Abstract
Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race1,2,3,4,5. While the mechanisms of many bacterial antiphage defence systems are known1, how these systems avoid toxicity outside infection yet activate quickly after infection is less well understood. Here we show that the bacterial phage anti-restriction-induced system (PARIS) operates as a toxin–antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The E. coli B185 AriA(EQ) cryo-EM reconstruction has been deposited at the Electron Microscopy Data Bank (EMDB) under accession number EMD-42969. Corresponding coordinates have been deposited at the RCSB PDB under accession number 8V49. The E. coli B185 AriA(EQ)–AriB(E90A) complex (form I) cryo-EM reconstruction has been deposited at the EMDB under accession number EMD-42966. Corresponding coordinates have been deposited at the PDB under accession number 8V46. The E. coli B185 AriA(EQ)–AriB(E90A) complex (form II) cryo-EM reconstruction has been deposited at the EMDB under accession number EMD-42967. Corresponding coordinates have been deposited at the PDB under accession number 8V47. The E. coli B185 AriA(EQ)–AriB(E90A) complex (form III) cryo-EM reconstruction has been deposited at the EMDB under accession number EMD-42968. Corresponding coordinates have been deposited at the PDB under accession number 8V48. The AriA–Ocr complex cryo-EM reconstruction has been deposited at the EMDB under accession number EMD-42965. Corresponding coordinates have been deposited at the PDB under accession number 8V45. Other structural data used in comparative analysis are also available at the PDB under accession numbers 3AV0, 8DK3, 6TPQ, 6NK8 and 1S7Z. Protein sequences used for multiple-sequence alignments are available online (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Source data are provided with this paper.
Code availability
This study did not involve the use of custom code.
References
Georjon, H. & Bernheim, A. The highly diverse antiphage defence systems of bacteria. Nat. Rev. Microbiol. 21, 686–700 (2023).
Hampton, H. G., Watson, B. N. J. & Fineran, P. C. The arms race between bacteria and their phage foes. Nature 577, 327–336 (2020).
Gao, L. A. et al. Prokaryotic innate immunity through pattern recognition of conserved viral proteins. Science 377, eabm4096 (2022).
Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018).
Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host Microbe 30, 740–753 (2022).
Gao, Z. & Feng, Y. Bacteriophage strategies for overcoming host antiviral immunity. Front. Microbiol. 14, 1211793 (2023).
Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat. Microbiol. 7, 1568–1579 (2022).
Makarova, K. S., Wolf, Y. I., Snir, S. & Koonin, E. V. Defense islands in bacterial and archaeal genomes and prediction of novel defense systems. J. Bacteriol. 193, 6039–6056 (2011).
Benler, S. et al. Cargo genes of Tn7-like transposons comprise an enormous diversity of defense systems, mobile genetic elements, and antibiotic resistance genes. mBio 12, e0293821 (2021).
Wu, Y. et al. Bacterial defense systems exhibit synergistic anti-phage activity. Cell Host Microbe 32, 557–572 (2024).
Lau, R. K. et al. Structure and mechanism of a cyclic trinucleotide-activated bacterial endonuclease mediating bacteriophage immunity. Mol. Cell 77, 723–733 (2020).
Lopatina, A., Tal, N. & Sorek, R. Abortive infection: bacterial suicide as an antiviral immune strategy. Annu. Rev. Virol. 7, 371–384 (2020).
Makarova, K. S., Anantharaman, V., Grishin, N. V., Koonin, E. V. & Aravind, L. CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems. Front. Genet. 5, 102 (2014).
Bondy-Denomy, J. et al. Prophages mediate defense against phage infection through diverse mechanisms. ISME J. 10, 2854–2866 (2016).
Atanasiu, C., Su, T.-J., Sturrock, S. S. & Dryden, D. T. F. Interaction of the ocr gene 0.3 protein of bacteriophage T7 with EcoKI restriction/modification enzyme. Nucleic Acids Res. 30, 3936–3944 (2002).
Walkinshaw, M. D. et al. Structure of Ocr from bacteriophage T7, a protein that mimics B-form DNA. Mol. Cell 9, 187–194 (2002).
Isaev, A. et al. Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence. Nucleic Acids Res. 48, 7601–7602 (2020).
