 2′,3′-cGAMP with a K_d of 12.4 ± 5.4 nM but did not bind other cGAMP isoforms (Figure 3C). This confirmed that BtCap14 is a direct receptor for 2′,3′-cGAMP. We then visualized BtCap14 using single particle cryo-electron microscopy (cryo-EM) to gain insight into its activation by 2′,3′-cGAMP. BtCap14 was exchanged into amphipol A8–35 to increase stability, and gel filtration suggested the presence of oligomers (Figure S2C, D). 2D classification confirmed that in absence of nucleotide, BtCap14 exists in assemblies composed primarily of dimers, with a small fraction of linearly stacked multimers (Figure 3D, S2E). Incubation with 2′,3′-cGAMP resulted in the formation of predominantly filamentous assemblies of parallel-stacked dimers (Figures 3D, S2F).

We determined a 3D reconstruction of the BtCap14 SAVED domain multimer bound to 2′,3′-cGAMP to ~3.4 Å (Figure 3E–F, Figure S3, Table S1). The protomer consists of two antiparallel Cap14 monomers forming a dimer that further stacks linearly through opposing faces of the SAVED domains above the anticipated plane of the membrane (Figure 3E–F). Density for the Saf-2TM domain was visible but unable to be resolved to high resolution (Figure 3E). A single 2′,3′-cGAMP molecule was identified in each SAVED domain at the interface between individual protomers (Figure 3F, G, S3). In the binding pocket, 2′,3′-cGAMP is cradled by aromatic residues Y345 and Y116, which ᴨ-stack with the adenosine and guanosine bases (Figure 3G, S3). The charged residues S322 and K213 appear to interact with the phosphate backbone from loops on opposing sides of the binding site (Figure 3G).

Supporting these findings, Cap14 mutations K213E, S322A, and Y345A disrupted 2′,3′-cGAMP binding in vitro, and ablated phage protection by BtCBASS in vivo (Figure 3H, S4A). We were unable to express Cap14^Y116A, however, the Y116A mutation compromised phage defense by over 10-fold. These mutations did not disrupt BtCap14 protein levels B. subtilis (Figure S4B). Collectively, these data indicate that BtCap14 binds 2′,3′-cGAMP which forms a filamentous assembly required for BtCBASS-mediated immunity.

Activated Cap14 disrupts artificial membranes

Cap14 filaments presumably cluster the Saf-2TM domains, which do not have a known biochemical activity but were previously hypothesized to form membrane pores.¹⁸ Alphafold models of BtCap14 multimers showed that the soluble SAVED domains form a linear filament strikingly similar to our experimentally determined structure, whereas the putative transmembrane domains cluster to form a predicted pore-like structure at higher multimeric assemblies (Figure 4A, S4C–E). These observations coupled with our findings of BtCBASS-induced cell lysis suggested that activated BtCap14 may directly disrupt cell membranes.

To test whether BtCap14 disrupts membranes directly, we reconstituted recombinant BtCap14 into artificial lipid bilayers and performed electrophysiology in symmetric salt solutions.^41,42 In this assay, the voltage (V) generates a driving force across the membrane and current is monitored as an output for membrane disruption events (Figure S5A). Reconstitution of BtCap14 alone did not result in any observable deviation of the current from baseline indicating intact membranes, but upon addition of 2′,3′-cGAMP to the cis chamber, we observed increases in current indicative of membrane disrupting events (Figure 4B).⁴¹ The current/voltage (I/V) relationship of BtCap14 in symmetric solutions of KCl confirmed that the measured currents were strictly dependent on 2′,3′-cGAMP, and not due to artifactual disruption of the membrane by BtCap14 reconstitution alone (Figure 4C). Furthermore, the BtCap14 variants that we determined to have disrupted 2′,3′-cGAMP binding did not exhibit activity in the presence of nucleotide, confirming that this membrane disrupting activity depends on the ability of BtCap14 to bind 2′,3′-cGAMP (Figure 4C). The observed current traces displayed fluctuations and variability that did not resemble classical single-channel behavior observed for ion channels or porins^42–44, suggesting that BtCap14 likely disrupts membranes through a different mechanism.

The current-voltage relationship demonstrated that BtCap14 currents were only observed at positive voltages (Figure 4C). This suggested that BtCap14-mediated membrane disruption may depend on ion selectivity and/or directionality.⁴² To test this, we substituted the recording solution in both cis and trans sides with cations and anions of varying sizes and measured BtCap14 activity with 2′,3′-cGAMP. The current/voltage profiles and current trances were largely unaffected when KCl was replaced with NaCl or tetraethylammonium chloride (TEA-Cl) (Figure S5B, C). The ionic radius of TEA⁺ (~ 3.85 Å) is a much larger than K⁺ (~ 1.3 Å) or Na⁺ (~ 1 Å) and is often impermeable to known cation channels.⁴⁵ In contrast, when KCl was substituted with potassium gluconate (K-Gluc) we observed a complete lack of activity for BtCap14 in the presence of 2′,3′-cGAMP (Figure S5B, C), which could be rescued by buffer exchange of KCl into the cis chamber (Figure S5D). BtCap14 inserts into the lipid bilayer in multiple orientations during these assays, however, 2′,3′-cGAMP was only added to the cis chamber and does not cross membranes due to its polar nature. Therefore, only BtCap14 with SAVED domains oriented on the cis side of the bilayer can be activated. Thus, the cis chamber represents the bacterial cytosol, where the SAVED domain would be accessible to 2′,3′-cGAMP produced by CdnB during infection.

Our data suggested that BtCap14 only disrupts membranes when activated by 2′,3′-cGAMP, which subsequently mediates Cl⁻ flux potentially resulting in osmotic destabilization. This would explain the cell lysis that we observed during phage infection of BtCBASS-containing bacteria (Figure 2). BtCap14-mediated Cl⁻ efflux from the cell is expected to depolarize the bacterial membrane. We tested this hypothesis by measuring changes in membrane potential of B. subtilis during SPP1 infection using 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃(5)). DiSC₃(5) integrates into hyperpolarized membranes, where its fluorescence is quenched. When membranes are depolarized, DiSC₃(5) is released from the membranes and into the medium resulting in a measurable fluorescence. SPP1-infected strains containing BtCBASS displayed an increase in DiSC₃(5) signal at 60 minutes post-infection whereas strains expressing BtCBASS^CD had no observable change (Figure 4D). This is consistent with the timing of membrane disruption and cell lysis that we observed by OD₆₀₀ and fluorescence microscopy (Figure 2). By 90 minutes post-infection both strains displayed high DiSC₃(5) release, which was expected as BtCBASS^CD strains undergo phage-mediated lysis.

We attempted to identify amino acid residues in the BtCap14 Saf-2TM domain required for membrane disruption, but most mutations either disrupted BtCap14 expression or had no effect on BtCBASS-mediated protection against SPP1 (Figure 4E, S5E). Variants within the predicted cytosolic loop between TM1 and TM2 (N24A), and within the predicted linker region between Saf-2TM and the SAVED domain (K103 and K105) resulted in disrupted protein expression (Figure 4E, S5F–H). Structure modeling of the BtCap14 dimer predicts these residues are at the interface of the protomers, and lack of mutant protein expression may highlight the importance of these linker regions in BtCap14 stability (Figure S5I).

A single variant, BtCap14^D54K, resulted in an intermediate phage protection phenotype against phage SPP1 without disrupting protein expression (Figure 4E, S4B). D54 is located on a predicted extracellular loop (Figure S5I–J). Structure predictions of BtCap14^D54K multimers demonstrated an assembly of the Saf-2TM domains that displayed altered transmembrane architecture from the wild-type BtCap14 (Figure S5J). These data suggest that D54 potentially plays a role in the assembly of the fully active BtCap14 complex.

Construction of a reprogrammed SAVED effector

The SAVED domain of BtCap14 adopts a similar structure to other SAVED domains, which are encoded as fusions with a wide range of enzymatic effector domains.¹⁸ Intriguingly, TIR-SAVED NADases³⁸, SAVED-CHAT proteases⁴⁰, and HNH-SAVED nucleases²⁷ also form nucleotide-activated multimers which is required for activation of their effector domains, similar to our hypothesis for activation of the Saf-2TM domain in cGAMP-activated BtCap14 filaments.

