rspcr什么时候用A viral ADP-ribosyltransferase attaches RNA chains to host proteins

Reagents were purchased from Sigma-Aldrich and used without further purification. Oligonucleotides, DNA and RNA were purchased from Integrated DNA Technologies (Supplementary Tables 7–10). Concentrations of DNA and RNA were determined by measurements using the NanoDrop ND-1000 spectrophotometer. Radioactively labelled proteins and nucleic acids were visualized using storage phosphor screens (GE Healthcare) and a Typhoon 9400 imager (GE Healthcare). Uncropped gel and blot images are provided (Supplementary Fig. 1).

DNA templates for Qβ RNA (100-nucleotide RNA) and E. coli RNAI were amplified by PCR (primer sequences are listed in Supplementary Table 9), and PCR products were analysed by 2% agarose gel electrophoresis and purified using the QIAquick PCR purification kit (QIAGEN). 5′-Triphosphate (ppp) Qβ RNA and RNAI were synthesized by in vitro transcription in the presence of 1× transcription buffer (40 mM Tris, pH 8.1, 1 mM spermidine, 10 mM MgCl₂, 0.01% Triton X-100), 5% DMSO, 10 mM DTT, 4 mM of each NTP, 20 μg T7 RNA polymerase (2 mg ml⁻¹, purified in our laboratory) and 200 nM DNA template. NAD–RNAI was made under similar conditions using 2 mM ATP and 4 mM NAD. The same conditions were applied for the synthesis of a mixture of α-³²P-labelled 5′-NAD and pppQβ RNAs, except we used 2 mM ATP, 80 μCi ³²P-α-ATP and 4 mM NAD instead of 4 mM ATP. The in vitro transcription reactions were incubated at 37 °C for 4 h and digested with DNase I (Roche). RNA was purified by denaturing PAGE, isopropanol-precipitated and resuspended in Millipore water. RNA sequences are listed in Supplementary Table 7.

To convert 5′ppp–RNAs into 5′-monophosphate–RNAs (5′p–RNAs), 250 pmol Qβ RNA was treated with 60 U RNA 5′-polyphosphatase (Epicentre) in 1× polyphosphatase reaction buffer at 37 °C for 70 min. Protein was removed from 5′p–RNAs by phenol–chloroform extraction and residual phenol–chloroform was removed by three rounds of diethyl ether extraction. 5′p–RNAs were isopropanol precipitated and resuspended in Millipore water.

We treated 120 pmol 5′p-Qβ RNA or 6.25 nmol 5′p–RNA 8-mer (Supplementary Table 7) with 50 U T4 polynucleotide kinase in 1× reaction buffer B and 1,250 μCi ³²P-γ-ATP. The reaction was incubated at 37 °C for 2 h. The resulting 5′-³²P-RNA 8-mer and 5′-³²P-Qβ RNA were separated from residual protein by phenol–chloroform extraction. The remaining ³²P-γ-ATP was removed by washing with three column volumes of Millipore water and centrifugation in 10 kDa (for Qβ RNA) or 3 kDa (for the 8-mer) Amicon filters (Merck Millipore) at 14,000 rpm at 4 °C four times. RNA sequences are listed in Supplementary Table 7. To convert the purified 5′-³²P-RNAs into 5′-³²P-NAD-capped RNA, 800 pmol 5′-³²P-RNA 8-mer or 30 pmol 5′-³²P-Qβ RNA was incubated in 50 mM MgCl₂ in the presence of a spatula tip of nicotinamide mononucleotide phosphorimidazolide, synthesized as described⁵², at 50 °C for 2 h. RNAs were purified by washing with Millipore water and centrifugation in 10 kDa (Qβ RNAs) or 3 kDa (8-mer) Amicon filters at 14,000 rpm at 4 °C four times. The concentrations of the 5′-³²P-RNAs were measured using a NanoDrop ND-1000 spectrophotometer and were used to calculate the approximate concentrations of yielded 5′-NAD-capped ³²P-RNAs, assuming an approximate yield of the imidazolide reaction of 50% (ref. ⁵²). The 5′-³²P-ADPr–RNA 8-mer was synthesized by incubating 8 µM 5′-³²P-NAD–RNA 8-mer and 0.08 µM ADP-ribosyl cyclase CD38 (R&D Systems) in 1× degradation buffer at 37 °C for 4 h. The reaction was purified by P/C/I-diethyl ether extraction and filtration through 3 kDa filters and washing with four column volumes of Millipore water.

To amplify bacteriophage T4 genes modA (GeneID: 1258568; Uniprot: P39421), modB (GeneID: 1258688; Uniprot: P39423) and alt (GeneID: 1258760; Uniprot: P12726), a single plaque from bacteriophage T4 revitalization was resuspended in Millipore water and used in a ‘plaque’ PCR, analogous to bacterial-colony PCR. The gene encoding the ADP-ribosylhydrolase ARH1 (GeneID: 141; Uniprot: P54922) was purchased from IDT as gBlocks and amplified by PCR. E. coli genes coding for rS1 (GeneID: 75205313; Uniprot: P0AG67), rL2 (GeneID: 947820; Uniprot: P60422) and PNPase (GeneID: 947672; Uniprot: P05055) were PCR-amplified from genomic DNA of E. coli K12, which was isolated using a GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich). Nucleotide sequences are listed in Supplementary Table 8. XhoI and NcoI restriction sites were introduced during amplification using appropriate primers (Supplementary Table 9). The resulting PCR product was digested with XhoI and NcoI (Thermo Fisher Scientific) and cloned into the pET–28c vector (Merck Millipore). After Sanger sequencing, the resulting plasmids were transformed into E. coli One Shot BL21 (DE3) (Life Technologies). The ARH1 D55,56A, ModB(R73A) and rS1 mutants were generated by site-directed mutagenesis using a procedure based on the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The resulting plasmids were sequenced and transformed into E. coli One Shot BL21 (DE3). All strains used and generated in this work are summarized in Supplementary Table 10.

Isopropyl beta–thiogalactoside (IPTG)-induced E. coli One Shot BL21 (DE3) containing the respective plasmid (Supplementary Table 10) was cultured in LB medium at 37 °C. Protein expression was induced at an optical density at 600 nm (OD₆₀₀) of 0.8, bacteria were collected after centrifugation for 3 h at 37 °C and lysed by sonication (30 s at 50% power, five times) in HisTrap buffer A (50 mM Tris-HCl, pH 7.8, 1 M NaCl, 1 M urea, 5 mM MgSO₄, 5 mM β-mercaptoethanol, 5% glycerol, 5 mM imidazole, one tablet per 500 ml complete EDTA-free protease inhibitor cocktail (Roche)). The lysate was cleared by centrifugation (37,500g for 30 min at 4 °C) and the supernatant was applied to a 1 ml Ni-NTA HisTrap column (GE Healthcare). The protein was eluted with an imidazole gradient using an analogous gradient of HisTrap buffer B (HisTrap buffer A with 500 mM imidazole added) and analysed by SDS–PAGE.

