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Functional amyloid proteins confer defence against predatory bacteria

Nature volume 644, pages 197–204 (2025)Cite this article

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Abstract

Bdellovibrio bacteriovorus is a predatory bacterium that non-selectively preys on Gram-negative bacteria by invading the prey-cell periplasm, leaching host nutrients and ultimately lysing the infected cell to exit and find a new host1,2. The predatory life cycle of B. bacteriovorus is, in many ways, comparable to a bacteriophage. However, unlike phage defence, defence against B. bacteriovorus has not been widely investigated. Here we screened a collection of diverse Escherichia coli strains for resistance to B. bacteriovorus and identified that roughly one-third of strains robustly defended against predation by producing curli fibres. Curli fibres are oligomers of the functional amyloid protein CsgA, which is exceptionally durable3. Using genetics and microscopy, we demonstrate that curli fibres provide a barrier that protects susceptible cells independent of genes required for biofilm formation. This barrier further protected E. coli against attack by the predatory bacterium Myxococcus xanthus and select phages. Bioinformatic analysis of bacterial amyloids showed these systems are diverse and widespread in diderm bacteria (those with both inner and outer membranes). One of these, an evolutionarily distinct amyloid encoded by Pseudomonas aeruginosa, also protected against B. bacteriovorus. This work establishes that functional amyloids defend bacteria against a wide range of threats.

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Fig. 1: Defence against B. bacteriovorus is widespread in E. coli.
Fig. 2: Curli production is necessary and sufficient for defence against B. bacteriovorus.
Fig. 3: Curli form an extracellular mesh-like network to protect bacteria.
Fig. 4: Curli production is required for B. bacteriovorus defence in many strains of E. coli and protects against other diverse threats.
Fig. 5: Functional amyloids are found in diverse bacteria and contribute to defence against B. bacteriovorus.

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Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. The accession numbers for proteins appearing in this study can be found in Supplementary Table 3. Source data are provided with this paper.

Code availability

All code and software used in this study are available or described at https://github.com/AravindLab/operons-script, and/or within the paper and its Supplementary Information.

References

  1. Sockett, R. E. Predatory lifestyle of Bdellovibrio bacteriovorus. Annu. Rev. Microbiol. 63, 523–539 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Lai, T. F., Ford, R. M. & Huwiler, S. G. Advances in cellular and molecular predatory biology of Bdellovibrio bacteriovorus six decades after discovery. Front. Microbiol. 14, 1168709 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Evans, M. L. & Chapman, M. R. Curli biogenesis: order out of disorder. Biochim. Biophys. Acta 1843, 1551–1558 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Santin, Y. G., Lamot, T., Van Raaphorst, R., Kaljević, J. & Laloux, G. Modulation of prey size reveals adaptability and robustness in the cell cycle of an intracellular predator. Curr. Biol. 33, 2213–2222.e4 (2023).

    Article  PubMed  Google Scholar 

  5. Georjon, H. & Bernheim, A. The highly diverse antiphage defence systems of bacteria. Nat. Rev. Microbiol. 21, 686–700 (2023).

  6. Aharon, E. et al. Secretion systems play a critical role in resistance to predation by Bdellovibrio bacteriovorus. Res. Microbiol. 172, 103878 (2021).

    Article  CAS  PubMed  Google Scholar 

  7. Koval, S. F. & Hynes, S. H. Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus. J. Bacteriol. 173, 2244–2249 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Duncan, M. C. et al. High-throughput analysis of gene function in the bacterial predator Bdellovibrio bacteriovorus. mBio 10, e01040–19 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Shemesh, Y. & Jurkevitch, E. Plastic phenotypic resistance to predation by Bdellovibrio and like organisms in bacterial prey. Environ. Microbiol. 6, 12–18 (2004).

    Article  PubMed  Google Scholar 

  10. Mitchell, R. J., Mun, W., Mabekou, S. S., Jang, H. & Choi, S. Y. Compounds affecting predation by and viability of predatory bacteria. Appl. Microbiol. Biotechnol. 104, 3705–3713 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Mun, W. et al. Cyanide production by Chromobacterium piscinae shields it from Bdellovibrio bacteriovorus HD100 predation. mBio 8, e01370–17 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dwidar, M., Nam, D. & Mitchell, R. J. Indole negatively impacts predation by Bdellovibrio bacteriovorus and its release from the bdelloplast. Environ. Microbiol. 17, 1009–1022 (2015).