Although effector domains vary among bacteria, the overall structure and assembly of experimentally studied SAVED domains is highly conserved. We hypothesized that SAVED domains and effector domains are functionally modular and that a universal role for SAVED domains is coordinating ligand dependent oligomerization to cluster effector domains independent of their downstream biochemical activity. We tested this hypothesis by constructing chimeric proteins that replaced the SAVED domain of HNH-SAVED effectors with Clade B Cap14 SAVED domains. HNH nucleases were selected because nuclease activity is readily assayed by visualizing DNA substrates using gel electrophoresis and fluorescent reporters. Most chimeras displayed poor expression or solubility; however, we arrived on a functional chimera between the SAVED domain of Clostridium botulinum (CbCap14) from clade B02 and the HNH domain of Parageobacillus thermoglucosidasius Cap5 (GsCap5) from clade B07 (Figure S6A, B) which natively responded to both 2′,3′-cGAMP and 3′,2′-cGAMP to degrade DNA (Figure S6C).^20,27 We named the resulting hybrid enzyme Chimera10, which was readily expressed and purified from E. coli (Figure 5A, S6A). In the absence of nucleotide, Chimera10 did not degrade a dsDNA substrate, however, when incubated with 2′,3′-cGAMP we observed DNA degradation (Figure 5B). No other cGAMP isomers or common cyclic dinucleotides activated DNA cleavage by Chimera10 even at a concentration as high as 1 μM, demonstrating the specificity of Chimera10 (Figure 5B). Chimera10 also displayed activity using as little as 1 nM 2′,3′-cGAMP (Figure 5C), demonstrating sensitivity similar to naturally occurring SAVED-domain containing proteins.^27,38–40

Figure 5. — (A) Strategy for engineering a 2′,3′-cGAMP-activated nuclease. The N-terminal HNH domain from GsCap5 was fused to the SAVED domain from CbCap14 to construct Chimera10. Numbers represent amino acid positions between proteins.

(B) Agarose gel electrophoresis of linear double stranded DNA incubated with Chimera10 and buffer alone (–) or 1 μM of the indicated cyclic dinucleotide for 30 minutes at 37°C. Data are representative of n = 3 biological replicates.

(C) Same as (B) but Chimera10 was incubated with the indicated concentration of synthetic 2′,3′-cGAMP. Data are representative of n = 3 biological replicates.

(D) DNAse alert activity assay of Chimera10 using the indicated synthetic nucleotides at 1 μM. Chimera10 activity is fold change over vehicle control (buffer only, no nucleotide added). Data are mean ± SEM for n = 3 technical replicates pooled from n = 2 biological replicates. A one-way ANOVA with Dunnett’s multiple comparison test was used, * P ≤ 0.05.

(E) Data as in (D) for the indicated concentrations of 2′,3′-cGAMP. Data are mean ± SEM for n = 3 technical replicates and are representative of n = 3 biological replicates. A one-way ANOVA with Dunnett’s multiple comparison test was used, * P ≤ 0.05.

(F) Chimera10-based detection of 2′,3′-cGAMP from bacterial cell lysates of *B. subtilis* expressing CBASS or CBASS^CD with or without infection with SPP1 or phiB002 using DNAse Alert. Chimera10 activity is fold change over CBASS^CD. Data are mean ± SEM for n = 3 technical replicates pooled from n = 2 biological replicates. A one-way ANOVA with Dunnett’s multiple comparison test was used, * P ≤ 0.05.

We adapted Chimera10 for a 96-well microplate assay using a fluorescent DNA oligonucleotide substrate (DNAse Alert) in which substrate cleavage decouples a fluorophore from a quencher and results in increased fluorescent signal, thus reporting nuclease activity (Figure S6D). Using this approach, Chimera10 displayed specificity for 2′,3′-cGAMP, was not responsive to other cyclic oligonucleotides at 1 μM and could detect as low as 1 nM of synthetic 2′,3′-cGAMP, similarly to our gel electrophoresis assays (Figure 5D–E). We next tested whether Chimera10 could be applied to detect 2′,3′-cGAMP in a biological sample and interrogated the phage-infected B. subtilis cell lysates used for our earlier ELISA-based measurements (Figure 2M). Chimera10 activity significantly increased in response to BtCBASS cell lysates compared to BtCBASS^CD lysates and suggested the final of 2′,3′-cGAMP concentration corresponded to ~1–10 nM (Figure 5E–F) similar to our measurements by ELISA (Figure 1G). These data demonstrate that SAVED domains can be functionally exchanged to activate alternative enzymatic effector domains.

STING-like effectors disrupt membranes in CBASS immunity

Our findings that BtCap14 mediates cell lysis motivated us to investigate membrane disruption by other transmembrane CBASS effectors. Recently mammalian STING, was proposed to function as a proton channel^46,47 and also forms 2′,3′-cGAMP-induced transmembrane filaments of stacked dimers^48–51, similar to BtCap14. Bacteria encode homologues of STING within CBASS operons.^15,17,52 The prokaryotic STING domain is predominantly fused to Toll/Interleukin-Like Receptor (TIR) domains (Cap12) or to 2TM transmembrane domains (Cap13).^17,52 Cap12 effectors bind nucleotide through the STING domain, which activates the TIR domain to hydrolyze NAD⁺.^17,53 Cap13 also binds cyclic dinucleotides, but the effector function is unknown.¹⁷ Structure predictions of Cap13 suggested an amphipathic arrangement of the 2TM domain with a charged central lumen (Figure S7). The structural parallels between Cap14 and human STING led us to hypothesize that Cap13 also mediates membrane disruption to restrict phage.

We selected a Type I CBASS system from Flavobacterium (FsCBASS) which encodes a Cap13 effector in an operon with a CdnE synthase.¹⁷ We expressed FsCBASS in B. subtilis and observed robust protection against phages SPP1 and phiB002 (Figure 6A–E). In liquid infection assays, FsCBASS appeared similar to BtCBASS and provided robust defense against phage SPP1 by initiating cell death and abortive infection (Figure 6F–G). At an MOI of 4, FsCBASS displayed a rapid drop in OD₆₀₀ at 60 minutes post-infection as compared to an empty vector and did not produce infectious SPP1 progeny (Figure 6F–G). Visualization of cells during infection revealed that FsCBASS activation resulted in membrane disruption as seen by gross morphological defects and increased propidium iodide uptake compared to the empty vector at 60 minutes post-infection (Figure 6H–K). Collectively these data reveal that membrane disruption is also a feature of Cap13 STING-like effectors in CBASS immunity.

Figure 6. — (A) Type I CBASS operon from *Flavobacterium sp*. (FsCBASS) encoding CdnE and a 2TM-STING (Cap13) effector.

(B–E) Double agar overlay assay of *B. subtilis* expressing empty vector (EV) or FsCBASS, challenged with serial dilutions of phage (B) SPP1, (C) phiB002, (D) phi29, or (E) SPβ. Images are representative of n = 3 biological replicates. Data are the mean ± SEM of n = 3 biological replicates.

(F) Growth of *B. subtilis* expressing the indicated systems in liquid culture measured by optical density (OD₆₀₀) after infection with phage SPP1 at the indicated MOI. Data are representative of n = 3 biological replicates.

(G) Time course of SPP1 replication during liquid culture infection of *B. subtilis* expressing the indicated systems at the indicated MOIs, as in (F). Data are the mean ± SEM of n = 3 biological replicates.

(H) Fluorescence microscopy of *B. subtilis* strains expressing the indicated systems after liquid-culture infection with SPP1 at an MOI of 4 at the indicated timepoint. FM143x (green) was used to stain the membrane, and propidium iodide (PI, false colored magenta) was used to probe membrane disruption. Dashed boxes are views shown in (I or J). Images are representative of > 10 fields of view and representative of n = 3 biological replicates. Scale bar = 10 μm.

(I and J) Zoomed-in fields of view (FOV) at 60 minutes post-infection with SPP1 for *B. subtilis* expressing the indicated system. FOV1 is from (H), FOV2 and 3 are additional representatives. Scale bar = 5 μm.

(K) Percentage of PI⁺ cells at 60 minutes post-infection from analysis as in (H). Data are the mean ± SEM from 18 fields of view analyzed from n = 3 biological replicates, 6 fields of view per replicate. A one-way ANOVA with Dunnett’s multiple comparison test was used, * P ≤ 0.05.

