Key Resources Table.
| Resource or Reagent | Source | Identifier |
|---|---|---|
| Bacterial and virus strains | ||
| P. aeruginosa BWHPSA011 (Pa011) WT | Deborah Hung Lab | NCBI: NZ_AXQR01000000.1 |
| P. aeruginosa ATCC 27853 (Pa278) WT | ATCC | NCBI: CP015117.1 |
| P. aeruginosa PAO1 WT | Joe Bondy-Denomy Lab | NCBI: NC_002516.2 |
| PAO1Pa011 (PAO1 attTn7::Pa011 Type II-A CBASS) | Joe Bondy-Denomy Lab 16 | N/A |
| PAO1Pa278 (PAO1 attTn7::Pa278 Type III-C CBASS) | This study | N/A |
| JBD67 | Alan Davidson Lab 30 | NCBI: NC_042135.1 |
| JBD67Δacb2 (Removal of 23005–23232 bp orf24 (acb2)) |
Joe Bondy-Denomy Lab 16 | N/A |
| JBD67 acb2K26A | This study | N/A |
| JBD67 acb2R82A | This study | N/A |
| JBD67 acb2R82A/T89A | This study | N/A |
| JBD67 acb2K26A | This study | N/A |
| Mammalian cell lines | ||
| HEK293T Dual-Null | Invivogen | Cat#: 293d-null |
| Recombinant DNA | ||
| pET28a-His6-SUMO- PaMx33-Acb2 | This study | N/A |
| pET28a-His6-SUMO- PaMx33-Acb2 Y11A | This study | N/A |
| pET28a-His6-SUMO- PaMx33-Acb2 K26A | This study | N/A |
| pET28a-His6-SUMO- PaMx33-Acb2 T74A | This study | N/A |
| pET28a-His6-SUMO- PaMx33-Acb2 R67A | This study | N/A |
| pET28a-His6-SUMO- JBD67-Acb2 | This study | N/A |
| pET28a-His6-CHI14-Acb2 | This study | N/A |
| pET28a-His6-CHI14-Acb2 E76T | This study | N/A |
| pET28a-His6-CHI14- Acb2 E76T/R67P | This study | N/A |
| pET28a-His6-SUMO-T4-Acb2 | This study | N/A |
| pET28a-His6-SUMO-T4-Acb2 D61R | This study | N/A |
| pUC18-mini-Tn7-LAC (pTn7) | 31 | N/A |
| pTn7-Pa278 Type III-C CBASS (6283357–6286926) | This study | N/A |
| pNTS3 | 32 | N/A |
| pMQ30 | 33 | N/A |
| pMQ30-HDR-JBD67 Acb2 WT | This study | N/A |
| pMQ30-HDR-JBD67 Acb2 K26A | This study | N/A |
| pMQ30-HDR-JBD67 Acb2 R82A | This study | N/A |
| pMQ30-HDR-JBD67 Acb2 T89A | This study | N/A |
| pHERD30T (p30T) | 34 | N/A |
| p30T-JBD67 WT Acb2 | This study | N/A |
| p30T-JBD67 Y11A Acb2 | This study | N/A |
| p30T-JBD67 K26A Acb2 | This study | N/A |
| p30T-JBD67 R82A Acb2 | This study | N/A |
| p30T-JBD67 T89A Acb2 | This study | N/A |
| p30T-Pa27853 Type III-C CBASS (6283357–6286926) | This study | N/A |
| pcDNA3 | Lingyin Li Lab | N/A |
| pcDNA3-hSTING-232R | Lingyin Li Lab | N/A |
| pcDNA3-Acb2 WT | This paper | N/A |
| pcDNA3-Acb2 Y11A | This paper | N/A |
| pcDNA3-Acb2 K26A | This paper | N/A |
| Chemicals, peptides, and recombinant proteins | ||
| Phusion High-Fidelity DNA polymerase | NEB | Cat #M0530S |
| dNTPs | NEB | Cat #N0447S |
| Phusion GC Buffer | NEB | Cat #B0519 |
