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Bacteria conjugate ubiquitin-like proteins to interfere with phage assembly

Nature volume 631, pages 850–856 (2024) Cite this article

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Abstract

Several immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defence1,2,3,4,5. Here we studied an antiphage defence system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fibre, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defence system release a mixture of partially assembled, tailless phage particles and fully assembled phages in which the central tail fibre is obstructed by the covalently attached ubiquitin-like protein. These phages show severely impaired infectivity, explaining how the defence system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.

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Fig. 1: Features of ubiquitin-like conjugation in the Bil antiphage defence system.
The alternative text for this image may have been generated using AI.
Fig. 2: The Bil system causes the production of non-infective phage particles.
The alternative text for this image may have been generated using AI.
Fig. 3: The phage CTF is the target of the Bil system.
The alternative text for this image may have been generated using AI.
Fig. 4: The Bil defence system targets tail tip proteins of distantly related phages.
The alternative text for this image may have been generated using AI.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium by means of the PRIDE partner repository61 with the dataset identifier PXD044622. The RNA-seq data have been deposited in NCBI’s Gene Expression Omnibus62 and are accessible through Gene Expression Omnibus series accession number GSE262579. Plasmid inserts of the constructs used are available in Supplementary Table 5. Uncropped images of gels and blots from all figures are presented in Supplementary Fig. 1. Source data are provided with this paper.

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Acknowledgements

We thank the Sorek laboratory members for comments on earlier versions of this paper. We thank A. Savidor and M. Kupervaser at the De Botton Protein Profiling Institute at the Weizmann Institute as well as S. Lamer at the Rudolf Virchow Center for help with mass spectrometry data generation and analysis. We thank the Core Unit SysMed at the University of Würzburg for technical support by E. Katzowitzsch and RNA-seq data generation. This work was supported in part by the Interdisciplinary Center for Clinical Research (IZKF) Würzburg (project no. Z-6). We also thank J. Vogel and the Helmholtz Institute for RNA-based Infection Research for help in difficult times. J.H. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), grant no. 466645764 and a fellowship from the Council for Higher Education and Israel Academy of Science and Humanities (CHE/IASH) Excellence Fellowship Program for International Postdoctoral Researchers. R.S. was supported, in part, by the European Research Council (grant no. ERC-AdG GA 101018520), Israel Science Foundation (MAPATS grant no. 2720/22), the Deutsche Forschungsgemeinschaft (SPP 2330, grant no. 464312965), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, the Dr. Barry Sherman Institute for Medicinal Chemistry, M. de Botton, the Andre Deloro Prize and the Knell Family Center for Microbiology.

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Author notes

  1. Jens Hör

    Present address: Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany

Authors and Affiliations

  1. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Jens Hör & Rotem Sorek

  2. Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

    Sharon G. Wolf

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Contributions

J.H. and R.S. conceived and designed the study. J.H. performed the experiments. S.G.W. performed electron microscopy. J.H. and R.S. analysed the data. J.H. visualized the data. R.S. acquired funding. J.H. and R.S. wrote the original draft of the manuscript. All authors reviewed and edited the manuscript and support the conclusions.

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Correspondence to Rotem Sorek.

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R.S. is a scientific cofounder and adviser of BiomX and Ecophage. The remaining authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Interactions between the Bil Ubl and the E1/E2 enzymes.

a, Multiple sequence alignment of human Ubiquitin and five BilA Ubl homologs. Residues highlighted in bold and yellow have a global similarity score of ≥ 0.7. Residues highlighted in bold and red are fully conserved. Visualized using ESPript56. Related to Fig. 1c. b, PFU quantification of plaque assays shown in Fig. 1d. Data, except for the Ubl G163L mutant, were taken from13. Bars represent the average of three biological replicates with individual data points overlaid. c, Enzymatic reaction in Ubl-conjugating pathways. The active site cysteine of the E1 protein forms a thioester with the C-terminal glycine of the Ubl, which is then transferred to the active site cysteine of the E2 to form another thioester. The E2 then transfers the Ubl to a lysine residue of the target protein. Deubiquitinases (DUBs) can reverse the conjugation by cleaving the Ubl off the target protein. d, Western blot of whole cell lysates of bacteria expressing the Bil system with a tagged E1. Two bands for the E1 are revealed. The upper band of ~70 kDa band is reduced by DTT, indicating a thioester bond between the E1 and the Ubl. GroEL was used as loading control on the same blot. Cells at OD600 of 0.8 are infected by phage SECphi27. A representative image of two biological replicates is shown. e, Mass spectrometry analysis of the ~70 kDa band observed in Ubl immunoprecipitation experiments verifies that it included the Ubl~E1 complex (related to Fig. 1e). The log10 of the sum of the label-free quantification (LFQ) intensity values obtained from wild-type and mutant Bil system for each detected protein was plotted against the log2 ratio of the LFQ intensities of the Bil system vs. the mutant Bil system. f, AlphaFold-Multimer19 prediction of the interaction between the Ubl and the E1. A high confidence structure (model confidence of 0.94 and overall low predicted alignment error, right side of the panel) shows G163 (indicated as spheres) of the Ubl to be positioned at the nucleotide-binding loop of the E1 (G217, G219 and G222; indicated as spheres). g, Mass spectrometry analysis of the ~50 kDa band observed in Ubl immunoprecipitation experiments verifies the E1 protein (related to Fig. 1e). Data analyzed and plotted as in e. h, Western blot of whole cell lysates of bacteria expressing the Bil system with a tagged E2. No covalent complex of the E2 could be observed. GroEL was used as loading control on the same blot. Cells at OD600 of 0.8 are infected by phage SECphi27. A representative image of three biological replicates is shown. i, Mass spectrometry analysis of the ~80 kDa band observed in Ubl immunoprecipitation experiments (related to Fig. 1e). Data analyzed and plotted as in e. MaeB is identified in this band, but as the molecular weight of MaeB is 82 kDa, it is likely pulled down unspecifically and not conjugated to the Ubl.