Discussion

In this study we discovered a 2′,3′-cGAMP signaling system in bacteria that restricts phage replication by triggering premature cell lysis via a Cap14 effector. These findings expand the role of 2′,3′-cGAMP-mediated immune signaling beyond metazoan cGAS-STING and extend it to bacterium-phage conflict. There are multiple correlations between the microbiome and human health.^54–57 An intriguing possibility is that bacteria serve as a reservoir of 2′,3′-cGAMP which may activate STING signaling in cancer, infection, or autoimmunity. It is plausible that bacterial 2′,3′-cGAMP could be released into the extracellular space during CBASS-mediated bacteriolysis. Extracellular 2′,3′-cGAMP is imported by mammalian cells and activates STING leading to amplified immune responses.^5–8,58 STING binds 2′,3′-cGAMP with high affinity (K_D of ~4 nM)⁵⁹, therefore even minute amounts of exogenously produced 2′,3′-cGAMP could have a significant impact in shifting the balance of STING-mediated signaling, which may be beneficial or detrimental to the host depending on cellular context. It will be important to understand the distribution and abundance of 2′,3′-cGAMP-generating CBASS systems in the constituents of the mammalian microbiome. It will also be of particular significance to identify the triggers that activate these CBASS systems in their native bacterial species during in vivo colonization of mammalian hosts.

Our investigation revealed that Cap14 is a membrane-disrupting effector protein activated by 2′,3′-cGAMP-induced oligomerization. The Cap14 effector was hypothesized to function as a poreforming domain.¹⁸ and subsequent studies observed gross membrane disruption for CBASS transmembrane effectors Cap14, Cap15 (S-2TMβ), and Cap16 (2TM+NUDIX) but direct evidence and mechanism of membrane disruption by Cap14 was not explored.^33,60 Our electrophysiological analysis of full-length BtCap14 showed that the protein directly disrupts membranes in response to 2′,3′-cGAMP and in the absence of other cellular components. We cannot conclude from these data whether activated BtCap14 forms a classically-defined channel or whether the observed currents represent membrane disruption and ion leakage as a result of the Saf-2TM domain rearranging in the membrane after cGAMP-induced oligomerization of the SAVED domains. The asymmetric current-voltage relationship and apparent dependence on Cl⁻ distinguishes Cap14 from other proteins that form large nonselective pores.^61,62 Our electrophysiology complements our observations of membrane depolarization, membrane blebbing, and cell lysis during phage infection. We hypothesize that activation of BtCap14 causes cell lysis directly through osmotic destabilization or indirectly through collapse of membrane potential. The exact mechanism of Cap14-mediated membrane disruption remains to be determined; however, our data suggests that it is likely distinct from known membrane-perforating cell death effectors. Cap14 effectors display sequence diversity and are found in a wide variety of bacteria, thus, homologs may exhibit different ion specificity, ligand selectivity, and additional regulatory mechanisms. Saf-2TM-SAVED effectors are also encoded within CRISPR loci, suggesting roles in bacterial immunity beyond CBASS.¹⁸

Filament formation by cGAMP-bound BtCap14 is consistent with a growing theme of supramolecular assembly in innate immunity.⁶³ Cyclic oligonucleotide binding to SAVED and STING domains initiate oligomerization, which is required for activity of fused effector domains such as TIR NADases and CHAT proteases by rearranging the effector domains to build a composite active site contributed by individual monomers.^{17,27,38,40,53} We propose that for Cap14, cGAMP-induced oligomerization of the SAVED domain initiates structural rearrangements of the Saf-2TM domain required to disrupt membrane integrity. Structure-guided Alphafold predictions suggest that multimerization may allow assembly of multiple small pores within the filament that may eventually mature into a larger complex that results in rapid lysis. It will be of particular importance to identify the minimal unit required to form a membrane disrupting complex, and whether this is conserved within the Cap14 family.

Our investigation enabled us to reprogram an HNH-SAVED CBASS effector with a SAVED domain from a Saf-2TM-SAVED effector. These data demonstrate that SAVED domains are modular and may provide a promising platform to design nucleotide-responsive proteins of choice. This portability of SAVED is likely key to the evolution of CBASS systems because it allows rapid generation of receptor/effector combinations and/or altering specificity for nucleotide second messengers, which may be an advantage for bacteria in their escalating arms race with phages.^18,52 Altering nucleotide preference for CBASS systems may also be necessary to accommodate competing cyclic nucleotides produced by other phage defense systems.^64,65 Chimera10 provides proof-of-principle for constructing programmable cyclic dinucleotide sensors. Detection of 2′,3′-cGAMP is limited to STING-based sensors⁶⁶, RNA-based probes⁶⁷, mass-spectrometry, and NMR.⁶⁸ Chimera10 enables measurement of 2′,3′-cGAMP that is exquisitely selective over other cGAMP isomers. We envision that Chimera10 could applied for high-throughput discovery of additional 2′,3′-cGAMP synthases, detection of 2′,3′-cGAMP production in unexpected contexts, and in mammalian cells to monitor cGAS activation in disease models or patient samples.

The structure and mechanism of Cap14 is distinct from other membrane depolarizing/disrupting immune effectors such as Cam1⁶⁹, Csx28⁶², gasdermins^61,70,71, which form structures with defined pore-like architectures. The mechanism of Cap14 shares some similarities with human STING, which also forms dimers, and multimerizes upon binding cGAMP; albeit by a different binding mechanism.^48,49 Recently, human STING was proposed to function as a proton channel.^46,47 Coupled with our observation for the bacterial STING homologue Cap13, membrane disruption may be a conserved feature of TM-STING receptors from bacteria to eukaryotes. We propose a model in which Cap14 and Cap13 effectors bind cyclic dinucleotides produced by CD-NTases during phage infection, form oligomeric assemblies, and mediate membrane disruption to prevent phage replication (Figure 7). The proposed proton channel activity of human STING is key for activation of non-interferon mediated pathways and may be an ancient mode of signaling.^46,47 While the nucleotide binding domains of human STING and Cap13 are unambiguously related, it is unclear if the two TM domains of Cap13 share a common ancestor with the four TM domains of human STING.^52,72 It will be interesting to determine how (and if) STING-mediated membrane disruption evolved from its bacterial counterparts as a primordial mechanism to initiate innate immune signaling.

Figure 7. — Phage infection triggers activation of the CD-NTase through an unknown mechanism and results in production of cyclic dinucleotides (CDNs). CDNs bind to Cap14 which initiates filament formation and subsequent membrane damage resulting in cell lysis. A similar model is postulated for Cap13 which also induces membrane damage through putative filament formation. Membrane damage likely results in premature cell lysis, preventing completion of the phage replication cycle.

Limitations of the study

A limitation of the study is defining whether Cap14 functions as a channel or instead disrupts membrane integrity in an alternative manner. Our in vitro data confirm that Cap14 reconstituted in planar lipid bilayers produces measurable currents in response to 2′,3′-cGAMP binding. However, our electrophysiology data did not show single channel traces with discrete “open” and “closed” steps with defined lifetimes as would be seen for classical channel-forming proteins. Further, we were unable to locate a channel-disrupting mutation that did not impact protein expression. Although we observed Cl⁻ selectivity using electrophysiology, it is possible that additional regulatory factors and/or ion selectivity occur in vivo. These features of Cap14 are challenging to investigate due to the rapid onset of BtCap14-induced cell lysis but remain an area for future investigation. For these reasons we define Cap14 as a membrane-disrupting receptor, as opposed to a channel or pore at this time. Further study on BtCap14 and its homologues in stable membrane mimetics in vitro and or cryo-electron tomography of in vivo complexes offer alternative approaches to resolve the structural basis for assembly.

Finally, our findings do not fully address the scope of SAVED domain modularity beyond Chimera10. In the future, it will be important to determine the range of compatibility for different SAVED domains with different effector domains in generating functional fusion proteins.

Resource Availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Aaron T. Whiteley (aaron.whiteley@colorado.edu).

Materials availability

Materials generated in this study will be made available on request, but we may require a completed materials transfer agreement if there is potential for commercial application.

Data and code availability

Coordinates of the BtCap14 + 2’3’-cGAMP structure are publicly available in the Protein Data Bank (PDB) under the accession number 9OM7, and the cryo-EM density map is available in the Electron Microscopy Data Bank (EMDB) under the accession number EMD-70609.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this work is available from the lead contact upon request.