    Article  CAS  PubMed  Google Scholar 

  13. Hoshiko, Y. et al. Quinolone signals related to Pseudomonas quinolone signal-quorum sensing inhibits the predatory activity of Bdellovibrio bacteriovorus. Front. Microbiol. 12, 722579 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Dwidar, M. et al. Diffusible signaling factor, a quorum-sensing molecule, interferes with and is toxic towards Bdellovibrio bacteriovorus 109J. Microb. Ecol. 81, 347–356 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  15. Fagan, R. P. & Fairweather, N. F. Biogenesis and functions of bacterial S-layers. Nat. Rev. Microbiol. 12, 211–222 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. Ochman, H. & Selander, R. K. Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157, 690–693 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Patel, I. R. et al. Draft genome sequences of the Escherichia coli Reference (ECOR) collection. Microbiol. Resour. Announc. https://doi.org/10.1128/mra.01133-18 (2018).

  18. Hoshiko, Y., Okuno, M., Yamamoto, T., Maeda, T. & Ogura, Y. Improved complete genome sequence of Bdellovibrio bacteriovorus 109J, a widely studied laboratory strain of predatory bacteria. Microbiol. Resour. Announc. https://doi.org/10.1128/mra.01296-23 (2024).

  19. Ogasawara, H., Yamamoto, K. & Ishihama, A. Regulatory role of MlrA in transcription activation of csgD, the master regulator of biofilm formation in Escherichia coli. FEMS Microbiol. Lett. 312, 160–168 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Ogasawara, H., Yamada, K., Kori, A., Yamamoto, K. & Ishihama, A. Regulation of the Escherichia coli csgD promoter: interplay between five transcription factors. Microbiology 156, 2470–2483 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Brown, P. K. et al. MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium. Mol. Microbiol. 41, 349–363 (2001).

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Serra, D. O., Richter, A. M. & Hengge, R. Cellulose as an architectural element in spatially structured Escherichia coli biofilms. J. Bacteriol. 195, 5540–5554 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Acheson, J. F., Derewenda, Z. S. & Zimmer, J. Architecture of the cellulose synthase outer membrane channel and its association with the periplasmic TPR domain. Structure 27, 1855–1861.e3 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Olsén, A., Arnqvist, A., Hammar, M., Sukupolvi, S. & Normark, S. The RpoS Sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli. Mol. Microbiol. 7, 523–536 (1993).

    Article  PubMed  Google Scholar 

  25. Reshamwala, S. M. S. & Noronha, S. B. Biofilm formation in Escherichia coli cra mutants is impaired due to down-regulation of curli biosynthesis. Arch. Microbiol. 193, 711–722 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Jubelin, G. et al. CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli. J. Bacteriol. 187, 2038–2049 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mun, W., Upatissa, S., Lim, S., Dwidar, M. & Mitchell, R. J. Outer membrane porin F in E. coli is critical for effective predation by Bdellovibrio. Microbiol. Spectr. 10, e03094–22 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Dueholm, M. S., Albertsen, M., Otzen, D. & Nielsen, P. H. Curli functional amyloid systems are phylogenetically widespread and display large diversity in operon and protein structure. PLoS ONE 7, e51274 (2012).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Smith, D. R. et al. The production of curli amyloid fibers is deeply integrated into the biology of Escherichia coli. Biomolecules 7, 75 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Sawaya, M. R., Hughes, M. P., Rodriguez, J. A., Riek, R. & Eisenberg, D. S. The expanding amyloid family: structure, stability, function, and pathogenesis. Cell 184, 4857–4873 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Olsén, A., Jonsson, A. & Normark, S. Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli. Nature 338, 652–655 (1989).

    Article  ADS  PubMed  Google Scholar 

  32. Barnhart, M. M. & Chapman, M. R. Curli biogenesis and function. Annu. Rev. Microbiol. 60, 131–147 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tursi, S. A. & Tükel, Ç. Curli-containing enteric biofilms inside and out: matrix composition, immune recognition, and disease implications. Microbiol. Mol. Biol. Rev. https://doi.org/10.1128/mmbr.00028-18 (2018).

  34. Maury, C. P. J. The emerging concept of functional amyloid. J. Intern. Med. 265, 329–334 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Evans, M. L. et al. The bacterial curli system possesses a potent and selective inhibitor of amyloid formation. Mol. Cell 57, 445–455 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gualdi, L. et al. Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli. Microbiology 154, 2017–2024 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Bian, Z. & Normark, S. Nucleator function of CsgB for the assembly of adhesive surface organelles in Escherichia coli. EMBO J. 16, 5827–5836 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Arnqvist, A., Olsén, A. & Normark, S. σS-dependent growth-phase induction of the csgBA promoter in Escherichia coli can be achieved in vivo by σ70 in the absence of the nucleoid-associated protein H-NS. Mol. Microbiol. 13, 1021–1032 (1994).

