Strains and growth conditions
All bacterial and phage strains used in this study are listed in Supplementary Table 2. Escherichia coli strains were routinely grown at 37 °C in Luria broth (LB) medium for cloning and maintenance. Phages were propagated by infecting a culture of E. coli MG1655 at an A600 of 0.1–0.2 with a MOI of 0.1. Cleared cultures were pelleted by centrifugation to remove residual bacteria and filtered through a 0.2-μm filter. Chloroform was then added to phage lysates to prevent bacterial growth. All phage infection experiments in liquid media and phage spotting experiments were performed in LB medium at 25 °C, except for spotting of T2 and T4 on strains producing CapRelSJ46 open reading frames were (6.4 g l−1 Na2HPO4-7H2O, 1.5 g l−1 KH2PO4, 0.25 g l−1 NaCl, 0.5 g l−1 NH4Cl medium supplemented with 0.1% casamino acids, 0.4% glycerol, 0.4% glucose, 2 mM MgSO4, and 0.1 mM CaCl2) at 30 °C. For liquid induction experiments from pBAD33 vectors, bacterial cells were grown in M9 medium. Antibiotics were used at the following concentrations (liquid; plates): carbenicillin (50 μg ml−1; 100 μg ml−1) and chloramphenicol (20 μg ml−1; 30 μg ml−1).
Plasmid construction
All plasmids are listed in Supplementary Table 3. All primers and synthesized gene sequences are listed in Supplementary Table 4.
pBR322-capRel constructs: DNA encoding CapRelSJ46, CapRelEbc, CapRelKp or CapRelEcHT open reading frames were expressed in E. coli and 100–200 bp of the upstream region from the source organism was added in each case for native expression (TZ-1 to TZ-5, TZ-92 and TZ-93). DNA was commercially synthesized by Integrated DNA Technology as gBlocks and assembled into a promoter-less backbone of pBR322 amplified with TZ-6 and TZ-7 by Gibson assembly. Mutations that produce the single amino acid substitutions CapRelSJ46(Y155A), CapRelEbc(Y153A), CapRelSJ46(L280Q), CapRelSJ46(L280P) and CapRelSJ46(L307A) were generated by site-directed mutagenesis using primers TZ-8 to TZ-11 and TZ-49 to TZ-54. To add an N-terminal His6-tag or a C-terminal Flag tag to CapRelSJ46, primers TZ-41 and TZ-42 or TZ-45 and TZ-46 were used to PCR-amplify pBR322-capRelSJ46 followed by Gibson assembly. pBR322-capRel-chimera was constructed by inserting capRelEbc(270–339) that had been PCR–amplified with TZ-22 and TZ-23 into pBR322-capRelSJ46 linearized with TZ-20 and TZ-21 using Gibson assembly. To add a C-terminal Flag tag to CapRelEbc or the CapRel chimera, primers TZ-90 and TZ-91, or TZ-45 and TZ-46 were used to PCR-amplify corresponding pBR322 vectors followed by Gibson assembly.
pBAD33-capRelSJ46 constructs: capRelSJ46(1–272) or full-length capRelSJ46 was PCR-amplified with TZ-14 and TZ-15, or TZ-14 and TZ-24, respectively, and inserted into pBAD33 linearized with TZ-12 and TZ-13 using Gibson assembly. pBAD33-capRelSJ46 variants (A77K, R116A, V338A, L339A, A341K, A351K, Y352A or Y355A) were constructed by site-directed mutagenesis using primers TZ-25 to TZ-40. pBAD33-capRelSJ46 variants (R78A, K311A, R314A, E319A and K346A) were constructed by site-directed mutagenesis using primers TZ-80 to TZ-89.
pEXT20-capRelSJ46 construct: capRelSJ46(273–373) was PCR-amplified with primers TZ-18 and TZ-19, and then inserted into linearized pEXT20 with TZ-16 and TZ-17 using Gibson assembly.
pBAD33-gp57 constructs: wild-type or mutant variant (L114P or I115F) gp57 was PCR-amplified from the corresponding wild-type or escape-mutant SECΦ27 phage using primers TZ-43 and TZ-44, and inserted into linearized pBAD33 using Gibson assembly. A C-terminal HA tag was added to wild-type or mutant gp57 using primers TZ-47 and TZ-48 to PCR-amplify the corresponding construct followed by Gibson assembly. The F113Y variant of gp57 was generated by site-directed mutagenesis using primers TZ-63 and TZ-64.
pBAD33-gp8: the genes encoding the major capsid protein homologues Gp8Bas4, Gp8Bas5 and Gp8Bas8 were PCR-amplified from the corresponding phage using primers TZ-55 to TZ-60 and inserted into linearized pBAD33 by Gibson assembly. The Y113F variant of gp8Bas4 was generated by site-directed mutagenesis using primers TZ-61 and TZ-62. The F120L and I124F variants of gp8Bas8 were cloned from the corresponding phage escape mutants using primers TZ-59 and TZ-60.
pET-gp57 constructs: a gp57 fragment was PCR-amplified with primers TZ-65 and TZ-66 and the TZ-67 template using Gibson assembly, the resulting linear DNA fragment was inserted into linearized pET24d (without tag) using TZ-68 and TZ-69. Template TZ-67 was synthesized as a gBlock by Integrated DNA Technology. To construct L114P and I115F-substituted gp57 variants, DNA fragments were PCR-amplified with primer pairs TZ-70 + TZ-71 and TZ-72 + TZ-73 (for L114P) or with pairs TZ-74 + TZ-71 and TZ75 + TZ-73 (for I115F) and pET-gp57 template. The resulting linear DNA fragments were assembled using Gibson assembly.
pET24d-His10-SUMO-capRelSJ46 constructs: the capRelSJ46 open reading frame was PCR-amplified using primers TZ-76 and TZ-77 as well as pBAD-capRelSJ46 as template, and, using Gibson assembly, inserted into a linearized pET24d-His10-SUMO plasmid using primers TZ-78 and TZ-79.
Strain construction
Plasmids described above were introduced into E. coli MG1655 or BW27783 by TSS transformation or electroporation.
A single copy of capRelSJ46, capRelSJ46(Y155A), His6-capRelSJ46, gp57 or gp57-HA with its native promoter was inserted onto the MG1655 chromosome at the HK022 attachment site using the CRIM system45, using the pAH69 helper plasmid with the pAH144 vector containing the desired insert.