STAR METHODS

Experimental model and subject details

Bacterial strains and culture conditions

All bacterial strains used in this study are listed in Table S3. E. coli was cultured in Lysogeny Broth (LB) consisting of 1% tryptone, 0.5% yeast extract and 0.5% NaCl shaking at 37 °C at 220 rpm in 4 ml media in a 14 mL culture tube unless otherwise indicated. For plasmid maintenance, the following concentrations of antibiotics were used: carbenicillin (100 μg/ml), chloramphenicol (20 μg/ml). Bacillus subtilis PY79 was grown in LB + 10 mM MgCl₂, 10 mM CaCl₂, and 0.1 mM MnCl₂ (LB phage medium) in a maximum volume of 2 ml in a 14 ml culture tube unless otherwise indicated. When needed, spectinomycin (100 μg/ml) was used for selection. E. coli Omnipir was used for molecular cloning as described previously,⁸¹ E. coli BL21(DE3) and E. coli C43⁸² were used for recombinant protein expression. All bacterial strains were stored at −70 °C in LB + 15% glycerol.

Bacteriophage isolation and amplification

All phages used in this study are listed in Table S3. Bacillus subtilis phage SPP1 was a kind gift from Daniel Kearns. SPβ and phi29 were obtained from the DSMZ stock collection. Phages were plaque purified on B. subtilis PY79 in LB phage medium, mixed with B. subtilis PY79, and amplified either using agar plates or liquid media. For agar plate amplification phage was scraped from the plates using a cell scraper and 100 μl of chloroform was added to lyse remaining bacteria, mixed, and allowed to settle. For liquid amplification of phage stocks, 50 ml of B. subtilis PY79 was grown in LB phage medium at 37 °C, 220 rpm to an OD of 0.2 and then infected at an MOI of 0.04 with the indicated phage and the culture was allowed to grow until the culture crashed indicating phage-mediated lysis. Phage stocks were stored at 4 °C. Isolation of the wild Bacillus subtilis phage phiB002 was performed as follows: soil from outside of the University of Colorado Boulder Jennie Smoly Caruthers Biotechnology Building was collected and mixed with LB for one hour and then B. subtilis PY79 at log phase (OD₆₀₀ = 0.4) was added to the mixture and allowed to grow for 3 hours. The mixture was then filter-sterilized and chloroform-treated, and single plaques were isolated on B. subtilis PY79. The phage phiB002 was isolated, plaque-purified three times, and then stored as described above. All phage stocks were validated using whole genome sequencing (SeqCoast) and were aligned to reference genomes in Geneious Prime. Phage phiB002 is 99.5% identical to Bacillus phage Grass (NC_022771.1), 99% identical to Bacillus phage SBSphiJ7 (OM982674.1), and 99% identical to Bacillus phage SBSphiJ (LT960608.1).

Method details

Cloning and plasmid construction

All plasmids used in this study are listed in Table S4. All DNA inserts encoding for the indicated proteins or operons were amplified using Q5 polymerase, separated by agarose gel electrophoresis, visualized by SYBR-SAFE, and purified by gel extraction. pLOCO2 vectors constructed for Bacillus subtilis expression used inserts containing SbfI / NotI cut sites, pET vectors for 6xhis-SUMO fusion proteins were cloned with BamHI / NotI sites, pET vectors for C-terminal-6x-his tag proteins were cloned with NdeI / BamHI sites.^20,81 Plasmids were assembled via NEB HiFi Assembly using the manufacturers protocol for 1 hour at 50 °C. The entire reaction was transformed into chemically competent E. coli OmniPir using heat shock, outgrown in SOC media for 1 hour at 37 °C at 200 RPM, followed by selection on LB agar supplemented with carbenicillin at 100 μg/ml (Carb100) + 1% glucose via incubation overnight at 37 °C. Clones were grown in LB + 1% glucose + Carb100 overnight and prepared as freezer stocks. Clones were validated using colony PCR, plasmids were isolated by miniprep and further validated using Sanger Sequencing (Quintara Biosciences or Genewiz / Azenta) or whole-plasmid sequencing (Plasmidsaurus). Plasmid map construction, primer design, and sequencing analysis was performed using the Geneious Prime software suite.

Thin layer chromatography and nuclease P1 treatment

Thin layer chromatography and radioactive CD-NTase enzymatic assays were performed as described previously.²⁰ Briefly, recombinant CD-NTase reactions consisting of 50 mM CAPSO pH 9.4, 50 mM KCl, 5 mM Mg(OAc)₂, 1 mM DTT, ≤5% glycerol, 250 μM individual NTPs, and trace amounts of [α³²P] NTP were started with addition of 1 μM enzyme. When indicated, Mn²⁺ was added. cGAS reactions were carried out with Tris at pH 7.5 and supplemented with 1 μM ISD45 dsDNA. Reactions were incubated for 2 h at 37 °C. Reactions were stopped by addition of 5 U of calf intestinal phosphatase, which removed triphosphates on remaining NTPs and converted the remaining nucleotide α³²P to ³²P_i enabling the visualization of cyclized species. For nuclease P1 digestion, 5 μL of the enzyme reaction was supplemented with 0.5 μL of 10× Nuclease P1 Reaction Buffer and treated with 50 U of Nuclease P1 (NEB), in addition to calf intestinal phosphatase. F-coated PEI–cellulose TLCs (Millipore) were developed in 1.5 M KH₂PO₄ (pH 3.8).

Bioinformatic identification of CD-NTases and phylogenetic tree construction

All CD-NTase numbers and clade assignments are based on their original description.²⁰ The amino acid sequence for CD-NTase005 (DaCdnB-02) was used as a BLAST seed in Geneious Prime to identify homologous sequences. Hits with an E-value of 1-e⁻¹⁰⁰ or lower and a % Pairwise identity over 40% were selected for a total of 417 AA sequences. A representative species of each unique organism was selected for tree construction and display. These results were then used to generate a global alignment in Geneious Prime with no outgroup, using Jukes-Cantor distance model with neighbor-joining. Species deemed relevant to human health, or agriculture were highlighted, and redundant species were removed for brevity. The full list of CD-NTases identified in this study are available in Table S2.

Recombinant protein expression and purification

MBP-CD-NTase and MBP-human cGAS proteins used in Figure 1A–B and S1A–C, were purified as described previously.²⁰ Elsewhere, for purification of MBP-human cGAS, the plasmid encoding MBP-TEV-hcGAS (pAW1385) was transformed into BL21(DE3) and selected on LB-agar supplemented with chloramphenicol at 20 μg/ml (Cm20) + 1% glucose in an incubator overnight at 37°C. A single colony was grown in 50 ml of LB Cm20 at 37 °C until the optical density at 600 nm (OD₆₀₀) reached 0.5, and then 0.2% arabinose was added to induce expression and the culture was moved to 20 °C for 24 hours. Cells were harvested and lysed as described above and were run over 5 ml of Amylose affinity resin (NEB), washed with 100 ml of 20 mM sodium phosphate pH 7.2, 500 mM NaCl, 1 mM DTT, and eluted in the same buffer + 30 mM maltose monohydrate. Samples were concentrated and stored in 50% glycerol in aliquots at −20 °C until needed.

CD-NTase005 and its homologues were expressed as 6x-his-SUMO fusion proteins in E. coli BL21 Rosetta cells. Plasmids were transformed using heat shock and plated on LB-agar Carb100 / Cm20 + 1% glucose and grown in an overnight at 37 °C. A single colony was then used to inoculate a 50 ml starter culture of LB Carb100/Cm20 + 1% glucose which was grown overnight at 37 °C. The next morning, 10 ml of the overnight culture was used to inoculate 1L of ZYP5052 Studier’s Autoinduction media in 2.5L Thompson flasks (500 ml / flask). Cultures were grown at 37 °C for 8 hours and then switched to 20 °C for 24 hours. Cells were harvested by centrifugation at 4,000 ×g and resuspended in Buffer 1: 50 mM sodium phosphate pH 7.2, 500 mM NaCl, 10 mM imidazole, 1% glycerol, 1 mM DTT + 1 mg/ml lysozyme. Cells were sonicated on ice at an amplitude of 70, for 30 seconds on / off for 10 minutes total. Sonicated lysates were centrifuged at 14,000 ×g for 1 hour at 4 °C to pellet debris, and the supernatant was run over 2 ml of Ni-NTa resin (Thermo Fisher Scientific) equilibrated with Buffer 1. The resin was washed with 100 ml of Buffer 2: 20 mM sodium phosphate pH 7.2, 500 mM NaCl, 20 mM imidazole, 1 mM DTT to remove nonspecific components bound to the resin. Proteins were eluted using Buffer 3: 20 mM sodium phosphate pH 7.2, 500 mM NaCl, 500 mM imidazole, 1 mM DTT. The 6xhis-SUMO tag was cleaved using 6xhis-hSENP2 (produced in-house) via dialysis overnight at 4 °C against 5L of Buffer 4: 20 mM sodium phosphate pH 7.2, 500 mM NaCl, 1 mM DTT using a 10 kDa MWCO dialysis membrane. The cleaved sample was run over 2 ml Ni-NTA to capture cleaved 6xhis-SUMO tag and uncleaved protein, whereas the flowthrough containing the recombinant, tag-free CD-NTase was collected. The purified CD-NTase samples were then concentrated using a 3kDa ultracentrifugation filter at 4,000 ×g at 4 °C to 1 mg/ml and were stored in 50% glycerol at −20 °C until needed. Protein purity was assessed via SDS-PAGE on a 4–20% gel (Genscript) followed by Colloidal Coomassie staining. 6xhis-SUMO-VacV Poxin was expressed in BL21(DE3), and purified and stored identically as described above, but was left as a fusion protein since this does not affect activity. The same protocol was used for expression, purification, and storage of GsCap5 and Chimera10, with the exception that BL21(DE3) was used as the expression host.

