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Bacteria deplete deoxynucleotides to defend against bacteriophage infection

Abstract

DNA viruses and retroviruses consume large quantities of deoxynucleotides (dNTPs) when replicating. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral lifecycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. Here, we report that bacteria use a similar strategy to defend against bacteriophage infection. We identify a family of defensive bacterial deoxycytidine triphosphate (dCTP) deaminase proteins that convert dCTP into deoxyuracil nucleotides in response to phage infection. We also identify a family of phage resistance genes that encode deoxyguanosine triphosphatase (dGTPase) enzymes, which degrade dGTP into phosphate-free deoxyguanosine and are distant homologues of human SAMHD1. Our results suggest that bacterial defensive proteins deplete specific deoxynucleotides (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Our study shows that manipulation of the dNTP pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.

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Fig. 1: A family of cytidine deaminases provide defence against phages.
Fig. 2: dGTPases defend against phages by depleting dGTP during phage infection.
Fig. 3: Distribution of homologues of nucleotide-depleting defence genes in bacterial genomes.
Fig. 4: Diversity of nucleotide-depleting defence genes in bacterial genomes.
Fig. 5: Phage mutants can overcome nucleotide-depletion defence.

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Data availability

Data that support the findings of this study are available in the Article and its Extended Data. Gene accessions appear in the Methods section of the paper. DNA and RNA sequencing data used in Extended Data Fig. 2 can be found in the European Nucleotide Archive (ENA) ID: ERA11772567. Source data are provided with this paper. Additional data are available from the corresponding authors upon request.

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Acknowledgements

We would like to thank P. Cossart for directing us to the SAMHD1 antiviral mechanism, and A. Bernheim and the Sorek laboratory members for comments on earlier versions of this manuscript and fruitful discussion. R.S. was supported, in part, by the European Research Council (grant no. ERC-CoG 681203), Israel Science Foundation (grant no. ISF 296/21), the Deutsche Forschungsgemeinschaft (SPP 2330, grant no. 464312965), the Ernest and Bonnie Beutler Research Programme of Excellence in Genomic Medicine, the Minerva Foundation with funding from the Federal German Ministry for Education and Research, the Knell Family Centre for Microbiology, the Yotam project and the Weizmann Institute Sustainability And Energy Research (SAERI) initiative, and the Dr Barry Sherman Institute for Medicinal Chemistry. A.M. was supported by a fellowship from the Ariane de Rothschild Women Doctoral Programme and, in part, by the Israeli Council for Higher Education via the Weizmann Data Science Research Centre, and by a research grant from Madame Olga Klein-Astrachan. Protein MS was performed at the Weizmann De Botton Protein Profiling Institute.

Author information

Authors and Affiliations

Authors

Contributions

N.T. and R.S. led the study and performed all analyses and experiments unless otherwise indicated. N.T. performed the genetic analyses and the in vivo experimental assays and analysed the data. A.M., E.Y. and N.T. performed the computational analyses of systems prediction. A.S-A. designed and executed the mutant-phage isolation experiments and their analysis. C.A. assisted with plaque assays and DNA isolation of mutant phages. T.F. assisted with isolation of mutant phages and in conducting plaque assays. A.L. and S.M. assisted in conducting plaque assays and preparing DNA sequencing libraries. G.A. assisted with sequence analysis and prediction of protein domain functions. A.B. and T.M. performed mass spectrometry and data analysis. The manuscript was written by N.T. and R.S. All authors contributed to editing the manuscript and support the conclusions.

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Correspondence to Rotem Sorek.

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R.S. is a scientific co-founder and advisor of BiomX and Ecophage. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 dCTP deaminases protect against phage infection.

(A) Superposition of the C-terminal region (residues 351-550) of the AlphaFold-predicted structure of E. coli AW1.7 dCTP deaminase (turquoise), aligned with Streptococcus mutans cytosine deaminase (PDB: 2hvw) (grey). Zn2+ ions are depicted as blue spheres. Alignment was performed by PDBeFOLD61, with a Q score = 0.33, Z score = 9.0 and RMSD = 2.72 Å between the two structures. (B) Representative instances of cytidine deaminase genes (in turquoise) and their genomic neighborhoods. Genes known to be involved in defense are shown in yellow. RM, restriction modification; TAs, toxin-antitoxin systems; Septu and Hachiman are recently described defense systems14. The bacterial species and the accession of the relevant genomic scaffold in the Integrated Microbial Genomes (IMG) database47 are indicated on the left. (C) Bacteria expressing dCTP deaminases from E. coli U09 or E. coli AW1.7, as well as a negative control that contains an empty vector, were grown on agar plates in room temperature. Tenfold serial dilutions of the phage lysate were dropped on the plates. Data represent plaque-forming units per milliliter for ten phages tested in this study. Each bar graph represents average of three replicates, with individual data points overlaid. (D) Growth curves of cells expressing E. coli AW1.7 dCTP deaminase (turquoise), and control cells (grey). Results of three replicates are presented as individual curves.