Bas4 mutant phage were generated using a CRISPR–Cas system for targeted mutagenesis as described46. In brief, sequences for RNA guides to target Cas9-mediated cleavage were designed using the toolbox in Geneious Prime 2021.2.2 and selected for targeting of gp8Bas4 but nowhere else in the Bas4 genome. The guides were inserted into the pCas9 plasmid and tested for their ability to restrict Bas4. An efficient guide was selected and the pCas9 guide plasmid was co-transformed into E. coli MG1655 with a high copy number repair plasmid containing gp8Bas4(Y113F) with the guide mutated to prevent self-cutting. The wild-type Bas4 phage was plated onto a strain containing both the pCas9 guide and the repair plasmid, and single plaques were screened by Sanger Sequencing. Two clones that produce the Y113F substituted Gp8 were propagated twice on strains containing only pCas9 guide for further selection and genomes were sequence verified by Illumina sequencing as described below.
External data
Previously published structures are available from the Protein Data Bank (PDB) with IDs 5DEC, 6S2T and 2LVH. The UniRef90 database is publicly available. Reference phage genomes are publicly available: SECΦ27 (NC_047938.1), Bas04 (MZ501069.1), Bas08 (MZ501059.1).
Toxicity assays on solid media
For producing the CapRelSJ46 N- and C-terminal domains, single colonies of E. coli MG1655 containing pBAD33-capRelSJ46(1–272) and pEXT20-capRelSJ46(273–373) or the corresponding empty vectors were grown for 6 h at 37 °C in LB-glucose to saturation. 200 μl of each saturated culture was then pelleted by centrifugation at 4,000g for 10 min, washed once in 1× phosphate-buffered saline (PBS), and resuspended in 400 μl 1× PBS. Cultures were then serially diluted 10-fold in 1× PBS and spotted on M9L plates (M9 medium supplemented with 5% LB (v/v)) further supplemented with 0.4% glucose, 0.2% arabinose or 0.2% arabinose and 100 μM IPTG. Plates were then incubated at 37 °C overnight before imaging.
For producing full-length CapRelSJ46, E. coli MG1655 containing pBAD33-capRelSJ46 or a mutant form of capRelSJ46 were grown to saturation and processed as above. Cultures were plated onto 0.4% glucose and 0.2% arabinose and incubated at 37 °C overnight.
For co-producing CapRelSJ46 and the major capsid proteins from SECΦ27, Bas4, Bas5, or Bas8, E. coli MG1655 harbouring pBR322-capRelSJ46 or MG1655 containing genomic capRelSJ46 and pBAD33-capsid protein were grown to saturation and processed as above. Cultures were plated onto 0.4% glucose and 0.2% arabinose and incubated at 37 °C overnight.
For co-producing CapRelSJ46 and variants of the major capsid protein from Bas8, E. coli BW27783 harbouring pBR322-capRelSJ46 and pBAD33-gp8Bas8 (wild-type or a mutant variant) were grown to saturation and processed as above. Cultures were plated onto 0.4% glucose and 0.0002% arabinose and incubated at 37 °C overnight.
Phage spotting assays and EOP measurements
Phage stocks isolated from single plaques were propagated in E. coli MG1655 at 37 °C in LB. To titre phage, dilutions of stocks were mixed with E. coli MG1655 and melted LB + 0.5% agar and spread on LB + 1.2% agar plates and incubated at 37 °C overnight. For phage spotting assays, 40 μl of a bacterial strain of interest was mixed with 4 ml LB + 0.5% agar and spread on an LB + 1.2% agar + antibiotic plate. Phage stocks were then serially diluted in 1× FM buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM MgSO4), and 2 μl of each dilution was spotted on the bacterial lawn. Plates were then incubated at 25 °C overnight before imaging. EOP was calculated by comparing the ability of the phage to form plaques on an experimental strain relative to the control strain. Experiments were replicated 3 times independently and representative images are shown.
For spotting phage T2 and T4 on strains producing CapRelSJ46 variants, 40 μl of a bacterial strain of interest was mixed with 4 ml M9 + 0.5% agar and spread on an M9 + 1.2% agar + antibiotic plate. Phage were serially diluted and spotted as described above. Plates were then incubated at 30 °C overnight before imaging.
Growth curves following phage infection in liquid culture
Single colonies of E. coli MG1655 pBR322 empty vector or pBR322-capRelSJ46 or pBR322-capRelSJ46(Y155A) were grown in LB overnight. Cultures were then back-diluted to A600 = 0.1 in fresh LB and 100 μl of cells were added into each well of a 96-well plate. Ten microlitres of serial diluted T4 phage were added to each well at the indicated MOI and growth following phage infection was measured at 15 min intervals with orbital shaking at 25 °C on a plate reader (Biotek). Data reported are the mean and standard deviation of eight plate replicates and the growth curve experiment was replicated three times independently.
One-step growth curves
Single colonies of E. coli MG1655 pBR322 empty vector or pBR322-capRelSJ46 were grown overnight in LB. Overnight cultures were back-diluted to A600 = 0.05 in 25 ml fresh LB and grown to A600 ~0.3 at 25 °C. Ten millilitres of each culture was infected with T4 phage at an MOI of 0.05 or SECΦ27 phage at an MOI of 0.01 in LB at 25 °C and phages were allowed to adsorb for 10 min before serial dilution in LB three times (1:100, 1:10, 1:10 serial dilution) to three flasks. Then, at indicated time points, 100 μl of infected cells from the corresponding dilution flask were mixed with 100 μl of indicator cells MG1655 pBR322 empty vector (A600 ~0.3), and the mixtures were mixed with 4 ml of LB + 0.5% agar and spread on LB + 1.2% agar plates. Plates were incubated overnight at 25 °C and plaques were enumerated the following day. PFU was calculated based on the dilution flask samples were taken from. Data reported are the mean and individual data points from 3 biological replicates.