Further protein purification was achieved by size-exclusion chromatography (SEC) through a Superdex 200 10/300 GL column (GE Healthcare) using SEC buffer containing 0.5 M NaCl and 25 mM Tris-HCl, pH 8. Fractions of interest were analysed by SDS–PAGE, pooled and concentrated in Amicon Ultra-4 centrifugal filters (molecular weight cut-off (MWCO) 10 kDa with centrifugation at 2,000 rpm and 4 °C). Protein concentration was measured with a NanoDrop ND-1000 spectrophotometer. Finally, proteins were stored in SEC buffer supplemented with 50% glycerol at −20 °C.

E. coli BL21 DE3 pET28-ARH1 and BL21-pET28-ARH1 D55A, D56A (Supplementary Table 10) were grown to an OD₆₀₀ = 0.6 at 37 °C and 175 rpm. Afterwards, bacteria were allowed to cool to room temperature for 30 min. Expression was induced with 1 mM IPTG, and bacteria were finally grown overnight at room temperature while shaking at 175 rpm. Bacteria were collected by centrifugation and proteins were purified in a similar way to rS1 variants.

E. coli BL21 DE3 pET28-ModA (Supplementary Table 10) was grown to an OD₆₀₀ = 1 at 37 °C with shaking at 175 rpm. Protein expression was induced with 0.5 mM IPTG and bacteria were collected by centrifugation after 3 h at 37 °C. Pelleted bacteria were resuspended in 50 mM NaH₂PO₄, pH 8, 300 mM NaCl, 1 mM DTT with one tablet per 500 ml complete EDTA-free protease inhibitor cocktail (Roche) and lysed by sonication (3× 1 min at 5% power). Lysates were centrifuged at 3,000g at 4 °C for 20 min. Sediments were washed by resuspension in 30 ml 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 M urea, 1 mM DTT and one tablet EDTA-free protease inhibitor (Roche), and centrifuged at 10,000g at 4 °C for 20 min. Pellets containing inclusion bodies were resuspended in 40 ml 100 mM Tris, pH 11.6, 8 M urea, transferred to 12–14 kDa MWCO dialysis bags (Roth) and dialysed overnight against 50 mM NaH₂PO₄, 300 mM NaCl. Protein solutions were centrifuged at 20,000g at 4 °C for 30 min. Supernatants were batch purified using disposable 10 ml columns (Thermo Fisher Scientific) packed with 2 ml Ni-NTA agarose (Jena Bioscience) and equilibrated with 10 column volumes of 50 mM NaH₂PO₄ (pH 8), 300 mM NaCl. Proteins were purified by washing the columns with 30 column volumes of 50 mM NaH₂PO₄, 300 mM NaCl, 15 mM imidazole, eluted with 5 ml 50 mM NaH₂PO₄, 300 mM NaCl, 300 mM imidazole and concentrated in Amicon (Merck Millipore) filters (MWCO 10 kDa with centrifugation at 2,000 rpm and 4 °C). Finally proteins were purified by SEC, as described for rS1.

E. coli BL21 DE3 pET28–ModB and E. coli BL21 DE3 pET28–ModB(R73A, G74A) (Supplementary Table 10) were grown to OD₆₀₀ = 2.0 at 37 °C with shaking at 185 rpm and cooled to 4 °C while being shaken at 160 rpm for at least 30 min. Protein expression was induced by the addition of 1 mM IPTG. The cultures were then incubated for 120 min at 4 °C, with shaking at 160 rpm and bacteria were collected by centrifugation (4,000 rpm at 4 °C for 25 min). The ModB protein was purified from the supernatant as described for rS1 variants.

The Alphafold prediction of ModB structure was performed with AlphaFold2.ipynb (v.1.3.0, https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb) with default parameters (use_templates = false, use_amber = false; msa_mode = MMseqs2 (UniRef+Environmental), model_type = “AlphaFold2-ptm”, max_msa = null, pair_mode = unpaired+paired, auto advanced settings). The ModB protein sequence was retrieved from Uniprot (primary accession: P39423). The ModB structure prediction model from rank_1 was further assessed using PyMol.

rS1 (0.3 µM) was ADP-ribosylated in the presence of 0.25 μCi μl^{−1 32}P-NAD or RNAylated in the presence of one of 0.6 µM ³²P-NAD–8-mer, 0.03 µM ³²P-NAD–Qβ RNA or 0.8 µM NAD–10-mer–Cy5 (Supplementary Table 7) by 1.4 µM ModB and in 1× transferase buffer (10 mM Mg(OAc)₂, 22 mM NH₄Cl, 50 mM Tris-acetate pH 7.5, 1 mM EDTA, 10 mM β-mercaptoethanol and 1% glycerol) at 15 °C for at least 120 min. Samples (5 μl) were taken before the addition of ModB and after 1, 2, 5, 10, 30, 60 and 120 min, and mixed with 5 μl 2× Laemmli buffer to stop the reaction. Reactions were assessed by 12% SDS–PAGE and gels were stained in Instant Blue solution (Sigma-Aldrich) for 10 min. Radioactive signals were visualized using storage phosphor screens and a Typhoon 9400 imager. The intensity of the radioactive bands was quantified using ImageQuant 5.2 (GE Healthcare). The RNAylation with NAD-capped Cy5-labelled RNA was visualized with the ChemiDoc (Bio-Rad) Cy5 channel. Gels were then stained by Coomassie solution and imaged using the same system. In some cases, stain-free imaging of proteins in SDS gels was performed by 2,2,2-trichloroethanol (TCE) incorporated in the gel. TCE binds to tryptophan residues of the proteins, which enhances their fluorescence under ultraviolet light and thereby enables their detection⁵³.

rL2 was ADP-ribosylated or RNAylated at the same settings using either 6.4 µM NAD or 6.4 µM NAD–8-mer as a substrate to modify 4.6 µM rL2 in the presence of 1.57 µM ModB for 4 h for LC–MS/MS measurements. For shift assays, 538 nM rL2 was RNAylated by 2.61 µM ModB in the presence of 6 µM NAD–8-mer. 12% SDS– PA gels were fixed with a solution of 40% ethanol and 10% acetic acid overnight and stained using Flamingo fluorescent protein dye (Bio-Rad) for up to 6 h and imaged with the ChemiDoc (Bio-Rad). Signal intensity was quantified in ImageLab (Bio-Rad). Where indicated, statistical tests were performed using two-sided t-tests in R (v.4.2.2) implemented in the ggpubr package (v.0.6.0) using a significance level of 0.05.