Source Data

Extended Data Fig. 2 The Bil system provides defense at expression levels relevant to physiological conditions.

a, PFU quantification of different phages infecting E. coli expressing the Bil system from a plasmid with a p15A origin (estimated ~10 copies per cell) under the control of a tetracycline-inducible promoter. Four different concentrations of inducer (anhydrotetracycline, aTc) were used. Bars represent the average of three biological replicates with individual data points overlaid. b, RNA-sequencing data of Bil-expressing E. coli with expression induced using 25 ng/ml of aTc (see a). Data show average log2 transcripts per kilobase million (TPM) of each gene with ≥ 10 average raw reads in three biological replicates, sorted by their log2 TPM values. Blue, plasmid-encoded Bil system transcripts (bilA, bilB, bilC, bilD), tetracycline repressor (tetR) and aminoglycoside phosphotransferase (kanR). Green, chromosomally encoded defense systems of E. coli MG1655, predicted using DefenseFinder60: RM type I (hsdM, hsdR, hsdS), RM type IV (mcrA, mcrB, mcrC, mrr), Lit (lit), RnlAB (rnlA, rnlB), Hachiman (abpA, abpB) and Druantia type III (yjiT). CRISPR genes are not shown for clarity. c, RNA-sequencing data of Caulobacter sp. Root343 at the indicated growth phases. Data show average log2 transcripts per kilobase million (TPM) of each gene with ≥ 10 average raw reads in three biological replicates, sorted by their log2 TPM values. Light blue, chromosomally encoded defense systems of Caulobacter sp. Root343, predicted using DefenseFinder60: Bil system (bilA, bilB, bilC, bilD), RM type II (MTase, REase), SanaTA (sanaA, sanaT), RosmerTA (rmrA, rmrT) and AbiZ (abiZ).

Source Data

Extended Data Fig. 3 Phage particle size measurements.

a-d, Particle size distributions of phages isolated from bacteria expressing the indicated systems, measured by nanoparticle tracking analysis. Bars represent the average of three biological replicates. The dashed areas of each graph are magnified on the right of each graph. High and low density indicate phages isolated from high- and low-density bands in the CsCl gradient. e, Overlay of the particle size distributions shown in a-d.

Source Data

Extended Data Fig. 4 The phage central tail fibre is the target of the Bil system.

a, Plaque assays showing that addition of an HA-tag to the Ubl of the Bil system does not interfere with phage defense. b, Immunoprecipitation of HA-tagged Ubl protein from SECphi27 isolated from bacteria expressing the Bil system, analyzed by western blotting. Phages from the low- and high-density bands of the CsCl gradient were immunoprecipitated separately. A representative image of two biological replicates is shown. c, Quantification of the percentage of Ubl-conjugated CTF from the total amount of CTF detected by western blotting (related to Fig. 3c). n.d., not detected. Bars represent the average of three biological replicates with individual data points overlaid.

Source Data

Extended Data Fig. 5 The Bil system modifies the tip of the phage tail.

Immunogold labeling transmission electron microscopy images of SECphi27 phages isolated from bacteria expressing the HA-Ubl Bil system (related to Fig. 3d). White arrowheads represent gold labeling of HA-Ubl. Scale bars represent 50 nm.