Source data

Extended Data Fig. 2 No evidence for editing of genome and transcriptome by the dCTP deaminase.

Cells expressing the deaminase from E. coli AW1.7 were infected by phage T7 at a multiplicity of infection (MOI) of 2 at 37 °C. Total DNA and total RNA were extracted after 15 minutes from the onset of infection, and were subjected to DNA-seq and RNA-seq, respectively. Panels A and B show the abundance of DNA reads with specific mismatches for reads aligned to the bacterial genome (A) or the phage genome (B). Panels C and D show the abundance of RNA-seq reads with specific mismatches for reads aligned to the bacterial genome (C) or the phage genome (D).

Source data

Extended Data Fig. 3 Cellular nucleotides during phage infection.

(A-H) Concentrations of various nucleotides in cell lysates extracted from T7-infected cells, as measured by LC-MS with synthesized standards. X axis represents minutes post infection, with zero representing non-infected cells. Cells were infected by phage T7 at an MOI of 2. Each panel shows data acquired for dCTP deaminase-expressing cells or for control cells that contain an empty vector. Bar graphs represent average of three biological replicates, with individual data points overlaid.

Source data

Extended Data Fig. 4 Mutated dCTP deaminase does not elicit dCTP depletion.

Relative abundance of dCTP in cell lysates extracted from T7-infected cells, as measured by LC-MS. X axis represents minutes post infection, with zero representing non-infected cells. Y axis represents the area under the peak for dCTP, in arbitrary units (AU). Cells were infected by T7 at an MOI of 2. Presented are data acquired for cells expressing the dCTP deaminase from E. coli AW1.7, control cells that contain an empty vector, or cell expressing mutated forms of the dCTP deaminase. Bar graphs represent average of two biological replicates, with individual data points overlaid.

Source data

Extended Data Fig. 5 A family of dGTPases in defense islands.

(A) Representative instances of dGTPase genes (in orange) and their genomic neighborhoods. Colors and annotations are as in Supplementary Fig. S1A. M, S and R designations within type I RM operon genes represent the methylase, specificity, and restriction subunits, respectively. (B) Superposition of the AlphaFold predicted structural model of Sp-dGTPase (orange) aligned with the N-terminus of the human SAMHD1 (PDB: 4bzb, chain D) (grey). Mg2+ ion is depicted as a yellow sphere. SAMHD1 catalytic site is depicted with grey sticks, and is bound to the dGTP ligand. Alignment was performed by PDBeFOLD, with a Q score = 0.11, Z score = 5.5 and RMSD = 2.89 Å between the predicted dGTPase AlfaFold2 model and the human SAMHD1. The presented structural alignment includes residues 23–161 & 248–452 from the dGTPase AlphaFold model, aligned to residues 129–275 & 299–439 of the human SAMHD1 structure.

Extended Data Fig. 6 dGTPases protect against phage infection.

(A) E. coli MG1655 cells expressing dGTPases cloned under an arabinose-inducible promoter from several species (Ec, E. coli G177; Ms, Mesorhizobium sp. URHA0056; Pl, Pseudoalteromonas luteoviolacea DSM6061; Sp, Shewanella putrefaciens CN-32), as well as a negative control, were grown on agar plates in room temperature in the presence of 0.2% arabinose. Tenfold serial dilutions of the phage lysate were dropped on the plates. Data represent plaque-forming units per milliliter for phages tested in this study. Each bar graph represents average of three replicates, with individual data points overlaid. (B) Growth curves of cells over-expressing the Sp-dGTPase gene from Shewanella putrefaciens CN-32 (orange) and control cells (grey) following 0.2% arabinose induction. Results of four replicates are presented as individual curves. Expression of the Sp-dGTPase protein was verified via protein mass spectrometry. (C-G) Concentrations of deoxynucleotides in cell lysates extracted from T7-infected cells, as measured by LC-MS with synthesized standards. X axis represents minutes post infection, with zero representing non-infected cells. Cells were infected by phage T7 at an MOI of 2. Each panel shows data acquired for dGTPase-expressing cells or for control cells that express GFP. Bar graphs represent average of three biological replicates (or two replicates for the dGTPase samples at t = 15 mins), with individual data points overlaid.

Source data

Extended Data Fig. 7 dGTPases cloned under native promoters protect against phage infection.