Samples were concentrated to ~ 2 mg/ml and buffer exchanged into reaction buffer: 10 mM Tris-HCl pH 7.4, 25 mM KCl, 1 mM DTT using a centrifugal filter with a 3 kDa MWCO (Pall Corporation) and experiments were performed immediately to avoid precipitation of Chimera10, and/or the proteins were stored in 50% glycerol at −20 °C which did not affect downstream activity.

BtCap14 was expressed with a C-terminal glycine-serine linker and 6x histidine epitope tag (GSHHHHHH). For expression of recombinant BtCap14 and its variants, the relevant plasmid was transformed into E. coli C43 competent cells via heat shock and was plated on LB agar + 100 μg/ml carbenicillin with 1% glucose and grown overnight at 37 °C. The following day a single colony was used to inoculate 50 ml of LB + 1% glucose + 100 μg/ml carbenicillin, and the culture was grown overnight for 16 hours at 37 °C at 200 RPM. The 50 ml culture was used to inoculate 4L of ZYP5052 autoinduction medium + 1X Vitamin Mix (Teknova) + 100 μg/ml carbenicillin (Note: the ZY base medium was modified to include 20 g tryptone, 10 g yeast extract, 5 g NaCl for 1 L). Cultures were split into 2.5L Thompson flasks (500 ml / flask) and were grown shaking for 8 hours at 37 °C, 200 RPM and then lowered to 20 °C and grown for an additional 24 hours at 200 RPM. Cultures were harvested at 4,000 ×g and resuspended in 40 ml lysis buffer per liter of culture (50 mM sodium phosphate, 500 mM NaCl, 1% glycerol, pH 7.0 + 1 mM PMSF, 2 μl benzonase, + 1 complete EDTA-free protease inhibitor tablet (Thermo Fisher Scientific) + 1 mg/ml lysozyme). Cells were sonicated on ice as described previously. The sample was centrifuged at 14,000 ×g for 2 hours at 4 °C and the supernatant was discarded. The pellet was resuspended in 20 ml of lysis buffer + 1% n-Dodecyl-B-D-maltoside (DDM / Gold Biosciences) + 20 mM imidazole and incubated for 2 hours at 4 °C on an end-over-end rotor to extract membrane proteins. The sample was then centrifuged at 14,000 ×g for 1 hour at 4 °C to separate cell debris from extracted membrane proteins. The supernatant was run over 5 ml of cobalt resin (Thermo Fisher Scientific) equilibrated with lysis buffer + 0.05% DDM + 20 mM imidazole in a gravity flow column. The column was washed with 100 ml of 20 mM sodium phosphate, 500 mM NaCl, pH 7.0 + 0.05% DDM + 20 mM imidazole. The protein was eluted in 20 mM sodium phosphate 500 mM NaCl pH 7.4 + 500 mM imidazole + 0.05% DDM and dialyzed with a 10 kDa MWCO dialysis tubing against 20 mM sodium phosphate 500 mM NaCl pH 7.0 overnight at 4 °C. BtCap14 variants were purified in the same manner. For gel filtration the sample was concentrated to 500 μl and run over a Superdex 200 column equilibrated with 20 mM sodium phosphate pH 7.0, 500 mM NaCl + 0.05% DDM. Sample quality was assessed by SDS-PAGE followed by Colloidal Coomassie staining or via immunoblotting. Proteins were stored in 50% glycerol at −20 °C until needed.

Analysis of proteins used in this study can be found in Figures S1F, S2B, S2D, S6A, and S6B.

Immunoblotting

For western blot analysis, samples were separated by SDS-PAGE on GenScript SUREPAGE Bis-Tris 4–20% gradient gels followed by transfer to a PVDF membrane using the Bio-Rad Trans-Blot Turbo transfer system, blocking with Li-Cor Odyssey blocking buffer (Bio-Rad) for 1 hour shaking at room temperature, then probing primary antibody overnight at 1:1,000 concentration in TBST overnight at 4 °C on an orbital shaker, followed by 3× washes for 15 minutes each with TBST, followed by secondary detection with secondary antibody at 1:10,000 in TBST + 0.01% SDS for one hour at room temperature, followed by 3× washes with TBST, and resuspension of the membrane in 1× TBS. The blot was then imaged using a Li-Cor Odyssey CLx Imager.

CD-NTase enzymatic assays and detection of 2′,3′-cGAMP via ELISA

1 μg of recombinant CD-NTases / CdnB enzymes or MBP-hcGAS (activated with 100 ng of dsDNA) were incubated at 37 °C for 18 hours with 250 nM ATP and 250 nM GTP in 10 mM Tris-HCl pH 7.4, 25 mM KCl, 20 mM MgCl₂, 1 mM MnCl₂, 1 mM DTT in 200 μl total volume in in triplicate in a 96 well plate sealed with parafilm. Samples were then split in half, and one set was treated with recombinant VacV poxin for 2 hours at 37 °C. The samples were then analyzed using the 2′,3′-cGAMP ELISA (Arbor Assays) according to the manufacturer’s instructions. The data was analyzed in GraphPad Prism using 4PLC analysis to interpolate the standard curve and unknowns as per the manufacturer’s instructions, and cross-validated using the manufacturer’s template. Synthetic cyclic oligonucleotides were used at 50 nM to determine cross-reactivity and agreed with the manufacturer’s observations. All synthetic oligonucleotides were obtained from BioLog/Axxora except for 2′,2′-cGAMP, which was obtained from Invivogen. The limits of detection and dynamic range were defined by the standard curve, from 20–0.08 nM of 2′,3′-cGAMP. Our results with synthetic nucleotides are consistent with the manufacturers’ observations. For measurement of cGAMP in cell lysates, 200 ml of B. subtilis expressing the indicated CBASS system was grown in a 1L culture flask at 30 °C until an OD of 0.2 was reached and then infected at an MOI of 0 or 4 with the indicated phage. The infection was allowed to continue for 30 minutes, and then cultures were spun down for 10 min at 3,000 ×g and resuspended in 500 μl of sterile water and left on the bench for an additional 30 minutes. Cells were then lysed by bead beating and then boiled for 30 minutes to denature any proteins that may be binding cGAMP, followed by centrifugation at 16,000 ×g for 10 minutes. The supernatant was then used for measurement via ELISA and/or for measurement via Chimera10.

Strain construction in Bacillus subtilis

Plasmids containing indicated defense systems were transformed into Bacillus subtilis PY79 as described previously^83,84 using an integrative vector (pLOCO2) with homology to the 3′ and 5′ ends of the amyE gene and selected on LB + 100 μg / ml Spectinomycin (Spec100) overnight at 37 ° C. Successful transformants were grown overnight in LB + Spec100 and frozen as 15% glycerol stocks for further use. Genomic DNA was isolated from successful transformants using the Qiagen DNAeasy blood and tissue kit and were confirmed for successful integration using PCR.

Bacteriophage challenge and plaque assays on solid media

Bacillus subtilis PY79 strains were grown overnight at 37 °C in LB supplemented with 100 μg/ml spectinomycin (Spec100). Overnight cultures were diluted 1:1,000 in B. subtilis infection media (LB + 10 mM MgCl₂, 10 mM CaCl₂, and 0.1 mM MnCl₂) and grown in 1 ml volume in a 12 ml culture tube (to allow sufficient aeration), at 30 °C at 200 RPM until mid-log phase (OD₆₀₀ = ~ 0.5). 400 μl of B. subtilis was mixed with 4.5 ml of 0.35% LB Lennox top agar (containing 10 mM MgCl₂, 10 mM CaCl₂, and 0.1 mM MnCl₂ stored at 55 °C until use), inverted, and overlaid on an LB agar plate and allowed to cool for 30 minutes to make the B. subtilis-containing top-agar. Serial dilutions of the indicated phages were spot-plated, and plates were grown overnight at 30 °C. Plaques were imaged and counted the next morning and quantified as plaque-forming units per milliliter (PFU/ml) and displayed as histograms in Graphpad Prism. Limit of detection for plaque enumeration assays was defined as 1 plaque at the lowest dilution tested, which corresponded to 50,000 PFU/mL. Limit of detection (LOD) is included on all histograms and represents the lowest dilution where no phage plaques were visible.