    Article  CAS  PubMed  Google Scholar 

  39. Weber, H., Pesavento, C., Possling, A., Tischendorf, G. & Hengge, R. Cyclic-di-GMP-mediated signalling within the σS network of Escherichia coli. Mol. Microbiol. 62, 1014–1034 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Pesavento, C. et al. Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli. Genes Dev. 22, 2434–2446 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lindenberg, S., Klauck, G., Pesavento, C., Klauck, E. & Hengge, R. The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control. EMBO J. 32, 2001–2014 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ahmad, I., Cimdins, A., Beske, T. & Römling, U. Detailed analysis of c-di-GMP mediated regulation of csgD expression in Salmonella typhimurium. BMC Microbiol. 17, 27 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Serra, D. O. & Hengge, R. A c-di-GMP-based switch controls local heterogeneity of extracellular matrix synthesis which is crucial for integrity and morphogenesis of Escherichia coli macrocolony biofilms. J. Mol. Biol. 431, 4775–4793 (2019).

    Article  CAS  PubMed  Google Scholar 

  44. Bian, Z., Brauner, A., Li, Y. & Normark, S. Expression of and cytokine activation by Escherichia coli curli fibers in human sepsis. J. Infect. Dis. 181, 602–612 (2000).

    Article  CAS  PubMed  Google Scholar 

  45. Reichhardt, C. et al. Congo Red interactions with curli-producing E. coli and native curli amyloid fibers. PLoS ONE 10, e0140388 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Serra, D. O. & Hengge, R. in c-di-GMP Signaling (ed. Sauer, K.) Vol. 1657, 133–145 (Springer, 2017).

  47. Lamprecht, O. et al. Regulation by cyclic di-GMP attenuates dynamics and enhances robustness of bimodal curli gene activation in Escherichia coli. PLoS Genet. 19, e1010750 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Contreras-Moreno, F. J., Pérez, J., Muñoz-Dorado, J., Moraleda-Muñoz, A. & Marcos-Torres, F. J. Myxococcus xanthus predation: an updated overview. Front. Microbiol. 15, 1339696 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  49. DePas, W. H. et al. Biofilm formation protects Escherichia coli against killing by Caenorhabditis elegans and Myxococcus xanthus. Appl. Environ. Microbiol. 80, 7079–7087 (2014).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  50. Bond, M. C., Vidakovic, L., Singh, P. K., Drescher, K. & Nadell, C. D. Matrix-trapped viruses can prevent invasion of bacterial biofilms by colonizing cells. eLife 10, e65355 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Gallardo, R., Ranson, N. A. & Radford, S. E. Amyloid structures: much more than just a cross-β fold. Curr. Opin. Struct. Biol. 60, 7–16 (2020).

    Article  CAS  PubMed  Google Scholar 

  52. Hammer, N. D., Schmidt, J. C. & Chapman, M. R. The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization. Proc. Natl Acad. Sci. USA 104, 12494–12499 (2007).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wittmers, F., Needham, D. M., Hehenberger, E., Giovannoni, S. J. & Worden, A. Z. Genomes from uncultivated pelagiphages reveal multiple phylogenetic clades exhibiting extensive auxiliary metabolic genes and cross-family multigene transfers. mSystems 7, e0152221 (2022).

    Article  PubMed  Google Scholar 

  54. Rouse, S. L., Matthews, S. J. & Dueholm, M. S. Ecology and biogenesis of functional amyloids in Pseudomonas. J. Mol. Biol. 430, 3685–3695 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Aravind, L., Iyer, L. M. & Burroughs, A. M. Discovering biological conflict systems through genome analysis: evolutionary principles and biochemical novelty. Annu. Rev. Biomed. Data Sci. 5, 367–391 (2022).

    Article  CAS  PubMed  Google Scholar 

  56. MacLellan, S. R., Wecke, T. & Helmann, J. D. A previously unidentified sigma factor and two accessory proteins regulate oxalate decarboxylase expression in Bacillus subtilis. Mol. Microbiol. 69, 954–967 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Papayannopoulos, V. Neutrophil extracellular traps in immunity and disease. Nat. Rev. Immunol. 18, 134–147 (2018).