Adsorption assays
Single colonies of E. coli MG1655 pBR322-capRelSJ46 or pBR322 empty vector were grown overnight in LB. Overnight cultures were back-diluted to A600 = 0.05 in 25 ml of fresh LB and grown to A600 = 0.2 at 25 °C. Cells or a control containing only media were infected with phage T4 or SECΦ27 at MOI = 0.1 and incubated at 25 °C for 10 min to allow for adsorption. Cells were pelleted at 9,000g for 2 min and unadsorbed phages from the supernatant were serial diluted, mixed with indicator cells MG1655 pBR322 empty vector + top agar and plated. Plates were incubated overnight at 25 °C and plaques were enumerated the following day. Percent adsorption was calculated by normalizing unadsorbed phages from each sample to media control. Data reported are the mean and individual data points from 3 biological replicates.
Western blot of CapRelSJ46 and Gp57 after phage infection
For immunoblotting of CapRelSJ46, single colonies of E. coli MG1655 pBR322-His6-capRelSJ46 or E. coli MG1655 genomic His6-capRelSJ46 under its native promoter were grown overnight in LB. Overnight cultures were back-diluted to A600 = 0.05 in 25 ml fresh LB and grown to A600 = 0.2 at 25 °C. Cells were infected with phage SECΦ27 at MOI = 100, and incubated at 25 °C during the experiment. At each indicated time point (0, 10, 20, 40, 60 min), A600 was measured and 1 ml of cells was pelleted at 21,000g for 2 min at 4 °C. Supernatant was removed and pellets were flash-frozen in liquid nitrogen. Pellets were thawed and resuspended in 1× Laemmli sample buffer (Bio-Rad) supplemented with 2-mercaptoethanol with A600 normalized. Samples were then boiled at 95 °C and analysed by 12% SDS–PAGE and transferred to a 0.45 μm PVDF membrane. Anti-His6 antibody (Invitrogen) was used at a final concentration of 1:1000, and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) was used to develop the blots. Blots were imaged by a ChemiDoc Imaging system (Bio-Rad). Blots were stained with Coomassie stain and imaged as loading control. Image shown is a representative of two independent biological replicates.
For immunoblotting of Gp57, single colonies of E. coli MG1655 containing genomic gp57-HA under its native promoter were grown overnight in LB. Overnight cultures were back-diluted to A600 = 0.05 in 25 ml of fresh LB and grown to A600 = 0.2 at 25 °C. Cells were infected with phage SECΦ27 at MOI = 100, and incubated at 25 °C during the experiment. At each indicated time point (0, 10, 20, 30, 40, 50 min), A600 was measured and 1 ml of cells was pelleted at 21,000g for 2 min at 4 °C. Samples were processed as described above with A600 normalized. Samples were then boiled at 95 °C and analysed by 12% SDS–PAGE and transferred to a 0.45 μm PVDF membrane. Anti-HA antibody (Cell Signaling Technology) was used at a final concentration of 1:1000, and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) was used to develop the blots. Blots were imaged by a ChemiDoc Imaging system (Bio-Rad), then stained with Coomassie stain and imaged as loading control. The experiment was performed three times independently and band intensities were quantified using Fiji. Relative band intensities were calculated by normalizing the summed intensity of both Gp57 bands to the intensity of total proteins by Coomassie stain.
Western blot of CapRelSJ46, CapRelEB or chimera expression levels
Single colonies of E. coli MG1655 pBR322–capRelSJ46, pBR322–capRelSJ46-Flag, pBR322–capRelEbc-Flag or pBR322–chimera-Flag were grown overnight in LB. Overnight cultures were back-diluted to A600 = 0.05 in 5 ml fresh LB and grown to A600 = 0.2 at 37 °C. A600 was measured and 5 ml of cells was pelleted at 4,000g for 5 min. Supernatant was removed and pellets were resuspended in 1× Laemmli sample buffer (Bio-Rad) supplemented with 2-mercaptoethanol with A600 normalized. Samples were then boiled at 95 °C and analysed by 12% SDS–PAGE and transferred to a 0.45 μm PVDF membrane. Anti-Flag antibody (Cell Signaling Technology) and anti-GyrA antibody (Inspiralis) were used at a final concentration of 1:1,000, and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) was used to develop the blots. Blots were imaged by a ChemiDoc Imaging system (Bio-Rad). Image shown is a representative of 2 independent biological replicates.
Error-prone PCR mutagenesis of CapRelSJ46
The C terminus of CapRelSJ46 was mutagenized using error-prone PCR-based mutagenesis as described previously47. In brief, primers TZ-94 and TZ-95 were used to amplify the C terminus of CapRelSJ46 using Taq polymerase (NEB) and 0.5 mM MnCl2 was added to the reaction as the mutagenic agent. PCR products were treated with Dpn I, column purified, and inserted into pBR322-CapRelSJ46 backbone amplified with primer TZ-96 and TZ-97 using Gibson assembly. Gibson products were transformed into DH5α and grown overnight in LB at 37 °C. Overnight cultures were miniprepped to obtain the mutagenized library. Individual colonies were Sanger sequenced to assess the number of mutations. To perform the selection, mutagenized library was electroporated into E. coli MG1655 pBAD33-gp57, and plated onto LB plates containing 0.2% arabinose to select for survivors. Colonies were picked and sequenced to identify mutations in CapRelSJ46, and further validated by constructing plasmid with only single mutants.
Isolation of phage escape mutants to infect CapRelSJ46
The phage evolution experiment was conducted as described previously48. In brief, 5 independent populations were evolved in a 96-well plate containing a sensitive host E. coli MG1655 pBR322 empty vector and a resistant host E. coli MG1655 pBR322-capRelSJ46. One control population was evolved with only the sensitive host. Overnight bacterial cultures were back-diluted to A600 = 0.1 in LB and 100 μl were seeded into each well. Cells were infected with tenfold serial dilutions of SECΦ27 phage with MOI from 102 to 10−4, with one well uninfected to monitor for contamination. Plates were sealed with breathable plate seals and incubated at 25 °C for 6 h in a plate shaker at 1,000 rpm. Cleared wells from each population were pooled, pelleted at 4,000g for 20 min to remove bacteria, and the supernatant lysates were transferred to a 96 deep-well block with 40 µl chloroform added to prevent bacterial growth. Lysates were spotted onto both sensitive and resistant hosts to check the defence phenotype. Thirteen rounds of evolution were performed to allow all five populations to overcome CapRelSJ46 defence. Evolved clones from each evolved population were isolated by plating to single plaques on lawns of resistant host, and control clones from the control population were isolated on a lawn of the sensitive host. Two clones from each population were propagated using the corresponding host and sequenced as described below.