Phage growth curve experiments in liquid culture and viral titer measurements

B. subtilis strains were grown overnight at 37 °C in LB + Spec100. Strains were back-diluted 1:100 in 25 ml infection medium (LB + 10 mM CaCl₂, 10 mM MgCl₂, 0.1 mM MnCl₂) and grown at 37 °C at 200 RPM until an OD₆₀₀ of 0.5 – 1 was reached. Strains were then normalized by OD₆₀₀ to 0.2 and distributed into a 96 well plate. Phages were added at the indicated multiplicity of infection (MOI) in identical volumes, and the OD₆₀₀ was measured every two minutes using a Tecan Spark plate reader at 30 °C shaking at 200 rpm. For phage replication measurements, samples were taken at the indicated time-point post-infection and treated with 1:10 volumes of chloroform to lyse bacteria, and the resulting sample was serially diluted and spot plated on B. subtilis PY79 top agar as described above.

BtCap14-cGAMP binding assays by Microscale Thermophoresis

Microscale Thermophoresis (MST) was performed using a NanoTemper Monolith. BtCap14 and related binding variants were diluted from a stock solution into 10 mM HEPES, 1M KCl, pH 7.2, + 0.5% DDM and was labeled using a His-Tag Labeling Kit RED-tris-NTA 2^nd Generation Kit (NanoTemper) via the manufacturers protocol in 1.7 ml low-adhesion tubes. The sample was then spun at 16,000 × g after labeling to remove aggregates. The samples were prepared with the required serial dilutions of cyclic dinucleotides in PCR tubes and were immediately transferred to Nanotemper glass capillaries and analyzed using the NanoTemper monolith. Replicates were analyzed using the MO-Control software to obtain fraction bound, and the resulting data was plotted using GraphPad Prism. Replicates are defined as three separate MST runs.

Domain analysis of BtCap14

The BtCap14 amino acid sequence was analyzed using TMHMM (https://dtu.biolib.com/DeepTMHMM) server³⁷ and MemSat Software on the PSIPRED server, (http://bioinf.cs.ucl.ac.uk/psipred/).⁸⁰

Planar lipid bilayer experiments

Voltage clamp electrophysiology measurements were performed on an Orbit Mini (Nanion Technologies) horizontal lipid bilayer system and data was collected using Elements Data Reader 3.0. Briefly, using the manufacturers’ recommendations: 100 μM MECA chips (Ionera) were bathed in recording solution (150 μl) at 2 nA gain, 1.25 kHz sampling rate at + 10 mV. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DphpC) from Avanti Polar Lipids was dissolved at 10 mg/ml in n-octane (Sigma, electronics grade) and saved at −20 °C until use. Recording buffer was prepared using 10 mM HEPES pH 7.2 as the buffering agent with 1M of KCl or the indicated salt solution, and was filter sterilized prior to use. Membranes were painted using a paintbrush and Teflon was used to remove excess lipid until the capacitance was between 15–30 pF (100 μM MECA chip) or 30–60 pF (150 μM MECA chip) as per the manufacturer’s recommendation. Membrane integrity was monitored for 5 minutes to assess stability. Approximately 10 ng of BtCap14 was added per aperture and each membrane was monitored for fusion spikes to indicate insertion of proteins with the membrane, the fusion pulse option was used to enhance fusion at + 40 mV. 1 μM of 2′,3′-cGAMP was added in proximity to the apertures and mixed carefully using a pipette. The current trace was monitored for openings at which point either single channel data was collected at the indicated voltage or current/voltage relationships were determined. Data was analyzed using Elements Data Analyzer 3.0 and plotted in Graphpad Prism. All experiments were conducted at +40 mV unless otherwise indicated. For current/voltage relationships the voltage was clamped at −100 mV and activity was recorded for 10 seconds at 20 mV increments systematically with a return to 0 mV between steps. For substitution measurements experiments were conducted as described above, but for K-Gluconate 10 mM KCl was included in the solution to account for the use of Ag/AgCl₂ electrodes in the experimental setup.

Membrane potential measurements with Disc₃(5) in B. subtilis

The indicated bacterial strains were grown and infected with phage in a 96 well plate as described in the phage growth curve section for liquid culture infection experiments. At the indicated timepoints, Disc₃(5) was added to a final concentration of 5 μM (from a 500 μM stock solution dissolved in DMSO) and allowed to equilibrate for 2 minutes. Fluorescence intensity was measured on a Tecan SPARK fluorescence plate reader at 620/670 ± 5 nm Ex/Em.

Gel electrophoresis nuclease assays

Linear double-stranded DNA products were generated by PCR. 1 μg of Chimera10 was incubated with 1 μM of the indicated cyclic dinucleotide and 500 ng DNA in a final buffer of 10 mM Tris-HCl pH 7.4, 25 mM KCl, 1 mM DTT for 1 hour at 37 °C. DNA degradation was visualized on 1% agarose gels stained with SYBR-Safe. Data was representative of n > 3 protein preparations and n > 3 biological replicates.

DNAse Alert nuclease assays

DNAse Alert nuclease detection assay kit was purchased from IDT. One tube was resuspended in 5 ml of reaction buffer (as described in the Gel electrophoresis nuclease assays section). In a 96 well plate, samples were prepared in 100 μl buffer + substrate with the indicated cyclic dinucleotide stock (10 μl volume was added total) + 3 μg of Chimera10. The reaction was monitored at an excitation / emission of 535 / 570 nm in a TECAN Spark plate reader at 37 °C. End point fluorescence at 60 min was used to measure DNA digestion. Chimera10 activity is reported as a fold change over a background sample to normalize for variability between assays. For nucleotide standards, the fold change of observed signal over no nucleotide added is reported; for bacterial lysates, the fold change of observed signaling over a cGAMP-null strain (CBASS^CD) is reported.

Fluorescence Microscopy

B. subtilis strains were grown as described previously and were infected at an OD of 0.2 with an MOI of 4 of phage SPP1 prior to imaging. Samples were removed 5 minutes prior to the indicated timepoint, and 200 μl of bacteria was incubated in a 96 well plate with 5 μg/ml of FM-143x to stain the membrane, 10 μg/ml of DAPI, and 5 μM propidium iodide for 2 minutes. 5 μl of each sample was allowed to dry on an agar pad of MMCG minimal medium agar prepared as described⁸¹. A spinning disk microscope (Nikon Eclipse Ti/ Yokogawa CSU-X1) was used to image the cells. Samples were plated on a 4-chamber slide (Ibidi μ-slide) below a 1.5% agarose pad. At least nine locations were captured for each sample. For each location, three images were captured using the following optical configurations: DAPI (405 nm ex./428 – 466 nm em.; 200 ms exposure; 10% power), GFP (488 nm ex./ 500 – 550 nm em.; 150 ms exposure; 10% power), and TRITC (561 nm ex./ 575– 623 nm em.; 300 ms exposure; 20% power). Note that the exposure time and excitation power was adjusted from day-to-day to ensure the signal-to-noise ratio was consistent. Images were false colored and analyzed/quantified in Fiji to count cells that were positive for propidium iodide.

Cryo-EM sample preparation

BtCap14 in 0.05% DDM was incubated with 25 mg of A8–35 amphipols with Biobeads overnight on an end-over-end rotor. The exchanged sample was separated on a Superdex 200 column and the peak fraction between ~44 and ~158 kDa was pooled, concentrated to 1 mg/ml and vitrified on glow-discharged Quantifoil copper 400 mesh R1.2/1.3 using an FEI Vitrobot Mark IV at 100% humidity, 5° C, 8 seconds blotting time, blotting force 1.