Song, S. & Wood, T. K. A primary physiological role of toxin/antitoxin systems is phage inhibition. Front. Microbiol. 11, 1895 (2020).
Hopfner, K.-P. & Tainer, J. A. Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures. Curr. Opin. Struct. Biol. 13, 249–255 (2003).
Deep, A. et al. The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA. Mol. Cell 82, 4145–4159.e7 (2022).
Hopfner, K. P. et al. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell 101, 789–800 (2000).
Zawadzka, K. et al. MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin. eLife 7, e31522 (2018).
Schiltz, C. J., Adams, M. C. & Chappie, J. S. The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of class 1 OLD family nucleases. Nucleic Acids Res. 48, 2762–2776 (2020).
Oerum, S. et al. Structures of B. subtilis maturation RNases captured on 50S ribosome with pre-rRNAs. Mol. Cell 80, 227–236 (2020).
Ho, C.-H., Wang, H.-C., Ko, T.-P., Chang, Y.-C. & Wang, A. H.-J. The T4 phage DNA mimic protein Arn inhibits the DNA binding activity of the bacterial histone-like protein H-NS. J. Biol. Chem. 289, 27046–27054 (2014).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Nonejuie, P., Burkart, M., Pogliano, K. & Pogliano, J. Bacterial cytological profiling rapidly identifies the cellular pathways targeted by antibacterial molecules. Proc. Natl Acad. Sci. USA 110, 16169–16174 (2013).
Burman, N. et al. A virally-encoded tRNA neutralizes the PARIS antiviral defence system. Nature, https://doi.org/10.1038/s41586-024-07874-3 (2024).
Bregegere, F. Bacteriophage P2-lambda interference: inhibition of protein synthesis involves transfer RNA inactivation. J. Mol. Biol. 90, 459–467 (1974).
Ernits, K. et al. The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems. Proc. Natl Acad. Sci. USA 120, e2305393120 (2023).
Sironi, G. Mutants of Escherichia coli unable to be lysogenized by the temperate bacteriophage P2. Virology 37, 163–176 (1969).
Krishnan, A., Burroughs, A. M., Iyer, L. M. & Aravind, L. Comprehensive classification of ABC ATPases and their functional radiation in nucleoprotein dynamics and biological conflict systems. Nucleic Acids Res. 48, 10045–10075 (2020).
Deng, Y. M., Liu, C. Q. & Dunn, N. W. Genetic organization and functional analysis of a novel phage abortive infection system, AbiL, from Lactococcus lactis. J. Biotechnol. 67, 135–149 (1999).
Miller, W. G. et al. Diversity within the Campylobacter jejuni type I restriction-modification loci. Microbiology 151, 337–351 (2005).
Ogura, T. & Hiraga, S. Mini-F plasmid genes that couple host cell division to plasmid proliferation. Proc. Natl Acad. Sci. USA 80, 4784–4788 (1983).
Harms, A., Brodersen, D. E., Mitarai, N. & Gerdes, K. Toxins, targets, and triggers: an overview of toxin-antitoxin biology. Mol. Cell 70, 768–784 (2018).
Bobonis, J. et al. Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems. Nature 609, 144–150 (2022).
Zhang, T. et al. Direct activation of a bacterial innate immune system by a viral capsid protein. Nature 612, 132–140 (2022).
Fineran, P. C. et al. The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Proc. Natl Acad. Sci. USA 106, 894–899 (2009).
LeRoux, M. et al. The DarTG toxin-antitoxin system provides phage defence by ADP-ribosylating viral DNA. Nat. Microbiol. 7, 1028–1040 (2022).
Pecota, D. C. & Wood, T. K. Exclusion of T4 phage by the hok/sok killer locus from plasmid R1. J. Bacteriol. 178, 2044–2050 (1996).
Ledvina, H. E. et al. An E1-E2 fusion protein primes antiviral immune signalling in bacteria. Nature 616, 319–325 (2023).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
Deep, A. et al. Structural, functional and biological insights into the role of Mycobacterium tuberculosis VapBC11 toxin–antitoxin system: targeting a tRNase to tackle mycobacterial adaptation. Nucleic Acids Res. 46, 11639–11655 (2018).
Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).