    Article  CAS  PubMed  Google Scholar 

  58. Kumar, D. K. V. et al. Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease. Sci. Transl. Med. 8, 340ra72 (2016).

    Article  PubMed  Google Scholar 

  59. Ledvina, H. E. et al. An E1–E2 fusion protein primes antiviral immune signalling in bacteria. Nature 616, 319–325 (2023).

  60. Kibby, E. M. et al. Bacterial NLR-related proteins protect against phage. Cell https://doi.org/10.1016/j.cell.2023.04.015 (2023).

  61. Boyd, E. F., Hill, C. W., Rich, S. M. & Hartl, D. L. Mosaic structure of plasmids from natural populations of Escherichia coli. Genetics 143, 1091–1100 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Ferrières, L. et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J. Bacteriol. 192, 6418–6427 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Lee, P., Stopford, C. M., Svenson, A. G. & Rietsch, A. Control of effector export by the Pseudomonas aeruginosa type III secretion proteins PcrG and PcrV. Mol. Microbiol. 75, 924–941 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  65. Simon, R., Priefer, U. & Pühler, A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1, 784–791 (1983).

    Article  CAS  Google Scholar 

  66. Blattner, F. R. et al. The complete genome sequence of Escherichia coli K-12. Science 277, 1453–1462 (1997).

    Article  CAS  PubMed  Google Scholar 

  67. Jeukens, J. et al. Comparative genomics of isolates of a Pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients. PLoS ONE 9, e87611 (2014).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  68. Rahme, L. G. et al. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268, 1899–1902 (1995).

    Article  ADS  CAS  PubMed  Google Scholar 

  69. Stover, C. K. et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964 (2000).

    Article  ADS  CAS  PubMed  Google Scholar 

  70. Cain, A. K. et al. Complete genome sequence of Pseudomonas aeruginosa reference strain PAK. Microbiol. Resour. Announc. 8, e00865–19 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Daligault, H. E. et al. Draft genome assembly of Klebsiella pneumoniae type strain ATCC 13883. Genome Announc. 2, e00939–14 (2014).

    PubMed  PubMed Central  Google Scholar 

  72. Rendulic, S. et al. A predator unmasked: life cycle of Bdellovibrio bacteriovorus from a genomic perspective. Science 303, 689–692 (2004).

    Article  ADS  CAS  PubMed  Google Scholar 

  73. Lambert, C. & Sockett, R. E. Laboratory maintenance of Bdellovibrio. Curr. Protoc. Microbiol. 9, 7B.2.1–7B.2.13 (2008).

  74. Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat. Protoc. 10, 1820–1841 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Fenton, A. K., El Mortaji, L., Lau, D. T. C., Rudner, D. Z. & Bernhardt, T. G. CozE is a member of the MreCD complex that directs cell elongation in Streptococcus pneumoniae. Nat. Microbiol. 2, 16237 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  76. Chiang, S. L. & Rubin, E. J. Construction of a mariner-based transposon for epitope-tagging and genomic targeting. Gene 296, 179–185 (2002).

    Article  CAS  PubMed  Google Scholar 

  77. Cameron, D. E., Urbach, J. M. & Mekalanos, J. J. A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae. Proc. Natl Acad. Sci. USA 105, 8736–8741 (2008).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  78. Zemansky, J. et al. Development of a mariner-based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype. J. Bacteriol. 191, 3950–3964 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Zhou, Y., Smith, D. R., Hufnagel, D. A. & Chapman, M. R. Experimental manipulation of the microbial functional amyloid called curli. Methods Mol. Biol. 966, 53–75 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  81. Maffei, E. et al. Systematic exploration of Escherichia coli phage–host interactions with the BASEL phage collection. PLoS Biol. 19, e3001424 (2021).

  82. Matsui, Y. et al. Multilocus sequence typing of Escherichia coli isolates from urinary tract infection patients and from fecal samples of healthy subjects in a college community. MicrobiologyOpen 9, e1032 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  83. Hall, B. G. Building phylogenetic trees from molecular data with MEGA. Mol. Biol. Evol. 30, 1229–1235 (2013).

    Article  CAS  PubMed  Google Scholar 

  84. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Sayers, E. W. et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 52, D33–D43 (2024).

    Article  CAS  PubMed  Google Scholar 

  86. Steinegger, M. & Söding, J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026–1028 (2017).

    Article  CAS  PubMed  Google Scholar 

  87. Wootton, J. C. & Federhen, S. Analysis of compositionally biased regions in sequence databases. Methods Enzymol. 266, 554–571 (1996).