| Gibson Assembly HiFi DNA Master Mix | NEB | Cat #E2621 |
| SacI | NEB | Cat #R3156S |
| PstI | NEB | Cat #R3140S |
| HEPES sodium salt | Sigma-Aldrich | CAS: 7365–45-9 Cat #: V900477–500G |
| Tris base | Sigma-Aldrich | CAS: 77–86-1 Cat #: RDD008–2.5KG |
| Sodium dihydrogen phosphate dihydrate | Sigma-Aldrich | CAS: 13472–35-0 Cat #: 1063420250 |
| Disodium hydrogen phosphate dodecahydrate | Sigma-Aldrich | CAS: 10039–32-4 Cat #: 1065790500 |
| Bis-Tris propane | Sigma-Aldrich | CAS: 64431–96-5 Cat #: B6755–25G |
| Sodium bromide | Sigma-Aldrich | CAS: 7647–15-6 Cat #: 310506–100G |
| PEG 3350 | Biorigin | CAS: 25322–68-3 Cat #: BN33640 |
| Glycerol | Sigma-Aldrich | CAS: 56–81-5 Ca t#: V900122–500ML |
| Imidazole | CAS: 288–32-4 Cat #: V900153–500G |
|
| Sigma-Aldrich | Vazyme | Cat #: P515–03 |
| 2× Rapid Taq Master Mix | Vazyme | Cat #: P222-AA |
| KOD-Plus-Neo | TOYOBO | Cat #: KOD-401 |
| DpnI | NEB | Cat #: R0176S |
| Proteinase K | NEB | Cat #: P8107S |
| High Affinity Ni-NTA Resin | GenScript | Cat #: L00250–100 |
| α-tubulin (DM1A), Mouse mAb | Sigma-Aldrich | Cat# T6199; RRID: AB_477583 |
| STING (D2P2F) Rabbit mAb | Cell Signaling Technologies | Cat# 13647; RRID: AB_2732796 |
| Acb2 custom polyclonal antibody | GenScript | N/A |
| Goat Anti-Mouse IgG Antibody, IRDye® 680RD Conjugated | Li-COR Biosciences | Cat# 926–68070; RRID: AB_10956588 |
| IRDye 800CW Goat anti-Rabbit IgG (H+L) | Li-COR Biosciences | Cat# 925–32211; RRID: AB_2651167 |
| PaMx33-Acb2 WT recombinant protein | This study | N/A |
| PaMx33-Acb2Y11A recombinant protein | This study | N/A |
| PaMx33-Acb2R67A recombinant protein | This study | N/A |
| PaMx33-Acb2T74A recombinant protein | This study | N/A |
| PaMx33-Acb2K26A recombinant protein | This study | N/A |
| CHI14-Acb2 WT recombinant protein | This study | N/A |
| JBD67-Acb2 WT recombinant protein | This study | N/A |
| T4-Acb2 WT recombinant protein | This study | N/A |
| T4-Acb2 D61R recombinant protein | This study | N/A |
| Critical Commerical Assays | ||
| Plasmid Miniprep Kit | Vazyme | Cat #: DC201–01 |
| Gel DNA Extraction Mini Kit | Vazyme | Cat #: DC301–01 |
| QUANTI-Luc | Invivogen | Cat #: rep-qlc2 |
| Deposited Data | ||
| Structure of Acb2–2’,3’-cGAMP | This study | PDB: 8J8O |
| Structure of Acb2–3’,2’-cGAMP | This study | PDB: 8IXZ |
| Structure of Acb2-cA3 | This study | PDB: 8IY0 |
| Structure of Acb2-cAAG | This study | PDB: 8IY1 |
| Structure of Acb2–3’,3’-cGAMP-cA3 | This study | PDB: 8IY2 |
| Oligonucleotides | ||
| 3’,3’-cGAMP | Sigma-Aldrich | CAS: 849214–04-6 Cat #: SML1232-.5UMO |
