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Volume 171, Issue 11

Conflicts between the DNA replication and repair machineries promote cell death in Gram-positive bacteria Open Access

Abstract

Cellular proliferation relies on the successful coordination and completion of genome replication and segregation. To help achieve this, many bacteria utilize regulatory pathways that ensure DNA replication initiation only occurs once per cell cycle. When dysregulated, loss of DNA replication control can have severe consequences. In , it has been established that hyper-initiation of DNA synthesis leads to pleiotropic genome instability and cell death. Therefore, targeting DNA replication initiation proteins to promote hyper-initiation may be an approach to generate novel antimicrobials. However, the pathways and potential consequences of replication hyper-initiation in Gram-positive species remain enigmatic. To address this question, we devised genetic systems to artificially induce hyper-initiation in the model organism and the pathogen . In both species, hyper-initiation elicited cellular degeneration culminating in growth inhibition by cell death. During this process in , temporal analyses revealed the early onset of the DNA damage response, followed by membrane depolarization and cell lysis. This phenotype could be suppressed by removing pathways that repair damaged DNA, suggesting that cell death is a consequence of conflicts between DNA replication and repair. In cells quickly accumulated striking morphological changes associated with rapid loss of chromosomal DNA and death via a lysis-independent pathway. Moreover, inducing hyper-initiation in was observed to decrease bacterial survival during infection of murine macrophages. Taken together, the data suggest that stimulating initiation of bacterial DNA synthesis could be an alternative approach to inhibiting microbial growth, particularly in combination with compounds that inhibit or poison DNA repair, akin to cancer therapies.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Bacillus subtilis , DNA damage , DNA repair , DNA replication , Hyper-initiation and Staphylococcus aureus
Funding
This study was supported by the:
  • Medical Research Council (Award MR/X50290X/1)
    • Principal Award Recipient: JuliaHubbard
  • Medical Research Council (Award MR/W009587/1)
    • Principal Award Recipient: KatarzynaMickiewicz
  • Wellcome Trust (Award 225811/Z/22/Z)
    • Principal Award Recipient: HeathMurray
  • Wellcome Trust (Award 226338/Z/22/Z)
    • Principal Award Recipient: CharlesWinterhalter
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2025-11-06
2025-12-06

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References

  1. Gaimster H, Stevens D, Grimshaw J, Hubbard J, Mickiewicz K et al.Conflicts between the DNA replication and repair machineries promote cell death in gram-positive bacteria Microbiology Society Figshare 2025 [View Article]
     [Google Scholar]
  2. Haeusser DP, Levin PA. The great divide: coordinating cell cycle events during bacterial growth and division. Curr Opin Microbiol 2008; 11:94–99 [View Article] [PubMed]
     [Google Scholar]
  3. Cooper G. The Cell: A Molecular Approach Sunderland, Ma: ASM Press, Sinauer Associates; 2000
     [Google Scholar]
  4. Bell SP, Kaguni JM. Helicase Loading at Chromosomal Origins of Replication Cold Spring Harb Perspect Biol; 2013
     [Google Scholar]
  5. Yi L, Lü X. New strategy on antimicrobial-resistance: inhibitors of DNA replication enzymes. Curr Med Chem 2019; 26:1761–1787 [View Article] [PubMed]
     [Google Scholar]