Bas8 escape mutants were isolated by plating a population of phage onto CapRelSJ46- or CapRelEcHT-containing cells. Twenty microlitres of 1011 PFU ml−1 Bas8 phage mixed with 40 µl overnight culture of E. coli MG1655 pBR322-capRelSJ46 or pBR322-capRelEcHT were added to 4 ml LB + 0.5% agar and spread onto LB + 1.2% agar. Plates were incubated at 25 °C overnight. Single plaques were isolated and propagated using the same strain in LB at 25 °C. Amplified phage lysates were pelleted to remove bacteria, and then plated to single plaques and propagated similarly for a second round of isolation to improve purity and sequenced.
Phage DNA extraction and Illumina sequencing
To extract phage DNA, high-titre phage lysates (>106 PFU μl−1) were treated with DNase I (0.001 U µl−1) and RNase A (0.05 mg ml−1) at 37 °C for 30 min. 10 mM EDTA was used to inactivate the nucleases. Lysates were then incubated with proteinase K at 50 °C for 30 min to disrupt capsids and release phage DNA. Phage DNA was isolated by ethanol precipitation. In brief, sodium acetate pH 5.2 was added to 300 mM followed by 100% ethanol to a final volume fraction of 70%. Samples were incubated at −80 °C overnight, pelleted at 21,000g for 20 min and supernatant removed. Pellets were washed with 100 µl isopropanol and 200 µl 70% (v/v) ethanol, and then aired dried at room temperature and resuspended in 25 µl 1× TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8). Concentrations of extracted DNA were measured by NanoDrop (Thermo Fisher Scientific).
To prepare Illumina sequencing libraries, 100–200 ng of genomic DNA was sheared in a Diagenode Bioruptor 300 sonicator water bath for twenty 30 s cycles at maximum intensity. Sheared genomic DNA was purified using Ampure XP beads, followed by end repair, 3′ adenylation, and adapter ligation. Barcodes were added to both 5′ and 3′ ends by PCR with primers that anneal to the Illumina adapters. The libraries were cleaned by Ampure XP beads using a double cut to elute fragment sizes matching the read lengths of the sequencing run. Libraries were sequenced on an Illumina MiSeq at the MIT BioMicro Center. Illumina reads were assembled to the reference genomes using Geneious Prime 2021.2.2.
Mass spectrometry of phages
Wild-type or mutant (L114P in Gp57, evolved clone 1 from population 3) SECΦ27 phage were propagated in E. coli MG1655 for high-titre stocks. In brief, E. coli MG1655 (A600 = 0.2) in LB were infected with phages at MOI = 0.1 and incubated at 37 °C for 4 h. Cells were pelleted at 4,000g for 10 min and supernatant lysates were filtered through 0.2-μm filters. Five hundred microlitres of phage stocks (1010 PFU μl−1) were further concentrated with Amicon Ultra filter (MW 100 kDa) and washed twice with 1× FM buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM MgSO4). Concentrated phage lysates were boiled to denature virions and run on 4–20% SDS–PAGE. Each lane from the gel was excised. Proteins were reduced with 10 mM dithiothreitol (Sigma) for 1 h at 56 °C and then alkylated with 20 mM iodoacetamide (Sigma) for 1 h at 25 °C in the dark. Proteins were then digested with 12.5 ng µl−1 modified trypsin (Promega) in 50 μl 100 mM ammonium bicarbonate, pH 8.9 at 25 °C overnight. Peptides were extracted by incubating the gel pieces with 50% acetonitrile/5% formic acid then 100 mM ammonium bicarbonate, repeated twice followed by incubating the gel pieces with 100% acetonitrile then 100 mM ammonium bicarbonate, repeated twice. Each fraction was collected, combined, and reduced to near dryness in a vacuum centrifuge. Peptides were desalted using Pierce Peptide Desalting Spin Columns (Thermo) and then lyophilized. The tryptic peptides were separated by reverse phase HPLC (Thermo Ultimate 3000) using a Thermo PepMap RSLC C18 column over a 90 min gradient before nano-electrospray using an Exploris mass spectrometer (Thermo). Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile. Detected peptides were mapped to SECΦ27 protein sequences and the abundance of proteins were estimated by number of spectrum counts/molecular mass to normalize for protein sizes.
Co-immunoprecipitation analysis
For immunoprecipitation of CapRelSJ46 after phage infection, E. coli MG1655 containing pBR322-capRelSJ46-Flag were grown overnight in LB. Overnight cultures were back-diluted to A600 = 0.05 in 175 ml of LB and grown to A600 ~0.3 at 25 °C. Cells were infected with wild-type or mutant (L114P in Gp57, evolved clone 1 from population 3) SECΦ27 at MOI = 100 and incubated at 25 °C. At the indicated time points (15 min or 40 min), A600 was measured and 50 ml of cells were pelleted at 6,000g for 5 min at 4 °C. Uninfected cells were collected at 0 min before phage infection. Supernatant was removed and cells were resuspended in 900 μl lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 5% glycerol) supplemented with protease inhibitor (Roche), 1 μl per ml Ready-Lyse Lysozyme Solution (Lucigen) and 1 μl per ml benzonase nuclease (Sigma). Samples were lysed by two freeze-thaw cycles, and lysates were normalized by A600. Lysates were pelleted at 21,000g for 10 min at 4 °C, and 850 μl of supernatant were incubated with pre-washed anti-Flag M2 magnetic beads (Sigma) for 1 h at 4 °C with end-over-end rotation. Beads were then washed 3 times with lysis buffer containing 350 mM NaCl but free of detergent. On-bead reduction, alkylation and digestion were performed. Proteins were reduced with 10 mM dithiothreitol (Sigma) for 1 h at 56 °C and then alkylated with 20 mM iodoacetamide (Sigma) for 1 h at 25 °C in the dark. Proteins were then digested with modified trypsin (Promega) at an enzyme/substrate ratio of 1:50 in 100 mM ammonium bicarbonate, pH 8 at 25 °C overnight. Trypsin activity was halted by addition of formic acid (99.9 %, Sigma) to a final concentration of 5 %. Peptides were desalted using Pierce Peptide Desalting Spin Columns (Thermo) then lyophilized. The tryptic peptides were subjected to LC–MS/MS as described above. Experiments were performed two times independently and spectral counts are reported. Ratio of spectral counts between Gp57 and CapRelSJ46 were calculated and plotted for normalization.