    Article  CAS  PubMed  Google Scholar 

  88. Gabler, F. et al. Protein sequence analysis using the MPI bioinformatics toolkit. Curr. Protoc. Bioinform. 72, e108 (2020).

    Article  CAS  Google Scholar 

  89. Deorowicz, S., Debudaj-Grabysz, A. & Gudyś, A. FAMSA: fast and accurate multiple sequence alignment of huge protein families. Sci. Rep. 6, 33964 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  90. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Teufel, F. et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat. Biotechnol. 40, 1023–1025 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Cuff, J. A. & Barton, G. J. Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins 40, 502–511 (2000).

    Article  CAS  PubMed  Google Scholar 

  93. Baek, M. et al. Accurate prediction of protein structures and interactions using a three-track neural network. Science 373, 871–876 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  94. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  95. Ravi, J. et al. The phage shock protein (PSP) envelope stress response: discovery of novel partners and evolutionary history. mSystems 9, e0084723 (2024).

    Article  PubMed  Google Scholar 

  96. Kaur, G., Burroughs, A. M., Iyer, L. M. & Aravind, L. Highly regulated, diversifying NTP-dependent biological conflict systems with implications for the emergence of multicellularity. eLife 9, e52696 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Dueholm, M. S. et al. Functional amyloid in Pseudomonas. Mol. Microbiol. 77, 1009–1020 (2010).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank D. Kadouri (Rutgers School of Dental Medicine) and S. Lory (Harvard Medical School) for sharing of strains; Ç. Tükel (Temple University) for technical advice and helpful discussion; A. Erbse, the CU Boulder Department of Biochemistry Shared Instruments Pool core facility (RRID SCR_018986) and its staff; G. Morgan and S. A. Zimmerman in the University of Colorado Boulder Electron Microscopy Services Core Facility for electron microscopy sample preparation and imaging; the BioFrontiers Institute’s Advanced Light Microscopy Core (RRID SCR_018302) for confocal imaging and members of the Whiteley laboratory for their advice and helpful discussion. This work was funded by the National Institutes of Health (NIH) through the NIH Director’s New Innovator Award grant no. DP2AT012346 and the NIH Common Fund grant no. 3DP2AT012346-01S1 (A.T.W.), the PEW Charitable Trust Biomedical Scholars Award (A.T.W.), a Mallinckrodt Foundation Grant (A.T.W.) and the Boettcher Foundation’s Webb-Waring Biomedical Research Program (A.T.W.). H.E.L. was supported in part as a fellow of the Jane Coffin Childs Memorial Fund for Medical Research; R.S. was supported in part by the NIH T32 Signaling and Cellular Regulation training grant (no. T32GM142607); R.O.C. was supported in part by the Biological Sciences Initiative and an Undergraduate Research Opportunities Program Student Assistantship grant, both funded by the University of Colorado Boulder, and A.R.M. was supported in part by the Uplift Research programme through STEM ROUTES funded by the University of Colorado Boulder. A.M.B. and L.A. are supported by the Intramural Research Program of the National Library of Medicine at the NIH. This work used the NIH HPC Biowulf computer cluster (A.M.B. and L.A.). The Nikon AXR Laser Scanning Confocal is supported by NIH grant no. 1S10OD034320.

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Authors and Affiliations

  1. Department of Biochemistry, University of Colorado Boulder, Boulder, CO, USA

    Hannah E. Ledvina, Ryan Sayegh, Ricardo O. Carale, Alexa R. Macklin, Layla D. Borja Najera & Aaron T. Whiteley

  2. Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, CO, USA

    Ryan Sayegh & Ashley L. Azadeh

  3. Computational Biology Branch, Division of Intramural Research, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA

    A. Maxwell Burroughs & L. Aravind

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Experiments were designed and conceived by H.E.L. and A.T.W. Plate-based predation assays were performed by H.E.L., R.S., R.O.C., A.L.A., L.D.B.N. and A.R.M. Transposon mutagenesis of bacterial strains was performed by H.E.L., R.S., R.O.C. and A.L.A. M. xanthus predation assays were performed by H.E.L. and A.R.M. Liquid-based predation assays, transcomplementation studies, targeted mutagenesis of E. coli and P. aeruginosa, TEM preparation, Congo Red analysis and ECOR phylogenetic analysis were performed by H.E.L. TEM quantification was performed by H.E.L., A.R.M. and L.D.B.N. Comparative genomics and sequence-structure analysis of bacterial amyloid protein were performed by A.M.B. and L.A. Figures were prepared by H.E.L., A.T.W., A.M.B. and L.A. The manuscript was written by H.E.L., A.M.B., L.A. and A.T.W. All authors contributed to editing the manuscript and support the conclusions.