  6. Fujikawa N, Kurumizaka H, Nureki O, Terada T, Shirouzu M et al. Structural basis of replication origin recognition by the DnaA protein. Nucleic Acids Res 2003; 31:2077–2086 [View Article] [PubMed]
     [Google Scholar]
  7. Richardson TT, Harran O, Murray H. The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding. Nature 2016; 534:412–416 [View Article] [PubMed]
     [Google Scholar]
  8. Pelliciari S, Dong M-J, Gao F, Murray H. Evidence for a chromosome origin unwinding system broadly conserved in bacteria. Nucleic Acids Res 2021; 49:7525–7536 [View Article] [PubMed]
     [Google Scholar]
  9. Winterhalter C, Pelliciari S, Stevens D, Fenyk S, Marchand E et al. The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin. Nucleic Acids Res 2023; 51:4322–4340 [View Article] [PubMed]
     [Google Scholar]
  10. Jameson KH, Wilkinson AJ. Control of initiation of DNA replication in Bacillus subtilis and Escherichia coli. Genes 2017; 8:22 [View Article] [PubMed]
     [Google Scholar]
  11. Kaguni JM. Replication initiation at the Escherichia coli chromosomal origin. Curr Opin Chem Biol 2011; 15:606–613 [View Article] [PubMed]
     [Google Scholar]
  12. O’Donnell M, Langston L, Stillman B. Principles and Concepts of DNA Replication in Bacteria, Archaea, and Eukarya Cold Spring Harb Perspect Biol; 2013
     [Google Scholar]
  13. Skarstad K, Katayama T. Regulating DNA Replication in Bacteria Cold Spring Harb Perspect Biol; 2013 p a012922
     [Google Scholar]
  14. Wendel BM, Courcelle CT, Courcelle J. Completion of DNA replication in Escherichia coli. Proc Natl Acad Sci USA 2014; 111:16454–16459 [View Article] [PubMed]
     [Google Scholar]
  15. Rudolph CJ, Upton AL, Stockum A, Nieduszynski CA, Lloyd RG. Avoiding chromosome pathology when replication forks collide. Nature 2013; 500:608–611 [View Article] [PubMed]
     [Google Scholar]
  16. Darmon E, Leach DRF. Bacterial genome instability. Microbiol Mol Biol Rev 2014; 78:1–39 [View Article] [PubMed]
     [Google Scholar]
  17. Wagner JK, Marquis KA, Rudner DZ. SirA enforces diploidy by inhibiting the replication initiator DnaA during spore formation in Bacillus subtilis. Mol Microbiol 2009; 73:963–974 [View Article] [PubMed]
     [Google Scholar]
  18. Charbon G, Bjørn L, Mendoza-Chamizo B, Frimodt-Møller J, Løbner-Olesen A. Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli. Nucleic Acids Res 2014; 42:13228–13241 [View Article] [PubMed]
     [Google Scholar]
  19. Simmons LA, Breier AM, Cozzarelli NR, Kaguni JM. Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability. Mol Microbiol 2004; 51:349–358 [View Article] [PubMed]
     [Google Scholar]
  20. Anderson ME, Smith JL, Grossman AD. Multiple mechanisms for overcoming lethal over-initiation of DNA replication. Mol Microbiol 2022; 118:426–442 [View Article] [PubMed]
     [Google Scholar]
  21. Midgley-Smith SL, Dimude JU, Rudolph CJ. A role for 3’ exonucleases at the final stages of chromosome duplication in Escherichia coli. Nucleic Acids Res 2019; 47:1847–1860 [View Article] [PubMed]
     [Google Scholar]
  22. Goswami S, Gowrishankar J. Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli. Nucleic Acids Res 2022; 50:8643–8657 [View Article] [PubMed]
     [Google Scholar]
  23. Raghunathan N, Goswami S, Leela JK, Pandiyan A, Gowrishankar J. A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication. Nucleic Acids Res 2019; 47:5698–5711 [View Article] [PubMed]
     [Google Scholar]
  24. Anagnostopoulos C, Spizizen J. Requirements for transformation in Bacillus subtilis. J Bacteriol 1961; 81:741–746 [View Article] [PubMed]
     [Google Scholar]
  25. Grosser MR, Richardson AR. Method for preparation and electroporation of S. aureus and S. epidermidis. Methods Mol Biol 2016; 1373:51–57 [View Article] [PubMed]
