Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structure of Geobacter pili reveals secretory rather than nanowire behaviour

Nature volume 597, pages 430–434 (2021)Cite this article

Subjects

Abstract

Extracellular electron transfer by Geobacter species through surface appendages known as microbial nanowires1 is important in a range of globally important environmental phenomena2, as well as for applications in bio-remediation, bioenergy, biofuels and bioelectronics. Since 2005, these nanowires have been thought to be type 4 pili composed solely of the PilA-N protein1. However, previous structural analyses have demonstrated that, during extracellular electron transfer, cells do not produce pili but rather nanowires made up of the cytochromes OmcS2,3 and OmcZ4. Here we show that Geobacter sulfurreducens binds PilA-N to PilA-C to assemble heterodimeric pili, which remain periplasmic under nanowire-producing conditions that require extracellular electron transfer5. Cryo-electron microscopy revealed that C-terminal residues of PilA-N stabilize its copolymerization with PilA-C (to form PilA-N–C) through electrostatic and hydrophobic interactions that position PilA-C along the outer surface of the filament. PilA-N–C filaments lack π-stacking of aromatic side chains and show a conductivity that is 20,000-fold lower than that of OmcZ nanowires. In contrast with surface-displayed type 4 pili, PilA-N–C filaments show structure, function and localization akin to those of type 2 secretion pseudopili6. The secretion of OmcS and OmcZ nanowires is lost when pilA-N is deleted and restored when PilA-N–C filaments are reconstituted. The substitution of pilA-N with the type 4 pili of other microorganisms also causes a loss of secretion of OmcZ nanowires. As all major phyla of prokaryotes use systems similar to type 4 pili, this nanowire translocation machinery may have a widespread effect in identifying the evolution and prevalence of diverse electron-transferring microorganisms and in determining nanowire assembly architecture for designing synthetic protein nanowires.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Discovery and identification of G. sulfurreducens pili.
Fig. 2: PilA-N recruits PilA-C to form a heterodimer that polymerizes into a filament.
Fig. 3: Geobacter sulfurreducens PilA-N–C pili are involved in the translocation of OmcS and OmcZ nanowires.

Similar content being viewed by others

Data availability

The key relevant datasets generated during and/or analysed during the current study are publicly available. Cryo-EM data have been deposited with the Electron Microscopy Data Bank (accession code EMD-21225) and with the Protein Data Bank (PDB) (accession code 6VK9). All other relevant data are included in the Supplementary Information. An interactive 3D visualization is available at http://Proteopedia.org/w/Malvankar/3. Source data are provided with this paper.

Code availability

No special software code was used to collect data.

References

  1. Reguera, G. et al. Extracellular electron transfer via microbial nanowires. Nature 435, 1098–1101 (2005).

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Wang, F. et al. Structure of microbial nanowires reveals stacked hemes that transport electrons over micrometers. Cell 177, 361–369 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Filman, D. J. et al. Cryo-EM reveals the structural basis of long-range electron transport in a cytochrome-based bacterial nanowire. Commun. Biol. 2, 219 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Yalcin, S. E. et al. Electric field stimulates production of highly conductive microbial OmcZ nanowires. Nat. Chem. Biol. 16, 1136–1142 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Malvankar, N. S. et al. Tunable metallic-like conductivity in microbial nanowire networks. Nat. Nanotechnol. 6, 573–579 (2011).

    Article  ADS  PubMed  Google Scholar 

  6. Michel, G. P. & Voulhoux, R. in Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis (ed. Wooldridge, K.) 67–92 (Caister Academic, 2009).