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Correspondence to Aaron T. Whiteley.

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Extended data figures and tables

Extended Data Fig. 1 B. bacteriovorus lifecycle, interaction with ECOR14, and details for plaque assays.

(a) Model depicting the B. bacteriovorus predatory lifecycle. (b) Graphical representation of how modified double-agar overlay infection assays are performed. Briefly, prey and B. bacteriovorus strains are prepared separately. The prey is then mixed with serial dilutions of B. bacteriovorus and the mixture is embedded in soft agar (0.35 % agar) that is overlayed in 6-well dishes containing 1.6% agar underlays. (c) Efficiency of plating, calculated as PFU/mL, of B. bacteriovorus HD100 when infecting the indicated strain of E. coli or K. pneumoniae. Experiments were conducted as depicted in (b). Data are graphed as in Fig. 1b. (d) Efficiency of plating, calculated as PFU/mL, of B. bacteriovorus 109J when infecting the indicated strain of E. coli or K. pneumoniae. Experiments were conducted as depicted in (b). Data are graphed as in Fig. 1b. For c-d; two-sided Student’s t-test. *P < 0.05, **P < 0.001, *** P < 0.0001; precise P-values can be found in Source Data Table 5.

Source Data

Extended Data Fig. 2 ECOR14 defense against B. bacteriovorus in liquid culture.

(a) Graphical representation of how liquid-based infection assays are performed. Briefly, prey and B. bacteriovorus strains are prepared separately. The prey and B. bacteriovorus are then mixed in a liquid culture and at various times post infection prey cell colony formation was quantified. The initial ratio of B. bacterivorous plaque forming units to E. coli colony forming units (multiplicity of infection, MOI) used in these assays was ~ 0.01. (b–c) Quantification of colony forming units (CFU) for the indicated strain of prey bacteria. Prey were either uninfected or infected with B. bacteriovorus (b) HD100 or (c) 109J in a liquid culture as depicted in (a). Data are the mean ± S.E.M. for n = 3 biological replicates.

Source Data

Extended Data Fig. 3 ECOR14 signature-tagged mutagenesis uncovers that curli is required for B. bacteriovorus defense.

(a) Overview of ECOR14 transposon mutagenesis and screen for B. bacteriovorus defense. ECOR14 was transposon-mutagenized and 6,734 individual mutants were gridded into multi-well plates. Each mutant was individually challenged B. bacteriovorus HD100; in these conditions, wells with wild-type ECOR14 became turbid from E. coli growth while wells with newly-susceptible mutants were cleared by B. bacteriovorus. Mutants that failed to defend were rescreened by plaque assay with a B. bacteriovorus HD100 dilution series (indicated from 100–10−5), as in Extended Data Fig. 1b. The transposon insertion site was mapped for confirmed susceptible mutant strains. (b) Efficiency of plating, calculated as PFU/mL, of B. bacteriovorus HD100 when infecting the indicated strains of E. coli. Details of each Tnmut can be found in Supplementary Table 2. Data graphed as in Fig. 1b. (c) Organization of the two curli operons underlying curli production along with a graphical representation of how curli fibres are formed on the outer membrane. Briefly, CsgBAC and CsgEFG are secreted via Sec to the periplasmic space. Here CsgEFG form the secretion apparatus required for CsgA and CsgB to cross the outer membrane32. CsgC aids CsgA folding at the proper location35. CsgB is the membrane anchor that seeds CsgA oligomerization and curli fibre formation52. See Supplementary Table 3 for protein accession numbers. (d) Efficiency of plating, calculated as PFU/mL, of B. bacteriovorus 109J when infecting the indicated strain of ECOR14. Data graphed as in Fig. 1b.

Source Data

Extended Data Fig. 4 Curli-mediated defense in ECOR14 is visible across experimental variations.