Steinegger, M. & Söding, J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026–1028 (2017).
Eastman, P. et al. OpenMM 7: rapid development of high performance algorithms for molecular dynamics. PLoS Comput. Biol. 13, e1005659 (2017).
Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).
Holm, L. Dali server: structural unification of protein families. Nucleic Acids Res. 50, W210–W215 (2022).
Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–W350 (2016).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Schiltz, C. J., Lee, A., Partlow, E. A., Hosford, C. J. & Chappie, J. S. Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage. Nucleic Acids Res. 47, 9448–9463 (2019).
Acknowledgements
We thank A. Whiteley for sharing T4 and T7 phages and for reading the manuscript; Y. Gu for help with cryo-EM data collection; M. Herzik for suggestions on cryo-EM data processing; A. Desai for reading the manuscript; the members of the Corbett laboratory for discussions; the staff of the cryo-EM facility at UC San Diego, including M. Matyszewski and I. Kuschnerus, for facilities and scientific and technical assistance; and F. Ahmed and P. Ordoukhanian at The Scripps Research Institute Biophysics and Biochemistry Core Facility for assistance with mass photometry. All electron microscopy data were collected at the UCSD cryo-EM Facility, which was built and equipped with funds from UCSD and an initial gift from the Agouron Institute. Q.L. is a recipient of a predoctoral fellowship from the American Heart Association. We acknowledge funding from the National Institutes of Health R35 GM144121 (to K.D.C.), R01 GM129245 (to J.P.) and the Howard Hughes Medical Institute Emerging Pathogens Initiative (to J.P. and K.D.C.).
Author information
Authors and Affiliations
Contributions
The study was designed by A.D. and K.D.C. Cloning and protein purifications were performed by A.D. and Q.L. Bacterial cytological profiling and growth curve experiments were performed by E.E. with guidance from J.P. All the cell growth, biochemical assays and cryo-EM work were performed by A.D. Figures were prepared by A.D., Q.L., E.E. and K.D.C. The manuscript was written by A.D. and K.D.C. All of the authors contributed to editing the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature thanks Jinwei Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 PARIS is an antiphage toxin-antitoxin system.
a, Schematic representation of the domains in the E. coli B185 AriA and AriB subunits, with predicted domain annotations. b, Multiple sequence alignment showing conservation of amino acid residues involved in ATP binding and catalysis in AriA and Rad50 homologues. The conserved residues involved in catalysis are indicated. c, ATPase activity of AriA and its conserved residue mutants. K39I: a Walker-A motif mutant designed to disrupt ATP binding. D392A: a Walker-B motif mutant designed to disrupt ATP binding. E393Q: a Walker-B motif mutant designed to disrupt ATPase activity. ATP hydrolysis is expressed as moles of ATP hydrolysed per minute per mole of AriA hexamer. Error bars represent the average and standard deviation of three measurements (n = 3; open circles). d, A representative plaque-forming unit assay, demonstrating T7 phage plaques on the E. coli MG1655 bacterial lawn carrying either an empty vector (EV), the PARIS system (AriA-AriB, as an operon), the ATPase mutant AriAE393Q (AriAEQ-AriB), or a putative nuclease-dead mutant AriBE90A (AriA-AriBE90A). T7 phage 10-fold dilutions are spotted as indicated with a gradient. e, f, Growth curve assays depicting the effects of AriB or AriA-AriB overexpression in E. coli MG1655 bacterial cells. The presence and absence of inducers, as well as their relative concentrations, are indicated above. Curves and error bars represent average ± standard deviation from three independent replicates (n = 3), and are representative of two independent trials. g, h, i, Schematic representations illustrating expression strategies and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of AriA, AriA-AriBE90A, and AriA-AriBE90A + AriBE90A complexes. Blue lines depict protein concentration (dRI; change in refractive index from baseline), while orange lines indicate measured molecular mass in kDa. The theoretical molecular weights for each subunit and the inferred complexes are indicated within each SEC-MALS plot.