For co-producing CapRelSJ46 and Gp57, E. coli MG1655 containing pBR322-capRelSJ46 or pBR322-capRelSJ46-Flag (wild type or mutants) and pBAD33-gp57-HA (wild type or mutants) were grown overnight in M9-glucose. Overnight cultures were back-diluted to A600 = 0.05 in 50 ml of M9 (no glucose) and grown to A600 ~0.3 at 37 °C. Cells were induced with 0.2% arabinose for 30 min at 37 °C, then A600 was measured and cells were pelleted at 4,000 g for 10 min at 4 °C. Supernatant was removed and cells were resuspended in 900 μl lysis buffer as described above. Samples were lysed by two freeze-thaw cycles, and lysates were normalized by A600. Lysates were pelleted at 21,000g for 10 min at 4 °C, and 850 μl of supernatant were incubated with pre-washed anti-Flag M2 magnetic beads (Sigma) for 1 h at 4 °C with end-over-end rotation. Beads were then washed 3 times with lysis buffer containing 350 mM NaCl. Laemmli sample buffer (Bio-Rad) supplemented with 2-mercaptoethanol was added to beads directly to elute proteins. Samples were boiled at 95 °C and analysed by 12% SDS–PAGE and transferred to a 0.45 μm PVDF membrane. Anti-Flag and anti-HA antibodies (Cell Signaling Technology) were used at a final concentration of 1:1,000, and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) was used to develop the blots. Blots were imaged by a ChemiDoc Imaging system (Bio-Rad). Images shown are representatives of 3 independent biological replicates.
Incorporation assays
For co-producing CapRelSJ46 and Gp57, the SECΦ27 major capsid protein, single colonies of E. coli MG1655 containing pBR322-capRelSJ46 and pBAD33-gp57 (wild-type or L114P variant) or corresponding empty vectors were grown overnight in M9-glucose. Overnight cultures were back-diluted to A600 = 0.05 in 25 ml M9-glucose and grown to A600 ~0.3 at 37 °C. Cells were pelleted at 4,000g for 5 min at 4 °C and washed once with M9 (no glucose), and then back-diluted to A600 = 0.1 in 15 ml M9 (no glucose) and recovered for 45 min at 37 °C. At the beginning of the experiment, cells were induced with 0.2% arabinose. At the indicated time points (0, 10, 20, 30, 40 min), A600 was measured and an aliquot of 250 μl of cells was transferred to microcentrifuge tube containing [5,6-3H]uridine (PerkinElmer) (4 μCi ml−1) for transcription measurements or EasyTag EXPRESS-35S Protein Labeling Mix, [35S] (PerkinElmer) at 44 μCi ml−1 for translation measurements. Tubes were incubated at 37 °C for 2 min, then quenched by addition of nonradioactive uridine (1.5 mM) or cysteine and methionine (15 mM each) and incubated for an additional 2 min. Samples were then added to ice cold trichloroacetic acid (TCA) (10% w/v) and incubated at least 30 min on ice to allow for precipitation. Resulting samples were vacuum filtered onto a glass microfibre filter (Whatman, 1820-024) that had been pre-wetted with 5% w/v TCA. Filters were washed with 35× volume of 5% w/v TCA, then with 5× volume of 100% ethanol. Air dried filters were placed in tubes with scintillation fluid and measured in a scintillation counter (PerkinElmer). CPM (Counts Per Million) was normalized to A600 and percent incorporation at each time point was calculated by normalizing to t = 0. Data reported are the mean and individual data points from three independent biological replicates.
For producing the CapRelSJ46 N-terminal toxin domain, single colonies of E. coli MG1655 containing pBAD33-capRelSJ46(1–272) or an empty vector were grown overnight in M9-glucose. Transcription and translation experiments were done as described above. Data reported are the mean and individual data points from three independent biological replicates.
For phage infection experiments, single colonies of E. coli MG1655 harbouring pBR322 empty vector or pBR322-capRelSJ46 were grown overnight in LB. Overnight cultures were back-diluted to A600 = 0.05 in 25 ml fresh LB and grown to A600 ~0.3 at 25 °C. Cells were then diluted to A600 = 0.1 in 10 ml LB and infected with wild-type or mutant (L114P in Gp57, evolved clone 1 from population 3) SECΦ27 at MOI = 100 and incubated at 25 °C. At the indicated time points (0, 15, 30, 45 and 60 min), A600 was measured and an aliquot of 250 μl of cells was transferred to a microcentrifuge tube containing [5,6-3H]uridine (PerkinElmer) (32 μCi ml−1) for transcription measurements or EasyTag EXPRESS-35S Protein Labeling Mix, [35S] (PerkinElmer) at 88 μCi ml−1 for translation measurements. Tubes were incubated at 25 °C for 4 min, then quenched by addition of nonradioactive uridine (1.5 mM) or cysteine and methionine (15 mM) and incubated for an additional 2 min. Samples were then processed same as above. Data reported are the mean and individual data points from three independent biological replicates. Statistical significance was determined by unpaired, two-tailed Student’s t-test (P < 0.05).
Homology search, alignment, and conservation analysis
CapRelSJ46 was identified in the sequence database from our previous bioinformatic survey of RSH proteins24 that included gene neighbourhood analysis to identify toxin–antitoxin systems49. Bacterial strains containing CapRelSJ46, CapRelEbc or CapRelKp with 100% amino acid identity were found on NCBI database. Local genomic regions (±10 kb of CapRel) were extracted and annotated for all coding sequences. Prophage genes and intact prophage regions were identified by PHASTER50. Additional homologues of CapRelSJ46 were identified by ConSurf51 using PSI-BLAST (default settings) to search UniRef90 database, yielding 44 homologues. For Extended Data Fig. 1, sequences were aligned with MAFFT L-INS-i v7.453 (ref. 52) with manual curation of the C-terminal region guided by homology modelling of the stand-alone Phrann Gp30 antitoxin using Swiss-Model53, and with our CapRelSJ46 predicted structure as a template. For ConSurf analysis, 52 homologues were used to generate the multiple sequence alignment by MAFFT and used as input. Conservation scores were calculated using the Bayesian method and default settings. For gene neighbourhood analysis, previously identified CapRel and PhRel sequences24 were reduced to representatives with 65% amino acid identity using MMSeqs v13.45111 easy-cluster (default settings54), while retaining proteins of interest. The associated accession numbers were then used as input to FlaGs v1.2.6 (one flanking gene either side of the query and otherwise default settings)49.