(A) E. coli MG1655 cells containing dGTPases cloned, together with their native promoters, from two different E. coli strains (Ec S, E. coli STEC 2595; Ec 1303, E. coli 1303), as well as a negative control, were grown on agar plates in room temperature. Tenfold serial dilutions of the phage lysate were dropped on the plates. Data represent plaque-forming units per milliliter for tested phages. Each bar graph represents average of three replicates, with individual data points overlaid. (B) T7 defense by dGTPases expressed from native promoters. Shown are ten-fold serial dilution plaque assays, comparing the plating efficiency of T7 phage on bacteria that express the Ec STEC 2529, Ec 1303 or a control strain that lacks the gene. Images are representative of three replicates. (C) Growth curves of cells harboring the Ec S - dGTPase gene from E. coli STEC 2595 (orange) and control cells with an empty plasmid (grey). Results of three replicates are presented as individual curves. (D-J) Concentrations of deoxynucleotides in cell lysates extracted from T7-infected cells, as measured by LC-MS with synthesized standards. X axis represents minutes post infection, with zero representing non-infected cells. Cells were infected by phage T7 at an MOI of 2. Each panel shows data acquired for cells expressing the Ec S dGTPase or for control cells containing an empty pSG1 vector. Bar graphs represent average of three biological replicates, with individual data points overlaid. (K) Effect of native dGTPase expression on T7 DNA replication throughout infection. Cells were infected by phage T7 at an MOI of 2 at 37 °C. Total DNA was extracted from each sample and DNA was Illumina-sequenced. Each panel shows data acquired for Ec-S-expressing cells or for control cells that contain an empty vector. Y axis represents phage DNA sequence reads normalized to reads from spiked-in DNA. Bar graphs represent the average of three biological replicates, with individual data points overlaid.

Source data

Extended Data Fig. 8 Distant homologs of Sp-dGTPase protect against phage infection.

(A) Bacteria expressing dGTPase cloned from multiple species (Yersinia enterocolitica YE38/03, Desulfovibrio halophilus DSM 5663, Yersinia enterocolitica E701, Escherichia coli HMLN-1, Glaesserella parasuis SC1401, Alteromonas sp. Mac1, Shewanella sp. Sh95, Vibrio cholerae YB2A05, Haemophilus sp. C1), as well as a negative control, were grown on agar plates in room temperature in the presence of 0.2% arabinose. Tenfold serial dilutions of the phage lysate were dropped on the plates. Data represent plaque-forming units per milliliter for tested phages. Each bar graph represents average of three replicates, with individual data points overlaid. (B) A summary of the defense results from the presented bar graphs.

Source data

Extended Data Fig. 9 Mutation verification of Gp5.7 and rifampicin treatment.

(A) Verification of the absence of Gp5.7 in T7 mutant n. 5 using mass spectrometry. Peptide fragments of Gp5.7 identified by protein mass spectrometry of cells 15 minutes post infection by WT T7 and T7 mutant n.5 (MOI = 2). Multiple peptides of Gp5.7 are observed in the WT T7, but no peptides are detected in the mutant T7, supporting that the mutant does not express Gp5.7. Peptide fragments are as follows: peptide 1: GHISCLTTSGR, peptide 2: NGGAWEITASGTR, peptide 3: NNASLVAEAASR, peptide 4: TFQSNYVR. Bar graphs represent average of two biological replicates, with individual data points overlaid. (B) As control to the measurements in panel A, shown are peptide fragments of the T7 RNA polymerase identified by protein mass spectrometry in cells 15 minutes post infection. The T7 RNA polymerase is readily identified in both WT and mutant phages. Peptide 1: EQLALEHESYEMGEAR, peptide 2: MNTINIAK, peptide 3: SVMTLAYGSK, peptide 4: VLAVANVITK. Bar graphs represent average of two biological replicates, with individual data points overlaid. (C-H) Concentrations of dNTP nucleotides in cell lysates extracted from rifampicin-treated cells, as measured by LC-MS with synthesized standards. X axis represents minutes post treatment, with zero representing non-treated cells. (C-E) Each panel shows data acquired for dCTP deaminase-expressing cells or for control cells that contain an empty vector. Bar graphs represent average of three biological replicates, with individual data points overlaid. (F-H) Each panel shows data acquired for dGTPase-expressing cells or for control cells that express GFP. Bar graphs represent average of three biological replicates, with individual data points overlaid. In panels D-F, 60 minute data represent the average of two biological replicates.

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Supplementary information

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Supplementary Tables

Supplementary Table 1. Homologues of the dCTP deaminases in this study. Supplementary Table 2. Homologues of the dGTPases in this study. Supplementary Table 3. Phages used in this study. Supplementary Table 4. Mutations observed in phages that escape nucleotide-depletion defence. Supplementary Table 5. defence genes synthetized and cloned in this study. Supplementary Table 6. Primers used in this study.

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Tal, N., Millman, A., Stokar-Avihail, A. et al. Bacteria deplete deoxynucleotides to defend against bacteriophage infection. Nat Microbiol 7, 1200–1209 (2022). https://doi.org/10.1038/s41564-022-01158-0

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