Extended Data Fig. 2 Cryo-EM workflow for AriAEQ.
a, Representative micrograph (from 5,174 micrograph dataset) displaying the AriAEQ sample. Scale bar = 100 nm. b, Workflow outlining the cryo-EM reconstruction process for AriAEQ using cryoSPARC. c, Representative 2D classes displaying AriA samples from the final particle stack, with the highlighted yellow classes also depicted in Fig. 2b. Scale bar = 10 nm. d, Gold-standard FSC curve for the final global refinement of AriAEQ. e, Two views of the globally refined cryo-EM map for the AriAEQ hexamer, colour-coded by local resolution from <3 Å (blue) to >7 Å (red). f, Gold-standard FSC curve for the masked local refinement of AriAEQ. g, A view depicting the locally refined cryo-EM map for the AriAEQ hexamer, colour-coded by local resolution from <3 Å (blue) to >7 Å (red). h, Example cryo-EM density with a built atomic model demonstrating the model fit. Yellow and blue are two AriAEQ protomers.
Extended Data Fig. 3 Comparative analysis of AriA and its structural homologue.
a, Two views of the AriA HH dimer, with ATPase heads and coiled coils labelled. b, Two views of the M. jannaschii Rad50 dimer (PDB ID 3AV0; no associated publication) equivalent to the AriA views in panel a. c, Overlay of AriA and Rad50 homodimer depicting the distinct coiled-coil orientation. d, Closeup view of the AriA and Mj Rad50 ATPase active site, with conserved AriA active site residues shown as sticks and labelled (see multiple sequence alignment in Extended Data Fig. 1b). Q393* indicates the E393Q mutation. e, f, g, Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of AriAK39I, AriAD392A, and AriAS367R (see Extended Data Fig. 1g for SEC-MALS analysis of wild-type AriA). K39I: a Walker A motif mutant designed to disrupt ATP binding. D392A: a Walker B motif mutant designed to disrupt ATP binding. S367R: a signature motif mutant designed to disrupt HH interface. Blue lines depict protein concentration (measured as a change in refractive index, dRI), while orange lines indicate measured molecular weight (molar mass in kDa). The theoretical molecular weight for an AriA monomer is 53.2 kDa, and for a hexamer is 319.2 kDa.
Extended Data Fig. 4 Cryo-EM Workflow for AriAEQ-AriBE90A.
a, Representative micrograph (from 5,058 micrograph dataset) displaying the AriAEQ-AriBE90A complex. Scale bar = 100 nm. b, Workflow outlining the cryo-EM reconstruction process for E. coli AriAEQ-AriBE90A complex using cryoSPARC. c, Representative 2D classes displaying AriAEQ-AriBE90A samples from the final particle stack, with the highlighted yellow classes also depicted in Fig. 3b. Scale bar = 10 nm. d, Gold-standard FSC curve illustrating the final global refinement of AriAEQ-AriBE90A. e, Two views presenting the globally refined cryo-EM map for AriAEQ-AriBE90A, colour-coded by local resolution from <3 Å (blue) to >7 Å (red). f, Gold-standard FSC curve for the masked local refinement of AriAEQ-AriBE90A. g, A view showcasing the locally refined cryo-EM map for AriAEQ-AriBE90A, colour-coded by local resolution from <3 Å (blue) to >7 Å (red). h, Example cryo-EM density with a built atomic model demonstrating the model fit. Yellow and blue are two AriA protomer chains, and pink corresponds to AriB.
Extended Data Fig. 5 Structural and functional analysis of AriA-AriB complex.