We incubated 0.8 µM NAD–10-mer–Cy5 (Supplementary Table 7) with 0.5 µM of protein E. coli RNA polymerase (New England Biolabs) and 3 µM Alt or ModA in the presence of 1× transferase buffer at 15 °C for 60 min. Samples were taken before the addition of Alt or ModA and after 60 min incubation. The reactions were stopped by the addition of 1 volume of 2× Laemmli buffer. Reactions were analysed by 10% SDS–PAGE with rS1 RNAylated by ModB with NAD–10-mer–Cy5 as a reference protein. RNAylated proteins were visualized using the ChemiDoc (Bio-Rad) Cy5 channel. Afterwards, gels were stained in Coomassie solution and imaged using the same system.

In 20-μl reactions, 3.6 µM ³²P-ADPr–8-mer (Supplementary Table 7) was incubated with either 2.6 µM rS1, 3.9 µM ModB or both 2.59 µM rS1 and 3.9 µM ModB in 1× transferase buffer. As a positive control, equal amounts of protein rS1 and ModB were incubated with 0.6 µM ³²P-NAD–8-mer. All reactions were incubated at 15 °C for 60 min. Samples were taken before the addition of ModB or after 60 min, and reactions were stopped by adding one volume of 2× Laemmli buffer. Reactions were analysed by 12% SDS–PAGE and autoradiography imaging.

0.05 µM ³²P-NAD–Qβ RNA, 0.15 µM 5′-³²P-Qβ RNA or 0.15 µM 5′-³²PPP-Qβ RNA (Supplementary Table 7) was incubated with 2.3 µM rS1 and 1.4 µM ModB in the presence of 1× transferase buffer at 15 °C for 60 min. Samples were taken before the addition of ModB and after 60 min, and reactions were stopped by adding 1 volume 2× Laemmli buffer. Reactions were analysed by 10% SDS–PAGE, applying rS1–³²P-ADPr in 1× Laemmli buffer as a reference, and subsequent autoradiography imaging.

ADP-ribosylation or RNAylation reactions were performed with radio-labelled substrates, washed and equilibrated in 1× transferase or 1× degradation buffer for further enzymatic treatments. The reactions were washed with four column volumes of the corresponding buffer by centrifugation at 10,000g at 4 °C in 10 kDa Amicon (Merck Millipore) filters. Proteins RNAylated with Cy5-labelled RNA were equilibrated in the same buffers using Zeba Spin desalting columns (7 kDa MWCO, 0.5 ml) (Thermo Fisher Scientific) according to the manufacturer’s instructions.

An rS1–100-nucleotide-RNA (³²P) mixture (19 μl) was equilibrated in 1× transferase buffer and incubated with either 1 μl nuclease P1 or 1 μl Millipore water at 37 °C for 60 min. Samples were taken at the beginning and after 60 min, and reactions were stopped by adding one volume of 2× Laemmli buffer. Reactions were analysed by 10% SDS–PAGE, applying rS1–³²P-ADPr in 1× Laemmli buffer as a reference, and subsequent autoradiography imaging.

Mixtures (19 μl) of both rS1 and rS1–8-mer (³²P) and of rS1 and rS1–ADPr (³²P) in 1× degradation buffer were incubated with either 0.2 µg Trypsin (Sigma, EMS0004, mass-spectrometry grade) or Millipore water as a negative control at 37 °C. Samples were taken before the addition of Trypsin/Millipore water and after 120 min. Reactions were stopped by adding one volume 2× Laemmli buffer to samples and were analysed by 12% SDS–PAGE and autoradiography imaging.

Aliquots from washed and equilibrated ADP-ribosylated (1 μl) and RNAylated (2 μl) (³²P) rS1 were treated with either 10 mM HgCl₂ or 500 mM NH₂OH (refs. ^54,55) at 37 °C for 1 h. Reactions were stopped by adding 2× Laemmli buffer and analysed by 12% SDS–PAGE.

Aliquots from washed and equilibrated (in 1× degradation buffer) ADP-ribosylated (1 μl) and RNAylated (2 μl) rS1 (³²P) were treated with 0.5 U endonuclease P1 (Sigma-Aldrich)⁵⁶ or 0.95 µM ARH1 or ARH3 (human recombinant, Enzo Life Science)⁵⁷ in the presence of 10 mM Mg(OAc)₂, 22 mM NH₄Cl, 50 mM HEPES, 1 mM EDTA, 10 mM β-mercaptoethanol and 1% (v/v) glycerol in a total volume of 20 μl at 37 °C for 1 h. Enzymatic reactions were stopped by adding 2× Laemmli buffer and analysed by 12% SDS–PAGE.

Reactions (20 μl) of 1.4 µM ModB and 2.3 µM protein rS1 with either 1 µM ³²P-NAD–8-mer or 3 µM 5′-³²P–8-mer (Supplementary Table 7) were incubated in the presence of 2 mM 3-MB (50 mM stock in DMSO) or the absence of the inhibitor (DMSO only) at 15 °C (ref. ⁵⁸). Samples were taken before the addition of ModB and after 60 min. Reactions were stopped by the addition of 1 volume 2× Laemmli buffer and analysed by 12% SDS–PAGE.

We incubated 1.1 µM NAD–RNA–Cy5 (linear, 5′ overhang, 3′ overhang and blunt ends; Supplementary Table 7) with 0.9 µM rS1 and 0.4 µM ModB in 1× transferase buffer. Samples of 5 µl were taken before the addition of ModB protein and 2, 5, 10, 30, 60 and 120 min after the start of the reaction. The samples were directly mixed with one volume of 2× Laemmli buffer to stop the reaction. The conversion of the substrates was analysed by 12% SDS–PAGE, following visualization on ChemiDoc (Bio-Rad) in the Cy5 channel. The maximum observed signal intensity of RNAylated rS1 protein was used to determine the relative conversion for each of the analysed substrates at distinct time points.