Cryo-EM data collection and processing

BtCap14 + 2′,3′-cGAMP datasets were collected on an FEI Titan Krios G3 (CU BioKEM Cryo-electron microscopy facility) using the EPU software (Thermo Fisher Scientific) in EER format at 0.97 Å/pix, 50 E/Å² total dose, 7.85s exposure, frames: 270 with defocus values ranging from −0.3 to −2.5 μM. All processing was performed in CryoSparc v4.4. For the BtCap14 + cGAMP dataset: 8471 micrographs were preprocessed using patch motion correction, patch CTF, and then curated to remove movies with poor ice thickness or CTF fits worse than 7Å yielding 7413 movies. The blob picker was used for initial particle picking followed by particle extraction with a box size of 540, iterative 2D classification and template-based particle picking. 2D inputs were then used as templates for the filament tracer tool, followed by 2D classification, cleanup, ab-initio model generation (C1), heterogeneous refinements, non-uniform refinement (C2 symmetry applied) and 3D cleanup (C2), followed by non-uniform refinement and local refinement. For the BtCap14 apo state, 8415 movies were collected, and preprocessed identically to yield 7454 micrographs of suitable quality. The blob picker was used for particle picking followed by 2D classification on particles extracted with a box size of 540. Subsequent attempts at 3D reconstruction were unsuccessful.

Atomic model building

Model Building was performed ab initio in Phenix to generate an initial template, and then further built using Coot. Ligand coordinates for 2′,3′-cGAMP (1SY) were imported into Phenix to generate restraints using Elbow and were built into the density map followed close inspection of the density map, followed by iterative real-space refinement and manual fixing of outliers in Coot. Atomic model and map visualization was performed in UCSF ChimeraX (74).

AlphaFold 3 structure predictions

Structural predictions and models for BtCap14 and FsCap13 were generated using the AlphaFold 3 server (36). All predictions used in this study are included in Data S1 and S2.

Accession numbers

The accession numbers for genes used in this study are: DaCdnB, WP_031517737.1; BtCdnB, EEM25276.1; CbCdnB, WP_053342861.1; BtCap14, WP_001028553.1; CbCap14, WP_053342862.1; GsCap5; WP_013400843.1.

Quantification and Statistical Analysis

The relevant statistical test used, value and meaning of n, definition of data presented, and error bar are indicated in the figure legends and methods sections. Graphpad Prism v10 software was used for statistical analysis. Elements Data Analyzer software was used for analysis and quantification of bilayer recordings. Fiji software was used to quantify microscopy images.

Data deposition and availability

The BtCap14 + 2’,3’-cGAMP structure was deposited to the PDB (ID: 9OM7), the cryo-EM density map was deposited to the EMDB (ID: EMD-70609), and additional relevant cryo-EM data collection, processing, and model building data are provided in Table S1.

Supplementary Material

Data S3. Full gel images, related to Figure 5, S4, S5, and S6.

NIHMS2161427-supplement-1.zip^{(2.7MB, zip)}

Supplemental information. Figures S1–S7 and Table S1.

NIHMS2161427-supplement-2.pdf^{(21.3MB, pdf)}

Table S2. Bioinformatic analysis of CD-NTases from this study, related to Figure 1.

NIHMS2161427-supplement-3.xlsx^{(103.4KB, xlsx)}

Table S3. Bacteria and phages used in this study, related to STAR Methods.

NIHMS2161427-supplement-4.xlsx^{(12.7KB, xlsx)}

Table S4. Plasmids and DNA used in this study, related to STAR Methods.

NIHMS2161427-supplement-5.xlsx^{(36.7KB, xlsx)}

Data S1. AlphaFold models 1–5 used in this study, related to Figure 4, S4, S5, and STAR Methods.

NIHMS2161427-supplement-6.zip^{(119.2MB, zip)}

Data S2. AlphaFold models 6–8 used in this study, related to Figure S4, S7, and STAR Methods.

NIHMS2161427-supplement-7.zip^{(82.3MB, zip)}

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
IRDye 800CW Goat anti-Mouse IgG (RRID:AB_621842)	Li-Cor	926–32210
IRDye 800CW Goat anti-Rabbit IgG (RRID:AB_621843)	Li-Cor	926–32211
IRDye 680RD Goat anti-Mouse IgG (RRID:AB_10956588)	Li-Cor	926–68070
IRDye 680RD Goat anti-Rabbit IgG (RRID:AB_10956166)	Li-Cor	926–68071
Anti-HA Rabbit Polyclonal (RRID:AB_11042321)	Thermo Fisher	51064–2-AP
anti-RNAP Mouse Monoclonal (Clone 8RB13) (RRID:AB_2564524)	BioLegend	663903
Anti-6xhis Rabbit Polyclonal (RRID AB_1069891)	Thermo Fisher	PA1–983B
Bacterial and virus strains
See Table S3 for a complete list of bacterial strains	N/A	N/A
See Table S3 for a complete list of virus strains	N/A	N/A
Chemicals, peptides, and recombinant proteins
Carbenicillin	Gold Biotechnology	C-103–50
Chloramphenicol	Gold Biotechnology	C-105–25
Spectinomycin	Gold Biotechnology	S-140–5
n-Dodecyl-B-D-Maltoside (DDM)	Gold Biotechnology	DDM10
N-octane (electronic grade >99.999% purity)	Sigma Aldrich	657042
Amphipol A8–35	Thermo Fisher	A50943
Amylose Resin	NEB	E8021L
HisPur^™ Ni-NTA Resin	Thermo Fisher	88222
HisPur^™ Cobalt Resin	Thermo Fisher	89965
Nuclease P1	NEB	M0660S
Benzonase	Millipore Sigma	706643
Phenylmethylsulfonyl fluoride (PMSF)	Millipore Sigma	200005–694
Pierce EDTA-free protease inhibitor mini tablets	Thermo Fisher	A32955
1,2-diphytanoyl-sn-glycero-3-phosphocholine (DphpC)	Avanti Polar Lipids	850356C
4′,6′-Diamidino-2-phenylindole dihydrochloride (DAPI)	VWR	IC15757401
Propidium Iodide	VWR	IC19545810
FM-143x	Thermo Fisher	F35355
DiSC₃(5) (3,3′-Dipropylthiadicarbocyanine Iodide)	Thermo Fisher	D306
c[G(2′,5′)pA(3′,5′)p] / 2′3′-cGAMP	BioLog	C 161
c[A(2′,5′)pG(2′,5′)p] / 2′2′-cGAMP	BioLog	C 210–005
c[A(2′,5′)pG(3′,5′)p] / 3′2′-cGAMP	BioLog	C 238–005
c-(ApGp) / 3′3′-cGAMP	BioLog	C 117
pppG(2′,5′)pA	BioLog	T 051
3′,3′-c-di-AMP	BioLog	C 088
3,′3′-c-di-GMP	BioLog	C 057–01
Ribonucleotide Solution Set (ATP, GTP, CTP, UTP)	NEB	N0450S
Critical commercial assays
2′,3′-Cyclic GAMP ELISA Kit	Arbor Assays	K067-H5
DNAseAlert Substrate	IDT	11-04-03-04
His-Tag Labeling Kit RED-tris-NTA 2nd Generation	NanoTemper	MO-L018
Deposited data
BtCap14 + 2’,3’-cGAMP structure	This study	PDB 9OM7
BtCap14 + 2’,3’-cGAMP cryo-EM density map	This study	EMDB-70609
Oligonucleotides
For a full list of oligonucleotides see Table S4	N/A	N/A
Recombinant DNA
For a full list of recombinant DNA see Table S4	N/A	N/A
Software and algorithms
Geneious Prime	Biomatters Ltd.	N/A
GraphPad Prism 9.2.0	GraphPad Software	N/A
Adobe Illustrator V 29.6.1	Adobe	N/A
Fiji	Schindelin et al.⁷³	https://imagej.net/software/fiji/downloads
Cryosparc	Punjani et al.⁷⁴	https://cryosparc.com/
Coot	Emsley et al.^75,76	https://www2.mrc-lmb.cam.ac.uk/%20personal/pemsley/coot/
PHENIX	Liebschner et al.⁷⁷	https://phenix-online.org/
AlphaFold 3	Abramson et al.³⁶	https://alphafoldserver.com/
UCSF ChimeraX v 1.10	Meng et al.⁷⁸	https://www.rbvi.ucsf.edu/chimerax/
EPU Single Particle Collection Software Suite	Thermo Fisher	https://www.thermofisher.com/us/en/home/electron-microscopy/products/software-em-3d-vis/epu-software.html
NIS Elements Software 4.10.04	Nikon	https://www.microscope.healthcare.nikon.com/products/software/nis-elements
Image Studio	Li-cor	https://www.licorbio.com/image-studio
DeepTMHMM	Hallgren et al.,⁷⁹	https://dtu.biolib.com/DeepTMHMM
Elements Data Reader	Elements	https://elements-ic.com/edr3-eda/
Elements Data Analyzer	Elements	https://elements-ic.com/edr3-eda/
MO Affinity Analysis Software	NanoTemper	https://shop-us.nanotempertech.com/mo-affinity-analysis-3-software-1-license/
MemSat	Nugent et al.⁸⁰	https://bioinf.cs.ucl.ac.uk/psipred/
Other
Tecan SPARK Multimode Microplate Reader	Tecan	https://www.tecan.com/spark-overview
Nanodrop One	Thermo Fisher Scientific	ND-ONE-W
Azure Biosystems 200	Azure Biosystems	AZI200–01
Odyssey CLx	Li-cor	9140–09
Nikon Eclipse TI / Yokogawa CSU-X1	Nikon	https://advancedimaging.colorado.edu/
Nikon D7500 DSLR Camera with 18–140mm Lens	Nikon	NID7500KR
Nanion Orbit Mini	Nanion	https://www.nanion.de/products/orbit-mini/
MECA 4 recording chips (100 μm)	Nanion	132002
Superdex 200 Increase 10/300 GL column	Cytiva	Cat# 28990944
Quantifoil^® R 1.2/1.3 400 Mesh, Cu grids	Electron Microscopy Sciences	Q450CR1.3
Titan Krios G3i Cryogenic Electron Microscope	FEI	https://www.colorado.edu/facility/biokem/
Vitrobot Mark IV	FEI	https://www.colorado.edu/facility/biokem/
NanoTemper Monolith	NanoTemper	https://nanotempertech.com/monolith/
Monolith Capillaries	NanoTemper	SKU: MO-K022