a, b, Two similarly oriented views of AriAEQ-AriBE90A and P. aeruginosa PA14 Wadjet complex (PDB ID 8DK3)20, illustrating that the AriB binding interface is canonically conserved among other SMC ATPases. c, ATPase activity of AriA and AriA-AriB complex. ATP hydrolysis is expressed as moles of ATP hydrolysed per minute per mole of AriA (as AriA6) or AriA-AriB (as AriA6B2). Error bars represent the average and standard deviation of three independent measurements (n = 3; open circles). d, Top: Structural overlay presenting AriAEQ and AriAEQ-AriBE90A complexes. Bottom: a close-up view depicts changes in the AriA ATPase head region upon binding with AriB. The residues involved in AriB binding are shown in stick representation for side chains. AriB is omitted for clarity. e, Cartoon representation of AriB monomer, with the toprim domain in pink and helical bundle domain in white. At the bottom; Close-up view of the AriB active site. f, Overall structure of Geobacillus stearothermophilus (Gs) M5 RNase bound to the 50 S ribosome. M5 RNase is shown in a contrast clear window, zoomed in cartoon view below (PDB ID 6TPQ)24. Bottom: Close-up view of the active site of M5 RNase. g, Cartoon structure of OLD DNase from Burkholderia pseudomallei (Bp) (PDB ID 6NK8)55. Two bound metal ions are shown as green spheres. Bottom: Close-up view of the active site. h, Overlay of active site residues from AriB, M5 RNase, and OLD DNase. The active site residues are shown in ball-and-stick representation. i, Top: A representative plaque-forming unit assay, demonstrating T7 phage plaques on the E. coli MG1655 bacterial lawn carrying either an empty vector (EV), the PARIS system (Wt; AriA-AriB as an operon), or the RNA binding mutant AriBR28E (AriA-AriBR28E). Bottom: The data represent mean and standard deviation of plaque-forming units (PFU) mL−1 of phage T7 from three independent replicates, with individual data points shown (n = 3). j, Bacterial dilution spotting (10-fold) assay performed to test the toxicity comparison in AriB and its point mutants (E90A and R28E). Data shown are representative of three replicates (n = 3). k, SDS-PAGE analysis of coexpression of AriA-AriB and AriA-AriBR28E constructs followed by Ni2+ affinity purification of AriA and associated proteins. Data are representative of three independent experiments.
Extended Data Fig. 6 AriA and AriB interact to form compositional and conformationally heterogeneous complexes.
a, The final nine cryo-EM reconstructions for the AriAEQ-AriBE90A complex, colour-coded by local resolution from <3 Å (blue) to >7 Å (red). The final resolution and the number of particles corresponding to each complex form are noted at the bottom of each map (see Supplementary Fig. 4 for reconstruction and data processing). b, c, cryo-EM density map and cartoon structural representations of the AriA6-AriB2 and AriA6-AriB3 complexes. d, A cartoon model depicting steric clashes between Up and Down AriB monomers at the ATPase head dimer. Only one AriB can interact with a given AriA HH interface. e, Left: Cryo-EM structural representations of the AriA6-AriB3 complex showing the relative orientations (Up/Down) of AriB. Right: A simplified schematic illustrating AriA-AriB complex formation and AriB Up/Down designations.
Extended Data Fig. 7 Identification of T7 phage molecular pattern recognized by PARIS system.
a, Overview of the experimental evolution strategy used to obtain T7 phage escaper mutants against PARIS38. b, Summary of the mutations identified in three T7 phage escaper mutants. Close-up views below show the DNA sequence alignment and the impact of mutations in the noted phage genes. c, Representative plaque-forming unit assay demonstrating T7 escaper phage #3 plaques on an E. coli MG1655 bacterial lawn carrying either an empty vector (Vector) or the PARIS system. d, Analysis of the empty vector (Vector) and PARIS system for their ability to defend against T7 PARIS escaper phage #3. The data represent mean and standard deviation of plaque-forming units (PFU) mL−1 from three independent replicates, with individual data points shown as open circles (n = 3). e, SEC-MALS analysis of T7 Ocr (orange curve with red molar mass measurement) and OcrL82R (red curve with blue molar mass measurement). Theoretical molar mass of His6-tagged Ocr = 16.1 kDa (monomer)/32.2 kDa (dimer). f, Bacterial dilution spotting (10-fold) assay performed to measure the toxicity of AriA-AriB (from Fig. 1c), AriAEQ-AriB, and AriA-AriBE90A upon co-expression with Ocr. Uninduced: LB media + 0.4% glucose (suppressor) + antibiotics; AriA-AriB Induced: LB media + antibiotics + 0.2% arabinose. AriA-AriB + Ocr Induced: LB media + antibiotics + 0.2% arabinose + 0.1 mM IPTG. Cell growth inhibition observed when coexpressing AriA-AriB plus Ocr without Ocr induction (red outline) is likely due to low-level leaky expression of Ocr. g, h, Growth curves of E. coli MG1655 cells transformed with plasmids carrying AriB (pink; from Extended Data Fig. 1e,f), AriA-AriB (purple, from Extended Data Fig. 1e,f), AriA-AriB + T7 Ocr (red), or AriA-AriBE90A + T7 Ocr (green), in the absence (panel g) or the presence (panels h) of inducers. Curves represent the average and standard deviation from three independent replicates (n = 3). i, ATPase activity assay showing the effect of Ocr and OcrL82R on AriA or AriA-AriB complex ATPase activity. ATP hydrolysis is expressed as moles of ATP hydrolysed per minute per mole of AriA hexamer. Error bars represent the average and standard deviation of three independent measurements (n = 3; open circles).