Precultures of E. coli B strain pTAC-rS1 (Supplementary Table 10) were incubated in LB medium with 100 µg ml⁻¹ ampicillin at 37 °C and 185 rpm overnight. For the main cultures, 150 ml LB medium with 100 µg  ml⁻¹ ampicillin were inoculated with preculture to an OD₆₀₀ = 0.1. At OD₆₀₀ = 0.4, protein expression was induced by the addition of 1 mM IPTG. At OD₆₀₀ = 0.8, cultures were either infected with bacteriophage T4 at a multiplicity of infection (MOI) of 10 (20 ml phage solution) (DSM 4505, Leibniz Institute DSMZ) or not infected by adding 20 ml LB medium instead (negative control). Cultures were incubated for 20 min at 37 °C with shaking at 240 rpm. Bacteria were collected by centrifugation at 4,000g at room temperature for 15 min. Pellets were stored at −80 °C.

Bacterial pellets were resuspended in 10 ml buffer A and lysed via sonication (1× 5 min, cycle 2 at 50% power). Lysates were centrifuged at 37,500g at 4 °C for 30 min. The supernatant was filtered through 0.45-μm filters (Sarstedt). rS1 from bacteriophage T4-infected or non-infected E. coli B strain was purified from the supernatant by gravity Ni-NTA affinity chromatography. Ni-NTA agarose slurry (1  ml, Thermo Fisher Scientific) was added to a 10 ml propylene column and equilibrated in buffer A. The supernatant was loaded onto the column twice. The column was washed with a mixture of 95% buffer A and 5% buffer B containing 29.75 mM imidazole. Protein was eluted from the column with 10 ml buffer B.

His-tagged-protein rS1 from T4-infected or uninfected E. coli B strain pTAC-rS1 was washed with two filter volumes of 1× degradation buffer (12.5 mM Tris-HCl, pH 7.5, 25 mM NaCl, 25 mM KCl, 5 mM MgCl₂) by centrifugation in 10-kDa Amicon filters at 5,000g at 4 °C and concentrated to a final volume of 120 μl. The fractions were analysed by 12% SDS–PAGE analysis and the gel was stained in Instant Blue solution for 10 min and imaged immediately.

E. coli B strain with endogenously His-tagged rS1 and E. coli B strain expressing His-tagged rS1 WT, R139A or R139K were infected with T4 to an MOI of 5.0, as described above for 8 min. 100 ml culture was collected and the pellet resuspended in 1.5 ml Ni-NTA buffer A with 15 mM imidazole (50 mM Tris-HCl, pH 7.8, 1 M NaCl, 1 M urea, 5 mM MgSO₄, 5 mM β-mercaptoethanol, 5% glycerol, 15 mM imidazole, one tablet per 500 ml complete EDTA-free protease inhibitor cocktail (Roche)). Cells were lysed by sonication (three times for 2 min at 80% power) and supernatant was cleared by centrifugation at 17,000g at 4 °C for 30 min. The supernatant was incubated with 75 µl Ni-NTA magnetic beads (Jena Bioscience) equilibrated in Ni-NTA buffer A with 15 mM imidazole for 1 h at 4 °C. Magnetic beads were washed seven times with 1 ml Ni-NTA buffer A with 15 mM imidazole and three times with Ni-NTA buffer without imidazole but with 4 M urea. Finally, protein was eluted by addition of Ni-NTA elution buffer (50 mM Tris-HCl, pH 7.8, 1 M NaCl, 1 M Urea, 5 mM MgSO₄, 5 mM β-mercaptoethanol, 5% glycerol, 300 mM imidazole, one tablet per 500 ml complete EDTA-free protease inhibitor cocktail (Roche)). Protein was equilibrated in 1× transferase buffer with Zeba columns (7 kDa MWCO, 0.5 ml) according to the manufacturer’s instructions, and protein was digested with trypsin in a 1:20 ratio (w/w) at 37 °C for 3 h. Peptides were C18-purified using 50 mM triethylamine-acetate (pH 7.0) buffer in combination with 0–90% acetonitrile and Chromabond C18 WP spin columns (20 mg, Macherey Nagel). Purified peptides were dissolved in HPLC-grade H₂O and subjected to LC–MS/MS analysis (see below).

In vitro RNAylated rS1 (D2) reactions in 1× transferase buffer were directly digested (without further purification) with 1 µg RNase A (Thermo Fisher Scientific) and 100 U RNase T1 (Thermo Fisher Scientific) at 37 °C for 1 h, following tryptic digest at 37 °C for 3 h in the same buffer with trypsin (Promega) in a 1:30 ratio (w/w) relative to the total protein content per sample. Peptides were purified with Chromabond C18 WP spin columns as described above and used for LC–MS/MS analysis (see below).

In vitro RNAylation reactions of rL2 with NAD–8-mer and ADP-ribosylation reactions were purified at similar settings to the proteins from T4 phage-infected E. coli. Here, reactions (200 µl) were incubated with 100 µl Ni-NTA beads equilibrated in 800 µl Ni-NTA buffer A with 10 mM imidazole and 40 U murine RNase inhibitor (New England Biolabs) at 4 °C for 1 h. Beads were washed eight times with 1 ml streptavidin wash buffer (50 mM Tris-HCl, pH 7.4, 8 M urea) at room temperature and protein was eluted with 130 µl Ni-NTA elution buffer. Purified proteins were rebuffered in 100 mM NH₄OAc using Zeba spin desalting columns (7 kDa MWCO, 0.5 ml) according to the manufacturer’s instructions. rL2 samples were dissolved in 4 M urea in 50 mM Tris-HCl (pH 7.5) and incubated for 30 min at room temperature, followed by dilution to 1 M urea with 50 mM Tris-HCl (pH 7.5). 10 μg RNase A (Thermo Fisher Scientific) and 1 kU RNase T1 (Thermo Fisher Scientific) were added, following incubation for 4 h at 37 °C. For protein digestion, 0.5 µg trypsin (Promega) was added to each sample and digestion was performed overnight at 37 °C. Samples were adjusted to 1% acetonitrile (ACN) and to pH 3 using formic acid. Samples were cleaned up using C18 columns (Harvard Apparatus) according to the manufacturer’s instructions.

Cleaned-up rS1 and rL2 peptide samples were dissolved in 2% ACN, 0.05% trifluoroacetic acid and subjected to LC–MS/MS analysis using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex Ultimate 3000 RSLCnano system. Peptides were loaded on a Pepmap 300 C18 trap column (Thermo Fisher Scientific) (flow rate, 10 µl min⁻¹) in buffer A (0.1% (v/v) formic acid) and washed for 3 min with buffer A. Peptide separation was performed on an in-house-packed C18 column (30 cm; ReproSil-Pur 120 Å, 1.9 µm, C18 AQ; inner diameter, 75 µm; flow rate 300 nl min⁻¹) by applying a linear gradient of buffer B (80% (v/v) ACN, 0.08% (v/v) formic acid). The main column was equilibrated with 5% buffer B for 18 s, the sample was applied and the column was washed for 3 min with 5% buffer B.