     [Google Scholar]
  26. Hanahan D, Jessee J, Bloom FR. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 1991; 204:63–113 [View Article] [PubMed]
     [Google Scholar]
  27. Helle L, Kull M, Mayer S, Marincola G, Zelder M-E et al. Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus. Microbiology 2011; 157:3314–3323 [View Article] [PubMed]
     [Google Scholar]
  28. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676–682 [View Article] [PubMed]
     [Google Scholar]
  29. Cutler KJ, Stringer C, Lo TW, Rappez L, Stroustrup N et al. Omnipose: a high-precision morphology-independent solution for bacterial cell segmentation. Nat Methods 2022; 19:1438–1448 [View Article] [PubMed]
     [Google Scholar]
  30. van der Walt S, Schönberger JL, Nunez-Iglesias J, Boulogne F, Warner JD et al. scikit-image: image processing in Python. PeerJ 2014; 2:e453 [View Article] [PubMed]
     [Google Scholar]
  31. Berg S, Kutra D, Kroeger T, Straehle CN, Kausler BX et al. ilastik: interactive machine learning for (bio)image analysis. Nat Methods 2019; 16:1226–1232 [View Article] [PubMed]
     [Google Scholar]
  32. Murray H, Errington J. Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA. Cell 2008; 135:74–84 [View Article] [PubMed]
     [Google Scholar]
  33. Scholefield G, Errington J, Murray H. Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA. EMBO J 2012; 31:1542–1555 [View Article] [PubMed]
     [Google Scholar]
  34. Noirot-Gros M-F, Velten M, Yoshimura M, McGovern S, Morimoto T et al. Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis. Proc Natl Acad Sci USA 2006; 103:2368–2373 [View Article] [PubMed]
     [Google Scholar]
  35. Au N, Kuester-Schoeck E, Mandava V, Bothwell LE, Canny SP et al. Genetic composition of the Bacillus subtilis SOS system. J Bacteriol 2005; 187:7655–7666 [View Article] [PubMed]
     [Google Scholar]
  36. Roberts DM. A new role for monomeric ParA/Soj in chromosome dynamics in Bacillus subtilis. Microbiologyopen 2023; 12:e1344 [View Article] [PubMed]
     [Google Scholar]
  37. Hill TM, Sharma B, Valjavec-Gratian M, Smith J. sfi-independent filamentation in Escherichia coli Is lexA dependent and requires DNA damage for induction. J Bacteriol 1997; 179:1931–1939 [View Article] [PubMed]
     [Google Scholar]
  38. Trusca D, Scott S, Thompson C, Bramhill D. Bacterial SOS checkpoint protein SulA inhibits polymerization of purified FtsZ cell division protein. J Bacteriol 1998; 180:3946–3953 [View Article] [PubMed]
     [Google Scholar]
  39. Gozzi K, Ching C, Paruthiyil S, Zhao Y, Godoy-Carter V et al. Bacillus subtilis utilizes the DNA damage response to manage multicellular development. NPJ Biofilms Microbiomes 2017; 3:8 [View Article] [PubMed]
     [Google Scholar]
  40. Kawai Y, Moriya S, Ogasawara N. Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis. Mol Microbiol 2003; 47:1113–1122 [View Article] [PubMed]
     [Google Scholar]
  41. Reed TT, Kendal AH, Wozniak KJ, Simmons LA. DNA replication initiation timing is important for maintaining genome integrity. J Bacteriol 2025; 207:e0017525 [View Article] [PubMed]
     [Google Scholar]
  42. Te Winkel JD, Gray DA, Seistrup KH, Hamoen LW, Strahl H. Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Front Cell Dev Biol 2016; 4:29 [View Article] [PubMed]
     [Google Scholar]
  43. Köhler P, Marahiel MA. Association of the histone-like protein HBsu with the nucleoid of Bacillus subtilis. J Bacteriol 1997; 179:2060–2064 [View Article] [PubMed]
     [Google Scholar]
  44. Westers H, Dorenbos R, van Dijl JM, Kabel J, Flanagan T et al. Genome engineering reveals large dispensable regions in Bacillus subtilis. Mol Biol Evol 2003; 20:2076–2090 [View Article] [PubMed]