Homologues of the major capsid proteins in BASEL phages were identified by BLASTp55 searches against each phage genome. Homologues of Gp57 (Gp8Bas4, Gp8Bas5 and Gp8Bas8) were aligned by MUSCLE56.
CapRelSJ46 preparation for crystallization and HDX-MS
For the production of His10–SUMO-tagged CapRelSJ46 and CapRelSJ46 variants, E. coli BL21 (DE3) cells were transformed with pET24d plasmids containing the gene of interest and grown in LB medium to A600 of 0.6. Expression of the protein of interest was induced by addition of 0.5 mM IPTG, and cells were grown for 3 h at 30 °C. The culture was then centrifuged, and pellet was resuspended in resuspension buffer (50 mM Tris-HCl pH 8.0, 1.5 M KCl, 2 mM MgCl2, 1 mM TCEP, 0,002% mellitic acid and 1 pastil of protease inhibitors cocktail (Roche)).
Cells were disrupted using a high-pressure homogenizer (Emulsiflex) and the supernatant was separated from the pellet by centrifugation and filtered through 0.45 μm filters. Protein extracts were loaded onto a gravity-flow column (Cytiva) packed with HisPur Nickel resin (Thermo Fisher Scientific), washed with buffer A (50 mM Tris-HCl pH 8, 500 mM NaCl, 500 mM KCl, 1 mM TCEP, 0.002% Melitic acid) and stepwise eluted in buffer A supplemented with 500 mM imidazole. To remove remaining contaminants and imidazole, the elution fraction was immediately transferred to a size-exclusion chromatography (SEC) column Superdex 200 pgcolumn (Cytiva), previously equilibrated in the SEC buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 500 mM KCl, 2 mM MgCl2, 1 mM TCEP, 0.002% mellitic acid). The fractions containing the protein were concentrated to around 1 mg ml−1 and the His tag was removed by incubating with UlpI protease (1:50 molar ratio) at 4 °C for 30 min. The His10–SUMO tag and the protease were then removed by passing the samples over a gravity-flow column (Cytiva) packed with HisPur Nickel resin (Thermo Fisher Scientific). Purity of the sample preparation was assessed spectrophotometrically and by SDS–PAGE. For all the purified protein samples, A260/A280 ratio was below 0.6. Samples were stored at −20 °C or concentrated to 7 mg ml−1 and used directly in crystallization experiments.
For the purification of the complex containing His10–SUMO–CapRelSJ46 and His10–SUMO–Gp57, E. coli BL21 (DE3) strain containing freshly transformed pET24d-His10-SUMO-capRelSJ46(Y155A) and pET21a-His10-SUMO-gp57 were grown in LB medium to A600 of 0.2. This culture was then diluted in fresh LB media and grown until A600 of 0.6. Expression of the protein of interest was induced by addition of 0.5 mM IPTG, and cells were grown for overnight at 16 °C. The subsequent purification, Sumo tag cleavage and purity assessment steps were identical to the workflow described above for the all the CapRelSJ46 protein variants.
Crystallization of CapRelSJ46
The screening of crystallization conditions of CapRelSJ46 was carried out using the sitting-drop vapour-diffusion method. The drops were set up in Swiss (MRC) 96-well two-drop UVP sitting-drop plates using the Mosquito HTS system (TTP Labtech). Drops of 0.1 μl protein and 0.1 μl precipitant solution were equilibrated to 80 μl precipitant solution in the reservoir. Commercially available screens LMB and SG1 (Molecular Dimensions) were used to test crystallization conditions. The condition resulting in protein crystals (LMB screen position C9 for CapRelSJ46: 26% w/v PEG 2000 MME 0.1 M Bis-Tris 5.8) were repeated as 2 µl drops. The final crystallization drop was made by mixing in a 1:1 ratio the crystallization mother liquor at pH 5.8 and CapRelSJ46 at pH 8.0. Crystals were collected using suitable cryo-protecting solutions (mother liquor supplemented with 20% glycerol) and vitrified in liquid N2 for transport and storage before X-ray exposure. X-ray diffraction data was collected at the SOLEIL synchrotron (Gif-sur-Yvette, Paris, France) on the Proxima 1 (PX1) and Proxima 2A (PX2A) beamlines using an Eiger-X 16M detector. Because of the high anisotropic nature of the data from all the crystals we performed anisotropic cut-off and correction of the merged intensity data as implemented on the STARANISO server (http://staraniso.globalphasing.org/) using the DEBYE and STARANISO programs. The analysis of the data suggested a resolution of 2.31 Å (with 2.31 Å in a*, 2.85 Å in b* and 2.72 Å in c*).
Structure determination
The data were processed with the XDS suite57 and scaled with Aimless. In all cases, the unit cell content was estimated with the program MATTHEW COEF from the CCP4 program suite58. Molecular replacement was performed with Phaser59. The crystals of CapRelSJ46 diffracted on average to ~2.3 Å. We used the coordinates of RelTtNTD (PDB ID 6S2T) as a search model for the toxSYNTH domain60. The MR solution from Phaser was used in combination with Rosetta as implemented in the MR-Rosetta61 suit from the Phenix package62. After several iterations of manual building with Coot63 and maximum likelihood refinement as implemented in Buster/TNT64, the model was extended to cover all the residues (R/Rfree of 21.5/26.0). Extended Data Table 1 details all the X-ray data collection and refinement statistics.