A linear gradient of 10–45% buffer B over 44 min was applied to elute peptides, followed by 4.8 min washing at 90% buffer B and 6 min at 5% buffer B. Eluting rS1 and rL2 peptides were analysed for 58 min in positive mode using a data-dependent top-20 acquisition method. The resolution for MS1 and MS2 were set to 120,000 and 30,000 full-width at half-maximum, respectively, and automatic gain control (AGC) targets were set to 10⁶ (MS1) and 10⁵ (MS2). The MS1 scan range was set to m/z = 350–1,600. Precursors were fragmented using 28% normalized, higher-energy collision-induced dissociation fragmentation. Other analysis parameters were set as follows: isolation width, 1.6 m/z; dynamic exclusion, 9 s; maximum injection times for MS1 and MS2, 60 ms and 120 ms, respectively.

For all measurements, the lock mass option (m/z 445.120025) was used for internal calibration.

MS data were analysed and validated manually using the OpenMS pipeline RNPxl and OpenMS TOPPASViewer³⁰. Precursor mass tolerance was set to 6 ppm. MS/MS mass tolerance was set to 20 ppm. A neutral loss of 42.021798 Da (C₁H₂N₂) at Arg residues was defined, as well as adducts of ribose minus H₂O (78.010565 Da, C₅H₂O), ADP-ribose (541.06111 Da, C₁₅H₂1N₅O₁₃P₂) and ADPr without adenine (485.97295 Da; C₁₀H₁₇O₁₆P₃)³¹. Results were filtered for a 1% false discovery rate on peptide spectrum match level. Ion chromatograms for rS1 peptides were extracted and visualized using Skyline (v.21.2.0.369)⁵⁹.

LC–MS/MS analysis of protein digests was performed on an Exploris 480 mass spectrometer connected to an electrospray ion source (Thermo Fisher Scientific). Peptide separation was done using the Ultimate 3000 nanoLC-system (Thermo Fisher Scientific), equipped with a packed-in-house C18 resin column (Magic C18 AQ 2.4 µm, Dr. Maisch). The peptides were eluted from a precolumn in backflush mode with a gradient from 98% solvent A (0.15% formic acid) and 2% solvent B (99.85% ACN, 0.15% formic acid) to 35% solvent B over 40 min and 90 min, respectively. The flow rate was set to 300 nl min⁻¹. The data-dependent acquisition mode for label-free quantification was set to obtain one high-resolution MS scan at a resolution of 60,000 (m/z of 200) with scanning range from 350 to 1,650 m/z. MS/MS scans were acquired for the 20 most-intense ions (90 min gradient) and for the most-intense ions detected within 2 s (cycle 1 s, 40 min gradient). To increase the efficiency of MS/MS attempts, the charged-state screening mode was adjusted to exclude unassigned and singly charged ions. The ion accumulation time was set to 25 ms for MS and ‘auto’ for MS/MS scans. The AGC was set to 300% for MS survey scans and 200% for MS/MS scans.

Raw MS spectra were analysed using MaxQuant (v.1.6.17.0 and 2.0.3.0) using a fasta database of the targets proteins and a set of common contaminant proteins. The following search parameters were used: full tryptic specificity required (cleavage after Lys or Arg residues); three missed cleavages allowed; carbamidomethylation (C) set as a fixed modification; and oxidation (M; +16 Da), deamidation (N, Q; +1 Da) and ADP-ribosylation (K; +541 Da) set as variable modifications. MaxQuant was executed in the default setting. All MaxQuant parameters are listed in Supplementary Tables 1 and 2. The MS proteomics data have been deposited with the ProteomeXchange Consortium by the PRIDE partner repository under the dataset identifier PXD041714.

The E. coli B strain with endogenously His-tagged rS1 was created by homologous recombination of linear transforming DNA (tDNA) using the pRET/ET plasmid in the E. coli B strain. The linear tDNA was generated by fusion PCR aligning four fragments: 156 base pairs (bp) of the rpsA gene with an additional His-tag amplified from the pET28 rS1 vector (serving as the left homologous flank), a 70-bp fragment of the native rpsA terminator, the Flp-flanked kanamycin cassette from pKD4 and 140 bp of the 3′ flanking region of the rpsA gene (the right homologous flank). The primers used are indicated in Supplementary Table 9. The subsequent procedure for recombination is based on the protocol for the E. coli Gene Deletion Kit by RET/ET Recombination (Gene Bridges). In brief, E. coli B strain containing the pRED/ET plasmid was grown in LB medium supplemented with 100 µg ml⁻¹ ampicillin at 30 °C. At OD₆₀₀ = 0.35, L-arabinose was added to 0.33% (w/v) to induce expression of the RED/ET recombination system during growth at 37 °C for 1 h. Next, 1.4 ml culture was collected by centrifugation at 3,000g at 4 °C for 1 min, and cells were washed twice with 1 ml cold 10% glycerol and finally resuspended in 50 µl 10% glycerol. Cells were electroporated with 1 µg tDNA using a MicroPulser Electroporator (Bio-Rad) at standard settings (Ec1). Electroporated cells were immediately resuspended in 1 ml prewarmed LB medium and incubated at 37 °C with shaking at 600 rpm for 3 h. Finally, cells were plated on kanamycin (30 µg ml⁻¹) LB–agar plates. Cells took 2 days to recover and grow. Successful recombination was evaluated by Sanger sequencing and correct protein expression was validated by pull-down and proteomics.

Cultures (100 ml) of E. coli B strain with endogenously His-tagged rS1 (Supplementary Table 10) in LB medium supplied with 1 mM CaCl₂, 1 mM MgCl₂ and 30 µg ml⁻¹ kanamycin were grown at 37 °C in 250 ml baffled Erlenmeyer flasks to an OD₆₀ of around 0.8. T4 phage WT or T4 phage ModB(R73A, G74A) were added to an MOI of 5.0. For the uninfected negative control, the same volumes of LB medium were added to the cultures. Cultures were then incubated at 37 °C for 8 min and E. coli was collected by centrifugation at 3,000g for 13 min. Dried pellets were stored at −80 °C.