  7. Yalcin, S. E. & Malvankar, N. S. The blind men and the filament: understanding structures and functions of microbial nanowires. Curr. Opin. Chem. Biol. 59, 193–201 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu, X., Zhuo, S., Rensing, C. & Zhou, S. Syntrophic growth with direct interspecies electron transfer between pili-free Geobacter species. ISME J. 12, 2142–2151 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Richter, L. V., Sandler, S. J. & Weis, R. M. Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and biofilm formation. J. Bacteriol. 194, 2551–2563 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shu, C., Xiao, K., Yan, Q. & Sun, X. Comparative analysis of type IV pilin in Desulfuromonadales. Front. Microbiol. 7, 2080 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  11. O’Brien, J. P. & Malvankar, N. S. A simple and low-cost procedure for growing Geobacter sulfurreducens cell cultures and biofilms in bioelectrochemical systems. Current Protoc. Microbiology 43, A.4K.1–A.4K.27 (2017).

    Google Scholar 

  12. Liu, X., Zhan, J., Jing, X., Zhou, S. & Lovley, D. R. A pilin chaperone required for the expression of electrically conductive Geobacter sulfurreducens pili. Environ. Microbiol. 21, 2511–2522 (2019).

    Article  CAS  PubMed  Google Scholar 

  13. Vignon, G. et al. Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization, and surface presentation of peptides. J. Bacteriol. 185, 3416–3428 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Durand, E. et al. XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus. J. Biol. Chem. 280, 31378–31389 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Wang, F. et al. Cryoelectron microscopy reconstructions of the Pseudomonas aeruginosa and Neisseria gonorrhoeae type IV pili at sub-nanometer resolution. Structure 25, 1423–1435 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Richter, L. V., Franks, A. E., Weis, R. M. & Sandler, S. J. Significance of a posttranslational modification of the PilA protein of Geobacter sulfurreducens for surface attachment, biofilm formation, and growth on insoluble extracellular electron acceptors. J. Bacteriol. 199, e00716-16 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Parge, H. E. et al. Structure of the fibre-forming protein pilin at 2.6 Å resolution. Nature 378, 32–38 (1995).

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Harvey, H. et al. Pseudomonas aeruginosa defends against phages through type IV pilus glycosylation. Nat. Microbiol. 3, 47–52 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Vik, Å. et al. Insights into type IV pilus biogenesis and dynamics from genetic analysis of a C-terminally tagged pilin: a role for O-linked glycosylation. Mol. Microbiol. 85, 1166–1178 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Craig, L., Pique, M. E. & Tainer, J. A. Type IV pilus structure and bacterial pathogenicity. Nat. Rev. Microbiol. 2, 363–378 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. López-Castilla, A. et al. Structure of the calcium-dependent type 2 secretion pseudopilus. Nat. Microbiol. 2, 1686–1695 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Li, J., Egelman, E. H. & Craig, L. Structure of the Vibrio cholerae type IVb Pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus. J. Mol. Biol. 418, 47–64 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Liu, X. et al. A Geobacter sulfurreducens strain expressing pseudomonas aeruginosa type IV pili localizes OmcS on pili but is deficient in Fe(III) oxide reduction and current production Appl. Environ. Microbiol. 80, 1219–1224 (2014).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  24. Xiao, K. et al. Low energy atomic models suggesting a pilus structure that could account for electrical conductivity of Geobacter sulfurreducens pili. Sci. Rep. 6, 23385 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Shipps, C. et al. Intrinsic electronic conductivity of individual atomically resolved amyloid crystals reveals micrometer-long hole hopping via tyrosines. Proc. Natl Acad. Sci. USA 118, e2014139118 (2021).

    Article  CAS  PubMed  Google Scholar 

  26. Mehta, T., Childers, S. E., Glaven, R., Lovley, D. R. & Mester, T. A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens. Microbiology 152, 2257–2264 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Shi, L. et al. Direct involvement of type II secretion system in extracellular translocation of Shewanella oneidensis outer membrane cytochromes MtrC and OmcA. J. Bacteriol. 190, 5512–5516 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bouhenni, R. A. et al. The role of Shewanella oneidensis MR-1 outer surface structures in extracellular electron transfer. Electroanalysis 22, 856–864 (2010).

    Article  CAS  Google Scholar 

  29. Liu, X., Ye, Y., Xiao, K., Rensing, C. & Zhou, S. Molecular evidence for the adaptive evolution of Geobacter sulfurreducens to perform dissimilatory iron reduction in natural environments. Mol. Microbiol. 113, 783–793 (2020).