(a-f) Efficiency of plating, calculated as PFU/mL, of B. bacteriovorus HD100 when infecting the indicated strain of prey (K.p. corresponds to K. pneumoniae). Data graphed as in Fig. 1b. Conditions that were varied in the experiment are listed for each panel. For (a) LB* represents that the overnight media was LB but cells were transferred to dilute nutrient broth prior to the assay. For (c) the prey concentrations tested were: undiluted, 1:10, 1:100, 1:1,000, and 1:10,000 of the overnight culture, which correlates to the indicated multiplicity of infection (MOI). Of note, for 1:1,000 and 1:10,000 the prey cells did not form a complete lawn, however defined plaques were still observed in K. pneumoniae and ECOR14 csgA::kanR. (g) Unedited representative transmission electron microscopy image of B. bacteriovorus predating on E. coli. (h) The same image as in (g) with false coloring applied (green, E. coli ECOR14; orange, B. bacteriovorus HD100; purple, curli). B. bacteriovorus cells (orange arrow), curli positive E. coli (dark grey arrow), curli negative E. coli (light grey arrow), B. bacteriovorus that are in contact with an E. coli cell (red arrow), and the length of the curli barrier starting at the outer membrane (black bracket) are all indicated. These features were the basis of the quantification presented in Extended Data Fig. 6. For (g-h), this image was also presented in Fig. 3i–m and scale bars represent 500 nm.

Source Data

Extended Data Fig. 5 Electron microscopy of curli-mediated defense.

(a-f) Representative transmission electron microscopy images of the indicated strain of ECOR14 uninfected. (g-l) Representative cryogenic electron microscopy images of the indicated strain of ECOR14 uninfected. (m-o) Representative transmission electron microscopy images of the MG1655 overexpressing an empty vector (sfGFP). (p-r) Representative transmission electron microscopy images of the MG1655 overexpressing a synthetic csg operon (csgABCEFG). (s-x) Representative transmission electron microscopy images of the indicated strain of ECOR14 infected with B. bacteriovorus HD100 for 4 h. For all images, data represent three individual biological replicates, and the scale bar represents 500 nm.

Extended Data Fig. 6 Transmission electron microscopy quantification reveals heterogeneity in curli production and fibre length.

(a) The number of E. coli cells identified per image for the indicated strain of ECOR14. (b) The number of B. bacteriovorus cells identified per image for the indicated strain of ECOR14. For (a) and (b), data are the mean ± standard error of the mean (S.E.M.) for wild-type n = 37; csgA::kanR n = 34 individual images representing three individual biological replicates. These data demonstrate the similarity of the selected fields of view. (c-d) The distribution of the proportion of B. bacteriovorus in contact with (c) ECOR14 or (d) ECOR14 csgA::kanR. Data is binned by the percentage of B. bacteriovorus in contact with E. coli within each field of view quantified; bins correspond to 12.5%. (e-f) The number of B. bacteriovorus in contact or not in contact with (e) wild-type ECOR14 or (f) ECOR14 csgA::kanR. (g-h) The distribution of the percentage of (g) wild-type ECOR14 or (h) ECOR14 csgA::kanR cells producing curli within each field of view quantified; bins correspond to 12.5%. (i-j) The number of curli negative or positive E. coli observed for (i) wild-type ECOR14 or (j) ECOR14 csgA::kanR. (k) The length of the curli barrier produced by the indicated strains of ECOR14. Data are the mean ± standard error of the mean (S.E.M.) for the indicated n representing individual images derived from three independent biological replicates. (a-k) See Quantification of transmission electron microscopy for additional details. For all quantified images, see Supplementary Figs. 1 and 2.

Source Data

Extended Data Fig. 7 Differences in curli production correlate B. bacteriovorus defense patterns and low levels of curli production results in a turbid plaque phenotype.

(a) Representative image of each ECOR strain plated on media containing Congo Red. Strains are grouped based on susceptibility and/or defense against B. bacteriovorus HD100 and 109J. (b-c) Efficiency of plating, calculated as PFU/mL (left axis), of B. bacteriovorus HD100 superimposed with the Congo Red score (right axis). The Congo red score was determined based on the about of red within a colony as shown in (a) (see Methods for more details). Data are the mean ± S.E.M. for n = 3 biological replicates. (b) Depicts ECOR1-48 and (c) depicts ECOR49-72. (d) Efficiency of plating, calculated as PFU/mL, of B. bacteriovorus 109J when infecting the indicated strains of E. coli. Data are graphed as in Fig. 1b. (e-f) Representative transmission electron microscopy images of the indicated strain of E. coli from three individual biological replicates. Cells expressing curli are indicated with a purple arrow and cells not expressing curli are indicated with a white arrow. Scale bars represent 500 nm. (g) Representative image of a plaque assay where the indicated strain of MG1655 was infected with HD100. Plaques are indicated with a black arrow. Data are representative of n = 3 biological replicates.

Source Data

Extended Data Fig. 8 Functional amyloid proteins of Pseudomonas and defense against B. bacteriovorus along with evidence that functional amyloid systems display key characteristics of defense systems.

