Journal: bioRxiv

Article Title: Phage protease enzymes activate CBASS antiphage immunity

doi: 10.64898/2026.03.04.709575

Figure Lengend Snippet: (A) Schematic of the type II CBASS operon from E. coli strain TW10828 used in this study. (B) Representative plaque assay depicting serial dilutions of WT phage T4 or phage T4 Δacb1/Δacb2 plated on cells expressing either WT or dead Ec CdnG D82/84A CBASS operons. (C) Quantification of the data in B and . (D) Experimental schematic depicting biochemical reconstitution of CBASS sensing T4 Δacb1/Δacb2 phage infection by mixing CBASS and phage-infected lysates. Activation of CBASS signaling in the reconstitution reactions is then analyzed using the Cap5-based 3′2′-cGAMP biosensor assay. (E) Agarose gel depicting production of 3′2′-cGAMP and cleavage of indicator dsDNA by Cap5 when CBASS lysates are mixed with T4 Δacb1/Δacb2 infected lysates. For CBASS lysates, ‘d’ indicates cells expressing operon encoding an inactive Ec- CdnG (D82/84A) and ‘wt’ indicates cells expressing an operon encoding WT Ec CdnG and an inactive Cap5 (H56A). ‘–’ indicates buffer was added to the reaction and ‘+’ indicates that lysate was added to the reaction. (F) Agarose gel depicting production of 3′2′-cGAMP and cleavage of indicator dsDNA when CBASS Cap5 H56A lysates are mixed with T4 Δacb1/Δacb2 phage lysates harvested 14 minutes post infection or later.

Article Snippet: E. coli strain MG1655 (ATCC 47076) was used for all experiments involving phage infections, E. coli strain Top10 (Thermo) was used for all cloning steps, and E. coli strain BL21 (DE3) RIL (Agilent) was used for protein expression.

Techniques: Plaque Assay, Expressing, Infection, Activation Assay, Biosensor Assay, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Phage protease enzymes activate CBASS antiphage immunity

doi: 10.64898/2026.03.04.709575

Figure Lengend Snippet: (A) Representative toxicity assay depicting serial dilutions of E. coli co-transformed with the indicated CBASS operon and T4 prohead protease gp21 construct and plated on LB supplemented with 0.02% arabinose and 1 μM IPTG. For CBASS, ‘dead’ refers to Ec CdnG D82/84A mutations and for T4 gp21 ‘dead’ refers to H85A/S140A mutations. (B) Aga-rose gel depicting the Cap5 activation and cleavage of indicator dsDNA when Ec CdnG is treated with the indicated protease. (C) Representative agarose gel depicting activation of Cap5 and cleavage of indicator dsDNA when titrating amounts of Ec CdnG are treated with proteinase K. (D) Quantification of the data in C.

Article Snippet: E. coli strain MG1655 (ATCC 47076) was used for all experiments involving phage infections, E. coli strain Top10 (Thermo) was used for all cloning steps, and E. coli strain BL21 (DE3) RIL (Agilent) was used for protein expression.

Techniques: Transformation Assay, Construct, Activation Assay, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Phage protease enzymes activate CBASS antiphage immunity

doi: 10.64898/2026.03.04.709575

Figure Lengend Snippet: (A) Representative toxicity assay depicting serial dilutions of E. coli co-transformed with the indicated CBASS operon and phage T4 prohead protease gp21 construct and plated on LB supplemented with 1% glucose. For CBASS, ‘dead’ refers to Ec CdnG D82/84A mutations and for T4 gp21 ‘dead’ refers to H85A/S140A mutations. (B) Quantification of the data in and . (C) Representative agarose gel depicting no activation of Cap5 when Ec CdnG D82/84A is treated with the indicated proteases. (D) Representative agarose gel depicting activation of Cap5 and cleavage of indicator dsDNA when Ec CdnG is treated with thermolabile (TL) proteinase K but not when TL proteinase K has been heat-inactivated.

Article Snippet: E. coli strain MG1655 (ATCC 47076) was used for all experiments involving phage infections, E. coli strain Top10 (Thermo) was used for all cloning steps, and E. coli strain BL21 (DE3) RIL (Agilent) was used for protein expression.

Techniques: Transformation Assay, Construct, Agarose Gel Electrophoresis, Activation Assay

Journal: bioRxiv

Article Title: Phage protease enzymes activate CBASS antiphage immunity

doi: 10.64898/2026.03.04.709575

Figure Lengend Snippet: (A) AlphaFold3 model of Ec CdnG highlighting the unstructured loop and canonical features of CD-NTase proteins. Model is colored by pLDDT confidence score. The flexible C-terminal tail of Ec CdnG was removed for clarity. (B) Representative anti-FLAG western blot of either wild-type or trypsin-blind 3×FLAG- Ec CdnG treated with titrating amounts of trypsin or proteinase K. Red arrow indicates ~28 kDa species that co-occurs with 3′2′-cGAMP synthesis. (C) Agarose gel depicting Cap5 activation and cleavage of indicator dsDNA when either wild-type or trypsin-blind 3×FLAG- Ec CdnG is treated with titrating amounts of trypsin or proteinase K. (D) Efficiency of plaquing (EOP) of T4 Δacb1/Δacb2 phage plated on cells encoding CBASS operons with the indicated Ec CdnG mutation. Data are normalized to the Ec CdnG D82/84A dead operon. (E) Representative plaque assays of serial dilutions of T4 Δacb1/Δacb2 phage or Bas51 phage plated on cells encoding the indicated Ec CdnG point mutations. (F) Representative toxicity assay depicting serial dilutions of E. coli co-transformed with CBASS operons encoding the indicated Ec CdnG mutations and either wild type or catalytically dead T4 prohead protease plated on 0.02% arabinose and 1 μM IPTG. (G) Representative plaque assays of T4 Δacb1/Δacb2 phage or Bas51 phage plated on cells encoding either inactive Cap5 (H56A), wild type, or Ec CdnG E190Q, E194Q, E212Q, E215Q (EQ) CBASS operons. (H) Quantification of the data in G.

Article Snippet: E. coli strain MG1655 (ATCC 47076) was used for all experiments involving phage infections, E. coli strain Top10 (Thermo) was used for all cloning steps, and E. coli strain BL21 (DE3) RIL (Agilent) was used for protein expression.

Techniques: Western Blot, Agarose Gel Electrophoresis, Activation Assay, Mutagenesis, Transformation Assay

Journal: bioRxiv

Article Title: Phage protease enzymes activate CBASS antiphage immunity

doi: 10.64898/2026.03.04.709575

Figure Lengend Snippet: (A) Representative plaque assays depicting serial dilutions of phage T4 Δacb1/Δacb2 or phage Bas51 plated on cells expressing CBASS operons with the indicated Ec CdnG point mutations. (B) Quantification of Bas48–59 phage titers when plated on cells expressing the indicated CBASS operon. Data are normalized to titers when plated on the Cap5 H56A inactive operon. (C) Representative toxicity assay depicting serial dilutions of E. coli co-transformed with CBASS operons with the indicated Ec CdnG mutations and phage T4 prohead protease gp21 construct and plated on LB supplemented with 1% glucose. (D) Quantification of the data in and .

Article Snippet: E. coli strain MG1655 (ATCC 47076) was used for all experiments involving phage infections, E. coli strain Top10 (Thermo) was used for all cloning steps, and E. coli strain BL21 (DE3) RIL (Agilent) was used for protein expression.

Techniques: Expressing, Transformation Assay, Construct