Isothermal titration calorimetry
For all ITC measurements CapRelSJ46 samples were prepared from the pET24d-His10-SUMO-capRelSJ46 as detailed above. In the case of Gp57, E. coli BL21 (DE3) cells were transformed with pET21a-His10-SUMO-gp57 and grown in LB medium to A600 of 0.2. This culture was then diluted in fresh LB media and grown until A600 of 0.6. Expression of His10–SUMO–Gp57 was induced by addition of 0.1 mM IPTG, and cells were grown for overnight at 16 °C. The subsequent purification, SUMO-tag cleavage and purity assessment steps were identical to the workflow described above for the all the CapRelSJ46 protein variants. After removing the SUMO tag, samples were concentrated to 10 μM and used directly for ITC immediately after purification.
All titrations were performed with an Affinity ITC (TA instruments) at 25 °C. For the titration, CapRelSJ46 was loaded in the instrument syringe at 150 μM and Gp57 was used in the cell at 10 μM. The titration was performed in 50 mM HEPES pH 7.5; 500 mM KCl; 500 mM NaCl; 150 mM imidazole; 10 mM MgCl2; 1 mM TCEP; 0.002% mellitic acid. Final concentrations were verified by the absorption using a Nanodrop One (ThermoScientific). All ITC measurements were performed by titrating 2 µl of CapRelSJ46 (or a CapRelSJ46 variant) into Gp57 (or a Gp57 variant) using a constant stirring rate of 75 rpm. All data were processed, buffer-corrected and analysed using the NanoAnalyse and Origin software packages.
Multiwavelength light scattering
The sample of the CapRelSJ46-Gp57 complex used for the MALS measurements was prepared in the same way as the samples used for hydrogen–deuterium exchange mass spectrometry (HDX-MS): 50 mM HEPES pH 7.5; 500 mM KCl; 500 mM; NaCl; 10 mM MgCl2; 1 mM TCEP; 0.002% mellitic acid at a concentration of 5 mg ml−1. The measurement was performed in an HPLC Alliance system (Waters) connected to a 2998 PDA detector (Waters), a TREOS II MALS detector (Wyatt Technology) and a RI-501 refractive index detector (Shodex). The data were analysed with Astra 7 suite (Wyatt technology).
HDX-MS
HDX-MS experiments were performed on an HDX platform composed of a Synapt G2-Si mass spectrometer (Waters Corporation) connected to a nanoAcquity UPLC system. Samples of CapRelSJ46 and CapRelSJ46 complexed with Gp57 were prepared at a concentration of 20 to 50 µM. For each experiment 5 µl of sample (CapRelSJ46 or CapRelSJ46-Gp57) were incubated for 1 min, 5 min, 15 min or 60 min in 95 µl of labeling buffer L (50 mM HEPES, 500 mM KCl, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 0.002% mellitic acid, pH 7.5) at 20 °C. The non-deuterated reference points were prepared by replacing buffer L by equilibration buffer E (50 mM HEPES, 500 mM KCl, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 0.002% mellitic acid, pH 7.5). After labelling, the samples are quenched by mixing with 100 µl of pre-chilled quench buffer Q (1.2 % formic acid, pH 2.4). Seventy microlitres of the quenched samples are directly transferred to the Enzymate BEH Pepsin Column (Waters Corporation) at 200 µl min−1 and at 20 °C with a pressure 8.5 kPSI. Peptic peptides were trapped for 3 min on an Acquity UPLC BEH C18 VanGuard Pre-column (Waters Corporation) at a 200 µl min−1 flow rate in water (0.1% formic acid in HPLC water pH 2.5) before eluted to an Acquity UPLC BEH C18 Column for chromatographic separation. Separation was done with a linear gradient buffer (7–40% gradient of 0.1% formic acid in acetonitrile) at a flow rate of 40 µl min−1. Peptides identification and deuteration uptake analysis was performed on the Synapt G2-Si in ESI ± HDMSE mode (Waters Corporation). Leucine enkephalin was applied for mass accuracy correction and sodium iodide was used as calibration for the mass spectrometer. HDMSE data were collected by a 20–30 V transfer collision energy ramp. The pepsin column was washed between injections using pepsin wash buffer (1.5 M guanidinium HCl, 4% (v/v) methanol, 0.8% (v/v) formic acid). A blank run was performed between each sample to prevent significant peptide carry-over. Optimized peptide identification and peptide coverage for all samples was performed from undeuterated controls (five replicates). All deuterium time points were performed in triplicate.
Data treatment and statistical analysis of HDX-MS
The non-deuterated references points were analysed by PLGS (ProteinLynx Global Server 2.5.1, Waters) to identify the peptic peptides belonging to CapRelSJ46 or Gp57. Then, all the HDMSE data including reference and deuterated samples were processed by DynamX 3.0 (Waters) for deuterium uptake determination. We chose the following filtering parameters: minimum intensity of 1,000, minimum and maximum peptide sequence length of 5 and 20, respectively, minimum MS/MS products of 3, minimum products per amino acid of 0.27, minimum score of 5, and a maximum MH+ error threshold of 15 p.p.m. Data were analysed at peptidic and overall level and manually curated by visual inspection of individual spectra. The overall level is based on the relative fractional uptake (RFUa,t), which can be calculated by the following formula:
$${{\rm{RFU}}}_{a,t}( \% )=\frac{{\gamma }_{a,t}}{{{\rm{MaxUptake}}}_{a}\,\times D}$$
where \({\gamma }_{a,t}\) is the deuterium uptake for peptide a at incubation time t, and \({{\rm{M}}{\rm{a}}{\rm{x}}{\rm{U}}{\rm{p}}{\rm{t}}{\rm{a}}{\rm{k}}{\rm{e}}}_{a}\times D\) is the theorical maximum uptake in deuterium value that peptide a can take. The ΔRFU compared RFU value between two different experimental conditions, in this case, this is the comparison between CapRelSJ46 and CapRelSJ46 + Gp57. Heat maps have been generated in DynamX. All the raw data can be accessed at: https://doi.org/10.6084/m9.figshare.19745089.
CapRelSJ46 expression and purification for biochemical assays
Full-length capRelSJ46 was overexpressed in freshly transformed E. coli BL21(DE3) pET24d-N-His10-SUMO-capRelSJ46 pMG25-paSpo co-transformed with the plasmid encoding PaSpo small alarmone hydrolase (SAH) from Salmonella phage SSU5, which has been shown to neutralize the toxicity of other toxSAS toxins24. Fresh transformants were used to inoculate 800 ml of LB medium (final A600 of 0.03) supplemented with 50 μg ml−1 kanamycin, 20 μg ml−1 chloramphenicol and 0.2% arabinose. Bacterial cultures were grown at 37 °C until an A600 of 0.4–0.5 and protein expression was induced with 0.1 mM IPTG (final concentration). Cells were grown for additional 1 h at 30 °C and the biomass was collected by centrifugation (10,000 rpm, for 5 min, JLA-10.500 rotor (Beckman Coulter)).