Pellets from the 100 ml culture infected with either WT T4 phage, T4 phage ModB(R73A, G74A) or the uninfected control (LB) were resuspended in 2 ml Ni-NTA wash buffer (10 mM imidazole, 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1 M urea, 5 mM MgSO₄, 5 mM β-mercaptoethanol, 5% glycerol, pH 8.0, EDTA-free protease inhibitor (Roche, one tablet per 500 ml)) on ice and lysed by sonication (6 min, 50% power, 0.5 s pulse). The lysate was cleared from the cell debris by centrifugation at 21,000g at 4 °C for 30 min. Supernatant (1.9  ml), 50 µl Ni-NTA agarose beads (Jena Bioscience, equilibrated in Ni-NTA wash buffer), 80 U murine RNase inhibitor (New England Biolabs) and 4.72 µg rS1 D2 RNAylated with NAD-capped RNAI were combined and incubated at 4 °C in a rotary mixer for 30 min. Entire samples were transferred to Mobicol mini spin columns (MoBiTec). Beads were washed four times with 200 µl Ni-NTA wash buffer and subsequently eight times with 200 µl streptavidin wash buffer (50 mM Tris-HCl, pH 7.5, 8 M urea). Beads were equilibrated in standard ligation buffer (10 mM MgCl₂, 50 mM Tris-HCl, pH 7.4) and blocked with bovine serum albumine (BSA) before 3′ RNA-adapter ligation at 4 °C overnight in the presence of standard ligation buffer, 50 mM β-mercaptoethanol, 0.05 µg µl⁻¹ BSA, 15% (v/v) DMSO, 5 µM adenylated RNA-3′-adapter, 0.5 U µl⁻¹ T4 RNL1 (New England Biolabs) and 10 U µl⁻¹ T4 RNL2, tr. K227Q (New England Biolabs). Protein was rebound by the addition of NaCl to 1.5 M and incubation at 20 °C, with shaking at 400 rpm for 20 min. Beads were subsequently washed six times with streptavidin wash buffer and equilibrated in first strand buffer (50 mM Tris-HCl, pH 8.3, 3 mM MgCl₂, 75 mM KCl) and blocked with BSA. Reverse transcription of protein-bound RNA was done in a 30-µl scale for 1 h at 40 °C using 10 U µl⁻¹ Superscript IV Reverse Transcriptase (Invitrogen) in the presence of 5 µM RT primer, first strand buffer, 25 mM β-mercaptoethanol, 0.05 µg µl⁻¹ BSA and 0.5 mM dNTPs. After incubation, NaCl was added to 1.5 M and the solution was incubated at 20 °C, with shaking at 400 rpm for 1 h to rebind RNA–cDNA hybrids. Beads were subsequently washed five times with 0.25× streptavidin wash buffer (2 M urea, 50 mM Tris-HCl, pH 7.5), equilibrated in ExoI buffer (10 mM Tris-HCl, pH 7.9, 5 mM β-mercaptoethanol, 10 mM MgCl₂, 50 mM NaCl) and blocked with BSA. Residual RT primer was removed by ExoI digest with 1 U µl⁻¹ E. coli ExoI (New England Biolabs) in ExoI buffer at 37 °C for at least 30 min. Finally, beads were washed with 200 µl 0.25× streptavidin wash buffer five times and subsequently with 200 µl immobilization buffer (10 mM Na-HEPES, pH 7.2, 1 M NaCl) three times. cDNA was eluted by incubation of beads in 100 µl 150 mM NaOH at 55 °C for 25 min and by washing with 100 µl MQ water. Eluate pH was neutralized by the addition of 0.05 volumes of 3 M NaOAc, pH 5.5. cDNA was removed from the residual protein by phenol–chloroform extraction and precipitated with 2.5 volumes of ethanol in the presence of 0.3 M NaOAc, pH 5.5 overnight. Precipitated cDNA was C-tailed using 1 U µl⁻¹ TdT (Thermo Fisher) in the presence of 1.25 mM CTP and 1× TdT buffer at 37 °C for 30 min and subsequently inactivated at 70 °C for 10 min. 5 µM cDNA anchor (hybridization of forward and reverse anchor, Supplementary Table 9) was ligated to C-tailed cDNA in standard ligation buffer in the presence of 10 µM ATP and 1.5 U µl⁻¹ T4 DNA Ligase (Thermo Fisher Scientific) at 4 °C overnight. Ligation reactions were inactivated at 70 °C for 10 min and cDNA was ethanol precipitated.

For the preparation of the Illumina RNAylomeSeq library, cDNA was amplified by PCR using 2 U Phusion Polymerase (Thermo Fisher Scientific) in the presence of 5% (v/v) DMSO, 200 µM dNTPs and 2,500 nM New England Biolabs Next Universal and Index Primer each (Primer Set 1, New England Biolabs). PCR products were purified by native PAGE and ethanol-precipitated. The double-stranded DNA (dsDNA) concentration was determined using a Quantus fluorometer (Promega) and library size was determined with the Bioanalyzer (Agilent). Equimolar amounts of each library were sequenced on a MiniSeq system (Illumina) using the MiniSeq High-Output Kit (150 cycles, Illumina) generating 20 million 151-bp single-end reads.

Next-generation sequencing (NGS) data were demultiplexed using bcl2fastq (v.2.20.0, Illumina). Fastq files were assessed using FastQC (v.0.11.9) and Illumina sequencing adapters were trimmed from reads using cutadapt (v.1.18). Reads were aligned to a reference genome composed of an E. coli K12 (U00096.3), bacteriophage T4 (NC_000866.4) and RNAI (our design) with hisat2 (v.2.2.1). Primary alignments were selected using samtools (v.1.7) and reads per genomic feature were counted with featureCounts (v.2.0.1 from Subread package). The resulting counts table was subjected to further analysis and data visualization in R (v.4.1.2). Read counts were normalized to the overall number of mapped reads per sample and to the respective read counts for the RNAI spike-in as follows:

Data visualization was done with a custom R script⁶⁰ and alignments were manually inspected in Integrative Genomics Viewer (IGV v.2.4.9). Hits were identified based on the following criteria: log₂-transformed fold change (LFC) ≥ 1.5 comparing WT T4 and the T4 R73A, G74A mutant and log₂-normalized mean expression among WT and R73A, G74A sample of one replicate ≥ −0.5.

cDNAs from RNAylomeSeq were diluted 1:30 in Millipore water. Quantitative PCR was performed on 1 µl diluted cDNA in 10 µl scale in technical duplicates amplifying regions of 100–150 bp with the iTaq Universal SYBR Green Supermix (Bio-Rad), according to the manufacturer’s instructions, using the primers indicated in Supplementary Table 9. The log₂-transformed difference in cycle-threshold values for WT T4 and T4 R73A, G74A infected samples from corresponding replicates was computed and an LFC ≥ 1 was set as a threshold for cDNA enrichment.