Extended Data Fig. 8 Cryo-EM workflow for the AriA-Ocr complex.
a, Sample preparation strategy and a representative micrograph (from 6,821 micrograph dataset) displaying the PARIS + Ocr sample. Scale bar = 100 nm. b, Workflow outlining the cryo-EM data processing for the AriA-Ocr complex using cryoSPARC software. c, Representative 2D classes showing AriA-Ocr samples from the final particle stack. Scale bar = 10 nm. d, Gold-standard FSC curve illustrating the final global refinement of the AriA-Ocr complex reconstruction. e, Two views of the globally-refined cryo-EM map for the AriA-Ocr complex, colour-coded by local resolution from <3 Å (blue) to >7 Å (red). f, Gold-standard FSC curve for the masked (noted in panel e) local refinement of the AriA-Ocr interaction interface region. g, A view of the locally refined cryo-EM map for the AriA-Ocr interaction interface region, colour-coded by local resolution from <3 Å (blue) to >7 Å (red). h, Example cryo-EM density with a docked atomic model demonstrating the model fit. Yellow and blue represent two AriA protomers, and green represents Ocr. i, The final five cryo-EM reconstructions for the AriA-Ocr complex, colour-coded by local resolution from <3 Å (blue) to >7 Å (red). The final resolution and the number of particles corresponding to each complex form are noted at the bottom of each map (see Supplementary Fig. 5 for reconstruction and data processing).
Extended Data Fig. 9 Structural rearrangements in AriA upon interactions with Ocr and AlphaFold structure prediction of AriB dimer.
a-c, Structural comparison and overall rearrangement of AriA upon Ocr binding. Representative helices of AriA from AriA-AriB structure (semi-transparent) and AriA-Ocr (solid) and their overall bending/motions are shown with noted trajectories. Pink and green circles denote the position of AriA ATPase head dimers in the AriA-AriB complex (pink circles) and AriA-Ocr complex (green circles). For clarity, AriB and other AriA protomer chains are not shown. d, Structural overlays showing that upon binding with Ocr, AriA’s AriB interaction interface is disrupted. Side chains of residues involved in AriA-AriB interactions are shown in ball-and-stick representations. e, Representative plaque-forming unit assays, demonstrating T7 and T4 phage plaques on the E. coli MG1655 bacterial lawn carrying either an empty vector (EV), the PARIS system (WT; AriA-AriB as an operon), or AriA receptor pocket mutants 2RE and 4RE. 2RE and 4RE are the double and quadruple charge-reversal mutants of AriA in AriA-AriB operon (as shown in highlighted box at bottom). f, Cartoon view of the E. coli B185 AriB homodimer as predicted by AlphaFold2 (blue and cyan), with one protomer overlaid with the structure of AriBE90A determined by cryo-EM (pink). g, AlphaFold2 predicted aligned error (PAE) plot for the AriB dimer structure prediction. h, AlphaFold2 predicted distogram plot for the AriB dimer structure prediction. i, A representative plaque-forming unit assay, demonstrating T7 phage plaques on the E. coli MG1655 bacterial lawn carrying either an empty vector (EV), the PARIS system (WT; AriA-AriB as an operon), or AriB dimerization interface mutants: AriBR31E (AriA-AriBR31E), AriBK144E (AriA-AriBK144E), AriBR289E (AriA-AriBR289E), AriBK144E+R289E (AriA-AriBK144E,R289E). Data for empty vector and WT samples are the same as the experiment shown in Extended Data Fig. 5i.
Supplementary information
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Deep, A., Liang, Q., Enustun, E. et al. Architecture and activation mechanism of the bacterial PARIS defence system. Nature (2024). https://doi.org/10.1038/s41586-024-07772-8
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41586-024-07772-8
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