Cell mass was resuspended in buffer A (750 mM KCl, 500 mM NaCl, 5 mM MgCl2, 40 μM MnCl2, 40 μM zinc acetate, 1 mM mellitic acid, 20 mM imidazole, 10% glycerol, 4 mM β-mercaptoethanol and 25 mM HEPES:KOH pH 8) supplemented with 0.1 mM PMSF and 1 U ml−1 of DNase I, and lysed by one passage through a high-pressure cell disrupter (Stansted Fluid Power, 150 MPa). Mellitic acid was added to buffers as it was earlier shown to stabilize Thermus thermophilus Rel stringent factor65. Cell debris was removed by centrifugation (25,000 rpm for 1 h at 4 ºC, JA-25.50 rotor (Beckman Coulter)), the clarified lysate was filtered through a 0.22-μm syringe filter and loaded onto a HisTrap 5 ml HP column (Cytiva) pre-equilibrated in buffer A. The column was washed with 5 column volumes of buffer A, and the protein was eluted using a combination of stepwise and linear gradient (5 column volumes with 0–100% buffer B) of buffer B (750 mM KCl, 500 mM NaCl, 5 mM MgCl2, 40 μM MnCl2, 40 μM Zn(OAc)2, 1 mM mellitic acid, 1 M imidazole, 10% glycerol, 4 mM β-mercaptoethanol, 25 mM HEPES:KOH pH 8). Fractions enriched in CapRelSJ46 (approximately 40% buffer B) were pooled, totalling approximately 5 ml. The sample was loaded on a HiLoad 16/600 Superdex 200 pg column pre-equilibrated with a high-salt buffer (buffer C; 2 M NaCl, 5 mM MgCl2, 10% glycerol, 4 mM β-mercaptoethanol, 25 mM HEPES:KOH pH 8). The fractions containing CapRelSJ46 were pooled and applied on a HiPrep 10/26 desalting column (Cytiva) pre-equilibrated with storage buffer (buffer D; 720 mM KCl, 5 mM MgCl2, 40 mM arginine, 40 mM glutamic acid, 10% glycerol, 4 mM β-mercaptoethanol, 25 mM HEPES:KOH pH 8). Fractions containing CapRelSJ46 were collected (about 14 ml in total) and the His10–SUMO tag was cleaved off by addition of 10 µg of His6–Ulp1 per 1 mg CapRelSJ46 followed by a 30-min incubation on ice. After the His10–SUMO tag was cleaved off, the protein was passed through a 5 ml HisTrap HP pre-equilibrated with buffer D supplemented with 20 mM imidazole. Fractions containing CapRelSJ46 in the flow-through were collected and concentrated on an Amicon Ultra (Millipore) centrifugal filter device with a 10 kDa cut-off. The purity of protein preparations was assessed by SDS–PAGE. Protein preparations were aliquoted, frozen in liquid nitrogen and stored at –80 °C. Individual single-use aliquots were discarded after the experiment.
Cell-free translation
Experiments with PURExpress in vitro protein synthesis kit (NEB, E6800) were performed as per the manufacturer’s instructions. All reactions were supplemented with 0.8 U µl−1 RNase Inhibitor Murine (NEB, M0314S). Purified CapRelSJ46 protein was used at a final concentration of 250 nM, with gp57, gp57(L114P) or gp57(I115F) as template plasmid at 10 ng µl−1. As a mock control CapRelSJ46 was substituted for equal volume of HEPES:Polymix buffer66, pH 7.5. After a 10-min incubation at 37 °C, a 1.34 µl aliquot of the reaction mixture was taken and quenched by addition of 13.66 µl of 2× sample buffer (100 mM Tris:HCl pH 6.8, 4% SDS, 0.02% bromophenol blue, 20% glycerol, 20 mM DTT and 4% β-mercaptoethanol), and DHFR template plasmid was added to the remaining reaction mixture at a final concentration of 20 ng µl−1. After further incubation at 37 °C for 1 h, the reaction mixture was mixed with ninefold volume of 2× sample buffer, boiled at 98 ºC for 5 min, and 5 µl of the mixture was resolved by 18% SDS–PAGE. The SDS–PAGE gel was fixed by incubating for 5 min at room temperature in 50% ethanol solution supplemented with 2% phosphoric acid, washed three times with water for 20 min at room temperature, and stained with ‘blue silver’ solution (0.12% Brilliant Blue G250 (Sigma-Aldrich, 27815), 10% ammonium sulfate, 10% phosphoric acid, and 20% methanol) overnight at room temperature. After washing with water for 3 h at room temperature, the gel was imaged on an Amersham ImageQuant 800 (Cytiva) imaging system. For tRNA pyrophosphorylation experiments, Gp57, Gp57(L114P), or Gp57(I115F) was produced in a similar reaction mixture without CapRelSJ46 and DHFR template at 37 °C for 2 h.
tRNA pyrophosphorylation by CapRelSJ46
The reaction mixture containing 5 µM tRNA from E. coli MRE600 (Sigma-Aldrich, 10109541001), 500 µM [γ32P]ATP, 250 nM CapRelSJ46 and 1/10 volume of either wild-type Gp57, Gp57(L114P), or Gp57(I115F) product from the PUREsystem in HEPES:Polymix buffer, pH 7.5 (5 mM Mg2+ final concentration) supplemented with 1 mM DTT was incubated at 37 °C for 10 min. To visualize phosphorylated tRNA, the reaction sample was mixed in 2 volumes of RNA dye (98% formamide, 10 mM EDTA, 0.3% bromophenol blue and 0.3% xylene cyanol), tRNA was denatured at 37 °C for 10 min and resolved on urea-PAGE in 1× TBE (8 M urea, 8% PAGE). The gel was stained with SYBR Gold (Life technologies, S11494) and exposed to an imaging plate overnight. The imaging plate was imaged by a FLA-3000 (Fujifilm).
Reporting summary
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