(a) Organization of the operon underlying FapC amyloid production along with a graphical representation of how the fibres are formed on the outer membrane97. Briefly, FapBCDEF are secreted via Sec to the periplasmic space. Here FapF forms the secretion apparatus required for FapBCE to cross the outer membrane. Prior to secretion, FapC is proteolytically processed by FapD. FapB is the membrane anchor that seeds FapC oligomerization and fibre formation. FapE serves as the cap of the fibre to stop polymerization. The precise role of FapA is unclear. See Supplementary Table 3 for protein accession numbers. (b) Efficiency of plating, calculated as PFU/mL, of B. bacteriovorus HD100 when infecting the indicated strains of P. aeruginosa. Data are graphed as in Fig. 1b. (c) The length distribution and variance of the indicated families of functional amyloid proteins. For objective comparison, the lengths are normalized by the mean length of the family. The distribution is shown as a box and whisker plot (central measure: median; lower and upper extreme denoted by dashed lines), whereas the variance is shown using the mean (central blue point) with standard deviation bars in blue solid line. (d) The distribution and variance of the measure of exploration of sequence space of selected families of functional amyloid proteins. The exploration of sequence space is measured using two indices, the first of which measures the deviation from the mean amino acid composition and the second of which measures the skew in polarity. The distance from the family centroid in this distribution space is then computed for each sequence. The distribution is shown as a box and whisker plot (central measure: median; lower and upper extreme denoted by dashed lines), whereas the variance is shown using the mean (central point) with standard deviation bars in blue solid line. For (c-d) Grey dots depict outliers; purple numbers are the P-values from a two-sided F-test that compares variances across the two indicated samples. FapE, N = 84; FapC, N = 149; FapB, N = 183; CsgB, N = 305; CsgA, N = 374.

Source Data

Extended Data Fig. 9 Diverse functional amyloid systems identified from across bacteria.

(a) Operonic and architectural organization of related components of selected Curli and Fap-like systems. A key shows the predicted functions/features annotated by color and outline: blue, transcription factor; purple, amyloid; yellow, peptidase; green, accessory protein; red, amyloid inhibitor; grey, other and/or lineage specific proteins. The presence of a signal peptide within the protein is indicated by an outline. Accession numbers correspond to the protein denoted with an asterisk. Operons found within phage are denoted with a box. (b) Protein domains identified within Curli and Fap-like systems grouped based on their predicted function. Colors correspond to (a). RGG, Arginine-Glycine-Glycine motif characteristic of these proteins; CD-iREC, inactive receiver domain; REC, receiver domain; HTH, helix-turn-helix domain; PAS, Per-Arnt-Sim domain; SSB, single strand DNA binding domain; β-sand, β-sandwich domain; PGBD, peptidoglycan bindings domain; IG, immunoglobulin. (c) Select protein structures from Curli and Fap-like systems that share homology with each other: the iREC (inactive receiver) domain of CsgD and REC (receiver) domain of VspR, the major transcriptional factors that regulate their respective system; CsgA and FapC, the major amyloid subunits of each system. CsgF and FapA, CsgF is known to contribute to secretion across the outer membrane; however, the role of FapA is still unclear. CsgG and FapF are the outer membrane transporters for each system. Protein structures are predicted using AlphaFold2 unless otherwise indicated.

Extended Data Fig. 10 Functional amyloid system presence or absence in the genomes of representative strains and taxa.

A presence-absence matrix for selected taxa. Grey cells indicate presence and white cells indicate absence of the indicated gene. Cells outlined with a dashed line are those where the component displays an alternative architecture.

Source Data

Supplementary information

Supplementary Fig. 1

TEM images of wild-type ECOR14 used for quantification of curli.

Reporting Summary

Supplementary Fig. 2

TEM images of ECOR14 csgA::kanR used for quantification of curli.

Supplementary Fig. 3

Unedited transmission electron micrographs corresponding to the figures indicated.

Supplementary Table 1

Bacterial strains and plasmids, phages and oligonucleotides used in this study.

Supplementary Table 2

ECOR14 transposon mutants disrupted for defence against B. bacteriovorus.

Supplementary Table 3

Accession numbers for proteins appearing in this study.

Supplementary File 1

Code for bioinformatic analysis.

Peer Review File

Source data

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Ledvina, H.E., Sayegh, R., Carale, R.O. et al. Functional amyloid proteins confer defence against predatory bacteria. Nature 644, 197–204 (2025). https://doi.org/10.1038/s41586-025-09204-7

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