70S ribosomes (4.3 µg µl⁻¹) were RNAylated in transferase buffer in the presence of either 1 µM NAD–10-mer–Cy5 or 1 µM NAD–40-mer–Cy5 (Supplementary Table 7) by 0.05 µg µl⁻¹ ModB at 15 °C for 90 min. RNAylated and non-RNAylated control samples were analysed using 12% SDS–PAGE. To identify RNAylated proteins, SDS–PAGE-separated protein bands were excised and proteins were digested in gel as described previously⁶¹. LC-MS was carried out on an Exploris 480 mass spectrometer connected to an Ultimate 3000 RSLCnano system with a Proflow upgrade and a nanospray flex ion source (all Thermo Scientific). Peptide mixtures were then analysed on the LC-MS system described above with a peptide-separating gradient of 30 min from 2% to 35% buffer B. Peptide separation was performed on a reverse-phase HPLC column (75 μm × 42 cm) packed in-house with C18 resin (2.4 μm, Dr. Maisch). Peptides were ionized at 2.3 kV spray voltage with a heated capillary temperature at 275 °C and funnel RF level at 40. MS survey scans were acquired with a resolution of 120.000 at m/z 200 and full MS AGC target of 300% with a maximal IT of 50 ms. The mass range was set to 350–1,650. Fragment spectra were acquired in data-dependent acquisition mode with a quadrupole isolation window of m/z = 1.5, an AGC target value of 200% and a resolution of 15.000, and fragmentation was induced with a normalized higher-energy collision-induced dissociation collision energy of 27%. MS raw data were searched with SEQUEST embedded in Proteome Discoverer 2.2 (Thermo Scientific) against a Uniprot E. coli protein database containing the bacteriophage T4 protein ModB. Spectral counts were exported from Scaffold Viewer and total spectral counts per sample were used to normalize spectral counts for all other proteins by division in Microsoft Excel 2016 followed by calculation of the ratio of normalized spectral counts from modified and unmodified bands.

A fresh pellet from 40 ml E. coli B strain culture at an OD₆₀₀ of around 0.8 was resuspended in 2 ml transferase buffer (10 mM Mg(OAc)₂, 22 mM NH₄Cl, 50 mM Tris-acetate, pH 7.5, 1 mM EDTA, 10 mM 2-mercaptoethanol, 1% glycerol). Cells were lysed by sonication (3× 2 min at 50% power, 0.5 s pulse) and the lysate was cleared from the cell debris by centrifugation at 27,670g at 4 °C for 30 min. The supernatant was used in RNAylation assays.

Lysate (100 µl) was incubated in the presence of 0.93 µM NAD–10-mer–Cy5 (0.47 µM with reference to the NAD-capped) or 0.93 µM P–10-mer–Cy5 (Supplementary Table 7), 0.37 U murine RNase inhibitor (New England Biolabs) and 0.69 µM ModB at 15 °C. Samples of 10 µl were taken before the addition of ModB and after 2, 5, 10, 20, 30 and 60 min, and were immediately resuspended in one volume of 2× Laemmli buffer. Samples were analysed by 12% SDS–PAGE applying the same reference (rS1 RNAylated with NAD–10-mer–Cy5) to each gel. The Cy5 signal was recorded using the Cy5 blot option of the ChemiDoc Imaging System at a manual exposure of 90 s. Gels were then stained in Coomassie solution and imaged with the same system.

E. coli lysates with various concentrations of ModB were processed and analysed by proteomics as described previously³⁸.

A dilution series of E. coli cell lysate was prepared in PBS. NAD was diluted in PBS starting from a 100 mM stock creating NAD solutions of 1,000 nM to 3.125 nM. The NAD solutions, the lysate dilution series and a PBS blank were assessed for their NAD concentrations using the NAD/NADH-Glo Assay (Promega), according to the manufacturer’s instructions in triplicates. Luminescence measurements were carried out on a Tecan plate reader (Spark) in a 384-well flat white plate. A linear fit (R² = 0.9836) was performed for NAD concentrations between 400 nM and 4 nM with a linear correlation to intensity. The equation was used to calculate NAD concentrations for the E. coli lysate as the mean of the technical triplicates.

Proteins were separated by 10% SDS–PAGE and gels were equilibrated in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, 20 % (v/v) methanol). Polyvinylidene difluoride membranes with a pore size of 0.2 μm (GE Healthcare) were activated in methanol for 1 min and equilibrated in transfer buffer. Proteins were transferred from gels to the membranes in a semi-dry manner at 300 mA for 1.5 h, unless indicated differently. After the transfer, membranes were dehydrated by soaking in methanol and washed twice with TBS-Tween (TBS-T; 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20). Afterwards, 10 ml blocking buffer (5% (w/v) milk powder in TBS-T) were added to the membranes and incubated at room temperature for 1 h. To detect ADP-ribosylated proteins, membranes were incubated with a 1:10,000 dilution of anti-pan-ADPr binding reagent MABE1016 (Merck) in 10 ml washing buffer (1% (w/v) milk powder in TBS-T) at 4 °C overnight⁶². Membranes were washed and incubated with 10 ml of a 1:10,000 dilution of the horseradish peroxidase–goat-anti-rabbit IgG secondary antibody (Advansta) in washing buffer at room temperature for 1 h. Afterwards, membranes were washed with PBS. ADP-ribosylated proteins were visualized by chemiluminescence using the SignalFire ECL Reagent or the SignalFire Elite ECL Reagent (Cell Signaling Technology), according to the manufacturer’s instructions.