    Article  CAS  PubMed  Google Scholar 

  30. Craig, L., Forest, K. T. & Maier, B. Type IV pili: dynamics, biophysics and functional consequences. Nat. Rev. Microbiol. 17, 429–440 (2019).

    Article  CAS  PubMed  Google Scholar 

  31. Coppi, M. V., Leang, C., Sandler, S. J. & Lovley, D. R. Development of a genetic system for Geobacter sulfurreducens. Appl. Environ. Microbiol. 67, 3180–3187 (2001).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mehta, T., Coppi, M. V., Childers, S. E. & Lovley, D. R. Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens. Appl. Environ. Microbiol. 71, 8634–8641 (2005).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. Nevin, K. P. et al. Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells. PLoS ONE 4, e5628 (2009).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  34. Chan, C. H., Levar, C. E., Zacharoff, L., Badalamenti, J. P. & Bond, D. R. Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens. Appl. Environ. Microbiol. 81, 7178–7186 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lefort, V., Longueville, J.-E. & Gascuel, O. SMS: smart model selection in PhyML. Mol. Biol. Evol. 34, 2422–2424 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yu, G., Smith, D. K., Zhu, H., Guan, Y. & Lam, T. T. Y. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol. Evol. 8, 28–36 (2017).

    Article  Google Scholar 

  37. R Core Team. R: a language and environment for statistical computing (R Foundation for Statistical Computing, 2013).

  38. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protocols 1, 2856–2860 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Desfosses, A., Ciuffa, R., Gutsche, I. & Sachse, C. SPRING – an image processing package for single-particle based helical reconstruction from electron cryomicrographs. J. Struct. Biol. 185, 15–26 (2014).

    Article  CAS  PubMed  Google Scholar 

  43. He, S. & Scheres, S. H. W. Helical reconstruction in RELION. J. Struct. Biol. 198, 163–176 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Egelman, E. H. The iterative helical real space reconstruction method: surmounting the problems posed by real polymers. J. Struct. Biol. 157, 83–94 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  48. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    Article  CAS  Google Scholar 

  49. Nečas, D. & Klapetek, P. Gwyddion: an open-source software for SPM data analysis. Open Phys. 10, 181–188 (2012).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

We thank L. Craig, S. Lory and Y. Xiong for discussions; E. Martz for calling our attention to the flaps in PilA-N–C, for the Supplementary Videos and for the interactive 3D visualizations; D. Lovley, K. Inoue and B. Kazmierczak for providing strains; S. Wu and M. Llaguno for help with cryo-EM; T. Lam and J. Kanyo for help with mass spectrometry analysis; Yale West Campus Imaging and Material Characterization Core; and T. Croll for help with ISOLDE. This research was supported by a Career Award at the Scientific Interfaces from Burroughs Welcome Fund (to N.S.M.), the National Institutes of Health Director’s New Innovator award (1DP2AI138259-01 to N.S.M.) and an NSF CAREER award no. 1749662 (to N.S.M.). Research was sponsored by the Defence Advanced Research Project Agency (DARPA) Army Research Office (ARO) and was accomplished under Cooperative Agreement Number W911NF-18-2-0100 (with N.S.M). This research was supported by NSF Graduate Research Fellowship award 2017224445 (to J.P.O.) and NIH Training Grant T32 GM007223, which supported V.S. Research in the laboratory of N.S.M. was also supported by the Charles H. Hood Foundation Child Health Research Award, and The Hartwell Foundation Individual Biomedical Research Award.