Open in a new tab

Highlights.

CdnB enzymes in bacterial CBASS systems synthesize 2′,3′-cGAMP in response to phage
2′,3′-cGAMP activates Cap14 to form filaments that abort phage infection via lysis
A SAVED domain can be swapped between effectors to generate a 2′,3′-cGAMP sensor
A 2TM-STING (Cap13) CBASS system induces membrane disruption and restricts phage

Acknowledgements

We thank Dr. Kelly Doran and Dr. Alexander Horswill (University of Colorado Anschutz) for use of the Orbit Mini and for the E. coli C43 strain, Dr. Daniel Kearns (Indiana University) for phage SPP1, Dr. Deborah Wuttke (University of Colorado Boulder) for use of their FPLC, Dr. Annette Erbse and the CU Boulder Department of Biochemistry Shared Instruments core facility (RRID:SCR_018986) for the NanoTemper Monolith (National Institutes of Health Shared Instrumentation Grant S10OD21603) and Avanti floor centrifuges (National Institutes of Health grant R24OD033699), Charles Moe and the Biochemistry Krios Electron Microscopy (BioKEM) facility (RRID:SCR_019057) for data collection, Dr. Shawn Laursen for assistance with Cryosparc, Jeremy Bennett for assistance with sample vitrification, and Drs. Kevin Corbett and Amar Deep for helpful discussions. Cryo-EM data processing used the Blanca and Alpine high performance computing resource at the University of Colorado Boulder. Blanca is jointly funded by computing users and the University of Colorado Boulder. Alpine is jointly funded by the University of Colorado Boulder, the University of Colorado Anschutz, Colorado State University, and the National Science Foundation (Award 2201538). This work was funded by the National Institutes of Health through an NIH Director’s New Innovator Award DP2AT012346 (A.T.W), the National Institute of Allergy and Infectious Disease awards R01AI018045 and R01AI026289 to John J. Mekalanos, a Mallinckrodt Foundation Grant (A.T.W.), a Boettcher Foundation Webb-Waring Biomedical Research Award (A.T.W.), a Burroughs Wellcome Fund PATH Award 1186087 (A.T.W.), and a University of Colorado ABNexus Grant (joint between A.T.W and Dr. Kelly Doran). U.T was supported as a fellow of the Cancer Research Institute Irvington Postdoctoral Fellowship (CRI4043); KS and PHW were supported in part by the Undergraduate Research Opportunity Program (UROP) from University of Colorado Boulder.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declaration of interests

The University of Colorado Boulder has patents issued for 2′,3′-cGAMP producing CD-NTase technologies and 2′,3′-cGAMP biosensor technologies on which U.T. and A.T.W are listed as inventors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S3. Full gel images, related to Figure 5, S4, S5, and S6.

NIHMS2161427-supplement-1.zip^{(2.7MB, zip)}

Supplemental information. Figures S1–S7 and Table S1.

NIHMS2161427-supplement-2.pdf^{(21.3MB, pdf)}

Table S2. Bioinformatic analysis of CD-NTases from this study, related to Figure 1.

NIHMS2161427-supplement-3.xlsx^{(103.4KB, xlsx)}

Table S3. Bacteria and phages used in this study, related to STAR Methods.

NIHMS2161427-supplement-4.xlsx^{(12.7KB, xlsx)}

Table S4. Plasmids and DNA used in this study, related to STAR Methods.

NIHMS2161427-supplement-5.xlsx^{(36.7KB, xlsx)}

Data S1. AlphaFold models 1–5 used in this study, related to Figure 4, S4, S5, and STAR Methods.

NIHMS2161427-supplement-6.zip^{(119.2MB, zip)}

Data S2. AlphaFold models 6–8 used in this study, related to Figure S4, S7, and STAR Methods.

NIHMS2161427-supplement-7.zip^{(82.3MB, zip)}

Data Availability Statement

Coordinates of the BtCap14 + 2’3’-cGAMP structure are publicly available in the Protein Data Bank (PDB) under the accession number 9OM7, and the cryo-EM density map is available in the Electron Microscopy Data Bank (EMDB) under the accession number EMD-70609.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this work is available from the lead contact upon request.

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PERMALINK

Bacterial 2′,3′-cGAMP activates a SAVED effector to form membrane-disrupting filaments and restrict phage replication

Uday Tak

Kate Schinkel

Peace Walth

Jian Wei Tay

Erik W Hartwick

Aaron T Whiteley

Summary

Graphical Abstract

eTOC blurb

Introduction

Results

Discovery of bacterial CD-NTases that synthesize 2′,3′-cGAMP

Figure 1. Forward biochemical screen to identify bacterial 2′,3′-cGAMP synthases.

2′,3′-cGAMP signaling restricts phage replication by initiating bacterial cell lysis

Figure 2. A CdnB-Cap14 CBASS system signals via 2′,3′-cGAMP to restrict phage replication.

Architecture of Cap14 bound to 2′,3′-cGAMP

Figure 3. The Cap14 effector binds 2′,3′-cGAMP and forms oligomers.

Activated Cap14 disrupts artificial membranes

Figure 4. BtCap14 causes cGAMP-dependent membrane disruption and cell depolarization.

Construction of a reprogrammed SAVED effector

Figure 5. SAVED domains can be functionally exchanged between effectors.

STING-like effectors disrupt membranes in CBASS immunity

Figure 6. Membrane disruption is conserved in bacterial 2TM-STING-mediated immunity.

Discussion

Figure 7. Model for Cap14 and Cap13 transmembrane effectors in CBASS immunity.

Limitations of the study

Resource Availability

Lead contact

Materials availability

Data and code availability

STAR METHODS

Experimental model and subject details

Bacterial strains and culture conditions

Bacteriophage isolation and amplification

Method details

Cloning and plasmid construction

Thin layer chromatography and nuclease P1 treatment

Bioinformatic identification of CD-NTases and phylogenetic tree construction

Recombinant protein expression and purification

Immunoblotting

CD-NTase enzymatic assays and detection of 2′,3′-cGAMP via ELISA

Strain construction in Bacillus subtilis

Bacteriophage challenge and plaque assays on solid media

Phage growth curve experiments in liquid culture and viral titer measurements

BtCap14-cGAMP binding assays by Microscale Thermophoresis

Domain analysis of BtCap14

Planar lipid bilayer experiments

Membrane potential measurements with Disc3(5) in B. subtilis

Gel electrophoresis nuclease assays

DNAse Alert nuclease assays

Fluorescence Microscopy

Cryo-EM sample preparation

Cryo-EM data collection and processing

Atomic model building

AlphaFold 3 structure predictions

Accession numbers

Quantification and Statistical Analysis

Data deposition and availability

Supplementary Material

Highlights.

Acknowledgements

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Membrane potential measurements with Disc₃(5) in B. subtilis