If proteins in SDS–PAGE gels needed to be visualized before blotting, a TCE staining method⁵³ was used. Resolving gels were supplemented with 0.5% (v/v) TCE. For visualization, gels were activated by ultraviolet transillumination (with a wavelength of 300 nm) for 60 s. Proteins then showed fluorescence in the visible spectrum.

rS1 proteins were isolated from E. coli strain B pTAC rS1 bacteria (Supplementary Table 10) that were either uninfected or infected with bacteriophage T4. rS1 (1.5 µM) was treated with 1 µM ARH1 in the presence of 12.5 mM Tris-HCl, pH 7.5, 25 mM NaCl, 25 mM KCl and 5 mM MgCl₂. Alternatively, rS1 (1.5 µM) was treated with 0.5 U endonuclease P1 in 100 mM Mg(OAc)₂, 220 mM NH₄Cl, 500 mM HEPES, pH 7.5, 10 mM EDTA, 100 mM β-mercaptoethanol and 10% glycerol. Digests were incubated at 37 °C for 2 h. Afterwards, digests were precipitated by the addition of nine volumes of ethanol and precipitated by centrifugation (14,000 pm) at 4 °C for 1 h. Protein pellets were resuspended in 10 μl 1× Laemmli buffer and analysed by Western blotting. ADPr modifications were detected by the primary antibody MABE1016 (Merck) as described above. The pan-ADPr signals for ADP-ribosylated rS1 were normalized to the corresponding band intensities in the TCE stain. Normalized intensities for untreated rS1 were then divided by the intensity for P1-treated rS1 to yield the fractions of ADP-ribosylated and RNAylated rS1 for the two modifications.

The CRISPR–Cas9 spacer plasmids were generated by introducing the modB spacer sequence into the DS-SPCas plasmid (Addgene, 48645) (Supplementary Table 10). The modB-carrying vector pET28_ModB was used as a donor DNA for homologous recombination in CRISPR–Cas9-mediated mutagenesis. The pET28_ModB plasmid was modified by site-directed mutagenesis, during which point mutations R73A and G74A were exposed to modB. The R73A mutation led to the inactivation of ADP-ribosyltransferase activity. The G74A mutation was located in the protospacer adjacent motif and was required to prevent the cleavage of donor DNA by Cas9 nuclease. The applied primers are listed in Supplementary Table 9. The resulting plasmids were sequenced and transformed into E. coli BL21 (DE3).

The CRISPR–Cas9-mediated mutagenesis was based on previous work³³. The DS_SPCas_ModB plasmid with the target spacer sequence and the donor plasmid pET28a_ModB_R73A/G74A were co-transformed into E. coli DH5α. The cells were further infected by bacteriophage T4 (1:10,000 phages:cells), and the plaque assay was done. The plates were incubated overnight at 37 °C and the resulting plaques were screened for mutants. Single plaques were picked by sterile pipet tips and transferred into 200 µl Pi–Mg buffer (26 mM Na₂HPO₄, 68 mM NaCl, 22 mM KH₂PO₄, 1 mM MgSO₄, pH 7.5) supplied with 2 µl CCl₃H. The samples were incubated at room temperature for 1 h. Next, 2 µl of the sample was transferred to a new PCR tube and heated to 95 °C for 10 min. The sample was further used for DNA amplification using PCR (primers used are listed in Supplementary Table 9). The amplified DNA was purified by agarose gel electrophoresis and submitted for Sanger sequencing.

The E. coli culture of interest was grown to an OD₆₀₀ of around 0.8–1.0. Next, 300 µl of the culture was infected with 100 µl of WT T4 phage or T4 ModB(R73A, G74A) (Supplementary Table 10) mutant, with either defined or unknown MOI. The bacteria–phage suspension was incubated at 37 °C for 7 min and subsequently transferred to 4 ml LB soft agar (0.75%), mixed and poured onto an LB-agar plate (1.5% LB agar). The plates were incubated at 37 °C overnight and validated the following day.

LB medium (100 ml in 500-ml baffled flasks) was inoculated with E. coli B culture overnight to OD₆₀₀ = 0.1 and was then incubated at 37 °C with shaking at 180 rpm until OD₆₀₀ = 0.8 was reached. The culture was cooled to room temperature and infected by either WT T4 phages or T4 ModB(R73A, G74A) mutants (Supplementary Table 10) to an MOI of 5. The culture was further incubated at room temperature with shaking at 150 rpm. Cell lysis was tracked by measuring the OD₆₀₀ at different times of infection (0–200 min after infection). The experiment was run in biological triplicates.

LB medium (100  ml in 500-ml baffled flasks) was inoculated with E. coli B culture overnight to OD₆₀₀ = 0.1 and was then incubated at 37 °C with shaking at 180 rpm until OD₆₀₀ = 0.8 was reached, as above. The culture was infected either by WT T4 phages or T4 ModB(R73A, G74A) mutant (Supplementary Table 10) to an MOI of 0.01 and further incubated at 37 °C without shaking.

To determine the total number of infective centres, T₀ (comprising unadsorbed and already adsorbed phages), at 5 min after infection, 100 µl of infected culture was used to reinfect 300 µl E. coli B cells (OD₆₀₀ = 1.0) with a subsequent plaque assay. The number of unadsorbed phages (U) was determined by transferring 1 ml infected culture to 50 µl CCl₃H. In this way, E. coli cells were disrupted, after which the unadsorbed phages remained intact and were used for plaque assay. T₀−U, represents the number of initially infected centres. The number of unadsorbed phages (U_xmin) was continuously traced during infection and used to calculate the number of T4-phage progeny (T4-phage progeny = U_xmin/(T₀−U_5min). The time point at which the first increase in phage number was observed was treated as the first burst time point and was used to calculate the phage burst size (burst size  =  U_burst1/(T₀−U_5min)).

Data were plotted using OriginPro 2020b software. Error bars represent s.d. of the means for three biological replicates. For selected time points, statistical tests were done as two-sided t-tests in R (v.4.2.2) implemented in the ggpubr package (v.0.6.0) using a significance level of 0.05.

LB medium (100 ml in 500-ml baffled flasks) was inoculated with E. coli B culture overnight to an OD₆₀₀ = 0.1 and incubated at 37 °C with shaking at 180 rpm until OD₆₀₀ = 0.8 was reached, as above. The culture was cooled to room temperature and infected by either WT T4 phages or T4 ModB(R73A, G74A) mutants (Supplementary Table 10) to an MOI of 0.1. Immediately after infection, 100 µl of the culture was used to determine the number of total infective centres, T₀, by plaque assay. Then 100 µl of the culture was taken at different time points of infection (0–25 min after infection) and 5 µl CCl₃H was added to disrupt E. coli cells. This suspension was subsequently used to determine the number of unadsorbed phages (U_xmin) by plaque assay. The calculation of the adsorption rate was performed as follows: adsorption rate (%) = 100% − (U_xmin/T× 100%).

Data were plotted using OriginPro 2020b software. Error bars represent s.d. of the means for three biological replicates. For selected time points, statistical tests were done as two-sided t-tests in R (v.4.2.2) implemented in the ggpubr package (v.0.6.0) using a significance level of 0.05.

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