     [Google Scholar]
  45. Reuß DR, Thürmer A, Daniel R, Quax WJ, Stülke J. Complete genome sequence of Bacillus subtilis subsp. subtilis strain ∆6. Genome Announc 2016; 4:e00759-16 [View Article] [PubMed]
     [Google Scholar]
  46. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 1997; 390:249–256 [View Article] [PubMed]
     [Google Scholar]
  47. Goranov AI, Katz L, Breier AM, Burge CB, Grossman AD. A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA. Proc Natl Acad Sci USA 2005; 102:12932–12937 [View Article] [PubMed]
     [Google Scholar]
  48. Scholefield G, Veening JW, Murray H. DnaA and ORC: more than DNA replication initiators. Trends Cell Biol 2011; 21:188–194 [View Article] [PubMed]
     [Google Scholar]
  49. Richardson TT, Stevens D, Pelliciari S, Harran O, Sperlea T et al. Identification of a basal system for unwinding a bacterial chromosome origin. EMBO J 2019; 38:e101649 [View Article] [PubMed]
     [Google Scholar]
  50. Imlay JA. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol 2013; 11:443–454 [View Article] [PubMed]
     [Google Scholar]
  51. Foster PL, Lee H, Popodi E, Townes JP, Tang H. Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing. Proc Natl Acad Sci USA 2015; 112:E5990–9 [View Article] [PubMed]
     [Google Scholar]
  52. Kuzminov A. Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proc Natl Acad Sci USA 2001; 98:8241–8246 [View Article] [PubMed]
     [Google Scholar]
  53. Caldecott KW. Causes and consequences of DNA single-strand breaks. Trends Biochem Sci 2024; 49:68–78 [View Article] [PubMed]
     [Google Scholar]
  54. Winterhalter C, Stratton KJ, Fenyk S, Murray H. Rescuing the bacterial replisome at a nick requires recombinational repair and helicase reloading. Nat Commun 2025 [View Article]
     [Google Scholar]
  55. Sasaki M, Kurusu Y. Analysis of spontaneous base substitutions generated in mutator strains of Bacillus subtilis. FEMS Microbiol Lett 2004; 234:37–42 [View Article] [PubMed]
     [Google Scholar]
  56. Kurokawa K, Mizumura H, Takaki T, Ishii Y, Ichihashi N et al. Rapid exchange of bound ADP on the Staphylococcus aureus replication initiation protein DnaA. J Biol Chem 2009; 284:34201–34210 [View Article] [PubMed]
     [Google Scholar]
  57. Pang T, Wang X, Lim HC, Bernhardt TG, Rudner DZ. The nucleoid occlusion factor Noc controls DNA replication initiation in Staphylococcus aureus. PLoS Genet 2017; 13:e1006908 [View Article] [PubMed]
     [Google Scholar]
  58. Pidwill GR, Gibson JF, Cole J, Renshaw SA, Foster SJ. The role of macrophages in Staphylococcus aureus infection. Front Immunol 2020; 11:620339 [View Article] [PubMed]
     [Google Scholar]
  59. Fan X-Y, Tang B-K, Xu Y-Y, Han A-X, Shi K-X et al. Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria. Proc Natl Acad Sci USA 2018; 115:2210–2215 [View Article] [PubMed]
     [Google Scholar]
  60. Grimwade JE, Leonard AC. Blocking the trigger: inhibition of the initiation of bacterial chromosome replication as an antimicrobial strategy. Antibiotics 2019; 8:111 [View Article] [PubMed]
     [Google Scholar]
  61. Hicks ND, Giffen SR, Culviner PH, Chao MC, Dulberger CL et al. Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis. PLoS Pathog 2020; 16:e1009063 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001633
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Conflicts between the DNA replication and repair machineries promote cell death in Gram-positive bacteria
Microbiology 171, 001633 (2025); https://doi.org/10.1099/mic.0.001633
/content/journal/micro/10.1099/mic.0.001633
/content/journal/micro/10.1099/mic.0.001633
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