Extended Data Fig. 6 The central tail fiber is a conserved structure targeted by the Bil system.

a, Representative immunogold labeling transmission electron microscopy image of SECphi27 phages isolated from bacteria expressing mutant HA-Ubl Bil system. No gold labeling could be observed on the phages. Scale bar represents 100 nm. b, Efficiency of center of infection assays with phages propagated on either the wild-type or the mutant Bil systems. Wild-type E. coli cells were infected at MOI = 1 (numbers of phage particles were determined by NTA) and allowed to adsorb for 30 min. After washing, the bacteria with adsorbed phages were serially diluted and dropped on a lawn of wild-type E. coli. Centers of infection were counted the next day. Bars represent the average of four biological replicates with individual data points overlaid.

Source Data

Extended Data Fig. 7 The central tail fiber is a conserved structure targeted by the Bil system.

a, Plaque assays showing the defense phenotype of the Bil systems against SECphi4. Data are representative of three biological replicates with quantification of the three replicates shown on the right (quantification data were taken from13). b, Co-expression of the Bil system with the central tail fiber of SECphi4, analyzed by western blotting. GroEL was used as loading control on the same blot. A representative image of two biological replicates is shown. c, AlphaFold216 predictions of the structures of the central tail fibers of SECphi27 (NCBI ID: YP_009965952.1) and SECphi4 (NCBI ID: QJI52569.1). The predicted structure of the SECphi4 central tail fiber was superimposed on the SECphi27 central tail fiber using the following domains: Residues 1–251 (RMSD = 0.91 Å), residues 252–628 (RMSD = 1.30 Å), residues 629–736 (RMSD = 1.11 Å), residues 737–829 (RMSD = 1.10 Å), residues 830–1139 (RMSD = 2.69 Å).

Source Data

Extended Data Fig. 8 Tail structure proteins of different phages contain structurally similar domains.

a, AlphaFold216 prediction of residues 250–614 of the SECphi27 central tail fiber (related to Extended Data Fig. 7c) superimposed on the cryo-EM structure of T5 pb3 (PDB: 7ZQB31; RMSD = 2.01 Å). b, High exposure version of the western blot shown in Fig. 4c. c, AlphaFold216 prediction of residues 250–614 of the SECphi27 central tail fiber (related to Extended Data Fig. 7c) superimposed on the crystal structure of T4 gp27 (PDB: 1K2863; RMSD = 3.64 Å). d, Co-expression of the Bil system with either the central tail fiber (CTF) of SECphi27, the baseplate hub protein gp27 of T6, the large distal tail fiber subunit protein gp37 of T6 or the baseplate wedge subunit gp7 of T6, analyzed by western blotting. GroEL was used as loading control on the same blot. A representative image of two biological replicates is shown.

Extended Data Fig. 9 The DUB is essential for the function of the Bil system and is able to cleave a Ubl fusion protein.

a, PFU quantification of different phages infecting E. coli cells that co-express Bil system variants and Ubl, mutated Ubl or RFP control. Bars represent the average of three biological replicates with individual data points overlaid. b, Western blot of whole cell lysates of bacteria co-expressing a Ubl-GFP fusion protein and the DUB of the Bil system. GFP can be cleaved off the Ubl-GFP fusion protein by the DUB. GroEL was used as loading control on the same blot. A representative image of three biological replicates is shown. c, Growth curves of E. coli expressing Bil system variants either without (left) or with (right) the addition of expression inducer. The DUB E32* mutant is a single-nucleotide mutant replacing the amino acid at position 32 of the DUB protein with a stop codon. Data shown is the average of three biological replicates. Error bars show average with s.d.

Source Data

Supplementary information

Supplementary Fig. 1 (download PDF )

Uncropped gel and immunoblot images from main and Extended Data figures. For each gel or immunoblot image shown throughout the main and Extended Data figures, the uncropped images are shown. For immunoblot images, regular photographs are shown on the left to visualize the molecular weight marker transferred to the membrane. On the right, the corresponding immunoblots are shown, the used primary antibodies are indicated and the regions that were cropped are indicated by dashed lines. For all images, the sizes of the used molecular weight markers are indicated.

Reporting Summary (download PDF )

Supplementary Table 1 (download XLSX )

Mass spectrometry analysis of SECphi27 immunoprecipitation.

Supplementary Table 2 (download XLSX )

Mass spectrometry analysis of SECphi4 immunoprecipitation.

Supplementary Table 3 (download XLSX )

SECphi27 versus SECphi4 proteome comparison.

Supplementary Table 4 (download XLSX )

Bacterial strains and phages used in this study.

Supplementary Table 5 (download XLSX )

Plasmids used in this study.

Supplementary Table 6 (download XLSX )

BilA Ubl homologues.

Supplementary Table 7 (download XLSX )

Primers used in this study.

Source data

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Hör, J., Wolf, S.G. & Sorek, R. Bacteria conjugate ubiquitin-like proteins to interfere with phage assembly. Nature 631, 850–856 (2024). https://doi.org/10.1038/s41586-024-07616-5

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  • DOI: https://doi.org/10.1038/s41586-024-07616-5

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