| 2’,3’-cGAMP | Sigma-Aldrich | CAS:1441190–66-4 Cat #SML1229-.5UMO |
| c-di-AMP | Sigma-Aldrich | CAS: 54447–84-6 Cat #SML1231–1UMO |
| c-di-GMP | Sigma-Aldrich | CAS: 61093–23-0 Cat #SML1228–1UMO |
| 3’,3’-c-UMP-GMP | Biolog Life Science Institute | CAS: 232933–52-7 Cat #C371 |
| 3’,3’-c-di-UMP | Biolog Life Science Institute | CAS: 73120–97-5 Cat #C256 |
| 3’,3’-c-UMP-AMP | Biolog Life Science Institute | CAS: 83799–66-0 Cat #C357 |
| 3’,2’-cGAMP | Biolog Life Science Institute | CAS: 1615704–64-7 Cat #C238 |
| 3’,3’,3’-cAAG | Biolog Life Science Institute | CAS: 2365165–54-2 Cat #C361 |
| 3’,3’,3’-cAAA (cA3) | Biolog Life Science Institute | CAS: 54447–85-7 Cat #C362 |
| cA4 | Biolog Life Science Institute | CAS: 118004–81-2 Cat #C335 |
| cA6 | Biolog Life Science Institute | CAS: 232933–63-0 Cat #C332 |
| cAMP | MedChemExprss | CAS: 60–92-4 Cat #HY-B1511S |
| cUMP | Sigma-Aldrich | CAS: 56632–58-7 Cat #SML3286–50UMOL |
| Y11A_F 5’–gcacaagaaaatcaagggcgccagagatctgtctcagga–3’ |
This study | N/A |
| Y11A_R 5’–ctcctgagacagatctctggcgcccttgattttcttgtgc–3’ |
This study | N/A |
| K26A_F 5’–gacatgatgaacagagtggcggaactgggcagccagtt–3’ |
This study | N/A |
| K26A_R 5’–aactggctgcccagttccgccactctgttcatcatgtc–3’ |
This study | N/A |
| Software and algorithms | ||
| National Center for Biotechnology Information (NCBI) database | 35 | https://blast.ncbi.nlm.nih.gov/ |
| HKL2000 | 36 | http://www.hkl-xray.com/ |
| PHENIX | 37 | http://www.phenix-online.org |
| COOT | 38 | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot |
| PyMOL | The PyMOL Molecular Graphics System, Version 2.5.2., Schrodinger, LLC | https://pymol.org/2/ |
| DALI | 39 | http://ekhidna2.biocenter.helsinki.fi/dali/ |
| Foldseek | 24 | https://search.foldseek.com/search |
| MAFFT | 40 | N/A |
| MMSeq2 | 41 | N/A |
| FastTree | 42 | N/A |
| iTOL | 43 | https://itol.embl.de/ |
| OriginPro 8 | OriginPro Software | N/A |
| GraphPad Prism 9 | GraphPad Software | https://www.graphpad.com/ |
| Other | ||
| Amicon Ultra-0.5 centrifugal filter unit | Merck | Cat #: UFC500396 |
| Amicon concentrators (3 K) | Millipore | Cat #: UFC800308 |
| Amicon concentrators (10 K) | Millipore | Cat #: UFC901096 |
| Amicon concentrators (30 K) | Millipore | Cat #: UFC903024 |
| HisTrap FF (5 mL) | GE Healthcare | Cat #: 17–5255-01 |
| HiTrap Heparin HP (5 mL) | GE Healthcare | Cat #: 17–0407-03 |
| HiTrap Q Sepharose FF (5 mL) | GE Healthcare | Cat #: 17–5156-01 |
| Superdex 200 increase 10/300 GL | GE Healthcare | Cat #: 17517501 |