Abstract
Restriction-modification (R-M) systems protect against phage infection by detecting and degrading invading foreign DNA. However, like many prokaryotic anti-phage defences, R-M systems pose a major risk of autoimmunity, exacerbated by the presence of hundreds to thousands of potential cleavage sites in the bacterial genome. Pseudomonas aeruginosa strains experience the temporary inactivation of restriction endonucleases following growth at high temperatures, but the reason and mechanisms for this phenomenon are unknown. Here we report that P. aeruginosa type I restriction endonuclease is degraded and the methyltransferase is partially degraded, by two Lon-like proteases when replicating at >41 °C. This post-translational regulation prevents self-DNA targeting, which is a risk due to stable genomic hypomethylation, as demonstrated by single-molecule, real-time sequencing and TadA-assisted N6-methyladenosine sequencing. When cells grown at >41 °C are returned to 37 °C, full genomic methylation does not fully recover for up to 60 bacterial generations, and thus restriction activity remains off for the duration. Our findings demonstrate that type I R-M is tightly regulated post-translationally with a long memory effect that ensures genomic stability and mitigates autotoxicity.
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Data availability
All strains and plasmids used in this study are detailed in Supplementary Table 2 and the oligonucleotides used in this study are listed in Supplementary Table 3. The raw data from RNA-seq are available at the National Center for Biotechnology Information Gene Expression Omnibus (GEO) under accession number GSE301453. Raw data from eTAM-seq are available under GEO accession no. GSE280585. Raw data from SMRT-seq are available in the NCBI BioProject database under accession number PRJNA1293863. Proteomics data are available via ProteomeXchange with identifier PXD066156. Genomic analyses were performed using the P. aeruginosa PAO1 reference genome GCA_000006765.1. Source data are provided with this paper.
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Acknowledgements
This work is part of the PhD thesis of E.S. Partial funding for this work was through the Dyna and Fala Weinstock Foundation to E.B. and the President’s Scholarships and the Merit-Based Scholarships at the Institute of Nanotechnology of Bar-Ilan University to E.S. Experiments were funded by awards to J.B.D. by the Bowes Biomedical Investigator Award and the University of California, San Francisco Sanghvi-Agarwal Innovation Award research gifts. A.V. was supported by the National Science Foundation (NSF) and S.D.M. by a National Institutes of Health F31 award. We thank the Crown Genomics Institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine and the Weizmann Institute of Science for proteomics data and SMRT-seq processing and analysis support. We thank R. Lavigne for sharing the Luz24 phage. The eTAM-seq section is supported by a pilot award under grant no. RM1 HG008935. We thank Q. Dai for constructive suggestions on eTAM-seq library preparation. We thank the Single Cell Immunophenotyping Core Facility at the University of Chicago for sequencing support.
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E.S., A.V., S.D.M. and I.A. generated strains and conducted phenotypic experiments and analyses. E.S. drafted the initial manuscript. A.V., S.S., E.B. and J.B.-D. reviewed and edited the manuscript. Y.W. and H.Y. prepared the eTAM-seq libraries. C.Y. performed the next-generation sequencing and bioinformatics analysis, with additional analysis contributions from I.L.-L. W.T. supervised the eTAM-seq work. S.K.-L. contributed to the methodology. E.B. and J.B.-D. provided overall supervision and secured funding.
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J.B.-D. is a scientific advisory board member of SNIPR Biome, Excision Biotherapeutics and LeapFrog Bio, consults for BiomX and is a scientific advisory board member and co-founder of Acrigen Biosciences and ePhective Therapeutics. J.B.-D.’s lab received research support from Felix Biotechnology. Patent application no. 63/417,245 has been filed for eTAM-seq by the University of Chicago. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Dynamics and conditions required for tiREN.
(a) Examination of restriction activity following overnight growth at various temperatures and sub-culture for infection with unmodified JBD30 phages at 37 °C. Values were calculated relative to the hsdR deletion strain. (b) PAO1 was grown at 43 °C for varying amounts of time. At specific time points (x-axis), CFUs/mL were measured (right y-axis) and the culture was incubated at 37 °C. Once saturated, plaque assays measured restriction activity (left y-axis).
Extended Data Fig. 2 DNA recombination and breaks do not induce tiREN.
(a) Restriction activity recombination genes mutant strains at 37 °C (blue) and following 43 °C growth, compared to WT strain. (b) Restriction activity of WT strain following 6 hr growth with sub-inhibitory concentration Fluoroquinolone treatment of Ciprofloxacin and Norfloxacin, compared to untreated WT strain. For the above graphs, restriction activity was assessed by JBD24 non-modified phages infection. For (a) and (b) graphs, the data represent the mean of three independent biological replicates +/− SD.
Extended Data Fig. 3 tiREN re-induction.
Restriction activity following overnight (ON) growth at 43 °C, 100 generations recovery at 37 °C, and re-introducing ON growth at 43 °C. Data shows a representative replicate.
Extended Data Fig. 4 HsdR expression is toxic only at tiREN state.
(a) Full plate images from spot assays of strains expressing either sfCherry (left) or hsdR (right), following ON growth at 37 °C (top) or 43 °C (bottom). (b) Growth curve at 37 °C following ON growth at either 37 °C (left) or 43 °C (right) of WT and strains harboring an inducible copy of hsdR, treated with Rha inducer. (c) Restriction activity: Infection curves were analyzed at 37 °C for WT, the hsdR mutant, and strains carrying an inducible copy of hsdR. The strains were treated with the Rha inducer, and infections were performed using un-modified JBD24 phages at an MOI of 1. (d) Activity dependence: Growth curves were conducted at 37 °C after overnight growth at 43 °C for the hsdM mutant strain carrying an inducible copy of hsdR, with or without the addition of the Rha inducer. For all the graphs above, the inducer was added at t = 0. The data represent the mean of three independent biological replicates +/− SD.
Extended Data Fig. 5 Modification hot spots at 43 °C do not specifically affect tiREN.
Restriction activity following ON growth at 37 °C (blue) or 43 °C (pink) of WT strain and mutants. Mutants are strains lacking the complete ORF containing the modification site. Restriction activity was assessed with un-modified JBD24 phages, relative to the hsdR mutant strain. The data represent the mean of three independent biological replicates +/− SD.
Extended Data Fig. 6 Restriction endonuclease is not transcriptionally regulated.
(a) Global transcriptomics analysis: Samples taken 5 generations into recovery following the 43 °C growth and at the end of the recovery phase (45 generations) were compared to normal growth conditions. Marked dots indicate Hsd proteins: HsdR and HsdMS. (b) Real-time PCR analysis of hsd transcripts following growth at either 37 °C or 43 °C. expression levels are normalized to rpoD as a ctrl. (c) Fluorescence and absorbance measurements over time of a strain harboring the hsdR ORF replaced by mCherry, representing transcriptional reporter analysis. (d) Fluorescence and optical density measurements over time of a strain harboring hsdR fused to mCherry, representing translational reporter analysis. The (c) and (d) graphs represent the mean of three independent biological replicates +/− SD.
Extended Data Fig. 7 HsdR-specific custom antibody showing a similar reduction in HsdR levels.
Endogenous measurement of Type I R-M proteins, HsdR from WT cells at 37 °C, 43 °C, or recovery at 37 °C for 10 generations by Western blot analysis using custom polyclonal antibodies, RNA pol was used as a loading ctrl.
Extended Data Fig. 8 Proteases complementation and fitness cost for restriction activation in the proteases mutant.
Protease complementation restores tiREN: (a) WT and mutant strains containing either empty vectors or plasmid-expressed protease were examined for restriction activity following ON growth at 43 °C. (b) Proteases OE at 37 °C affect restriction: Restriction activity at 37 °C with single and double proteases overexpression. Restriction activity was assessed with un-modified JBD24 phages, relative to the hsdR mutant strain. Each dot represents a single biological replicate, with error bars indicating standard deviation. (c) Flow-cytometry analysis of population variation in mixed WT and ∆hsdR strains (top), or double-proteases mutant and double mutant lacking hsdR (bottom). Mixtures were grown for six hours before analysis. Representative histograms of the reverse tagging analysis are displayed (left), and population percentages are shown as an mean of three biological replicates +/− SD. (right).
Extended Data Fig. 9 Flow cytometry gating strategy.
Shown is a representative sample: Δ2prot (GFP) vs. Δ2protΔhsdR (mCherry) at T6, 43 °C. Gating was applied sequentially as follows: (i) FSC-A vs. DAPI-A to select cells, (ii) FSC-H vs. FSC-W and SSC-H vs. SSC-W to gate singlets, (iii) FITC-A vs. PE-mCherry-A to quantify GFP-positive and mCherry-positive populations. Percentages indicate the fraction of events within each gate. The same gating strategy was used for all samples.
Supplementary information
Supplementary Table 1
The tiREN examination in different P. aeruginosa strains after growth at 43 °C, using unmodified JBD30 phages, with indicated genomic type I R-M systems.
Supplementary Table 2
Bacterial strains, phages and plasmids used in this study.
Supplementary Table 3
Oligos used in this study.
Source data
Source Data Fig. 3
Unprocessed western blots.
Source Data Fig. 4
Unprocessed western blots.
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Unprocessed western blots.
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Unprocessed western blots.
Source Data Extended Data Fig. 7
Unprocessed western blots.
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Shmidov, E., Villani, A., Mendoza, S.D. et al. Multigenerational proteolytic inactivation of restriction upon subtle genomic hypomethylation in Pseudomonas aeruginosa. Nat Microbiol 10, 2498–2510 (2025). https://doi.org/10.1038/s41564-025-02088-3
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DOI: https://doi.org/10.1038/s41564-025-02088-3
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