Author information

Author notes

  1. These authors contributed equally: Yangqi Gu, Vishok Srikanth

Authors and Affiliations

  1. Microbial Sciences Institute Yale University, West Haven, CT, USA

    Yangqi Gu, Vishok Srikanth, Aldo I. Salazar-Morales, Ruchi Jain, J. Patrick O’Brien, Sophia M. Yi, Fadel A. Samatey, Sibel Ebru Yalcin & Nikhil S. Malvankar

  2. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA

    Yangqi Gu

  3. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Vishok Srikanth, Aldo I. Salazar-Morales, Ruchi Jain, J. Patrick O’Brien, Sophia M. Yi, Fadel A. Samatey, Sibel Ebru Yalcin & Nikhil S. Malvankar

  4. Proteomics and Macromolecular Crystallography Shared Resource, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA

    Rajesh Kumar Soni

Authors
  1. Yangqi Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vishok Srikanth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aldo I. Salazar-Morales
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ruchi Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. Patrick O’Brien
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sophia M. Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rajesh Kumar Soni
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fadel A. Samatey
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sibel Ebru Yalcin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Nikhil S. Malvankar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.G. prepared and optimized cryo-EM grids, collected data used to build the atomic model, performed the image analysis, reconstructed the pili filament structure, generated and refined the filament model with help from F.A.S. and V.S., biochemically analysed filaments, performed AFM, circular dichroism, conductivity measurements, electrode fabrication and negative-staining TEM images. V.S. identified and purified pili filaments. A.I.S.-M. performed adhesion and twitching motility assays. A.I.S.-M. and R.J. carried out biochemical analyses and genetic experiments. J.P.O. grew biofilms on electrodes in microbial fuel cell. Y.G., S.M.Y. and R.K.S. carried out mass-spectrometric analyses. S.E.Y. performed AFM imaging of cell-attached filaments. N.S.M. conceived, designed and supervised the project. Y.G., V.S. and N.S.M. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Nikhil S. Malvankar.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Andreas Schramm and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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 Discovery and identification of PilA-N and PilA-C in pili.

a, b, Immunoblot of filament preparation and whole cells lysate for PilA-N (a) and PilA-C (b). M, marker. For gel source data, see Supplementary Fig. 14 and Supplementary Fig. 5 for PilA-N and PilA-C, respectively. c, d, TEM image (c) and SDS–PAGE (d) gel of PilA-NC filaments purified from ΔomcS cells. M, marker. Scale bar, 200 nm (a). For gel source data, see Supplementary Fig. 6. e, f, Immunoblot and corresponding mass spectrometry analysis for PilA-C (e) and PilA-N (f) containing band. For gel source data, see Supplementary Fig. 7.

Extended Data Fig. 2 Overexpressing PilA-N and PilA-C in wild-type G. sulfurreducens yielded pili-like filaments on the bacterial surface.

a, Immunoblot of whole-cell lysate showing overexpression of PilA-N and PilA-C under induced conditions. For gel source data, see Supplementary Fig. 8. b, c, Negative-stain TEM images of wild-type cells under uninduced (b) and induced (c) conditions. d, Zoomed image of pili-like filament shown in c. Scale bars, 200 nm (b, c), 50 nm (d).

Extended Data Fig. 3 De novo atomic model of PilA-N–C filament fit into the cryo-EM map.

a, PilA-N and PilA-C sequences. b–f, Zoomed view of the regions indicated in a for PilA-N (b) and PilA-C (c–f). g, h, Zoomed view of electron microscopy density and aromatic amino acids in PilA-N–C filament.

Extended Data Fig. 4 Contacts between PilA-N (orange) and PilA-C (cyan).

a, Pilus model. b, PilA-C translucent, showing protrusions of PilA-N. c, Heterodimer showing C-terminal residues 57–61 of PilA-N protruding to the right into PilA-C. d, Flaps of PilA-C (thick coils) enclosing protrusion of PilA-N. Four glycines (red balls) could provide hinges that may enable flaps to open. Animations are presented in Supplementary Videos 1–3.

Extended Data Fig. 5 Comparison between K. oxytoca pseudopili (PDB code 5WDA), P. aeruginosa T4P (PDB code 5XVY), G. sulfurreducens PilA-N–C filament and G. sulfurreducens PilA-N-alone filament model.

a, Helical arrangement with P and P + 4 subunits shown in the same colour. b, Hydrophobicity surface coloured from yellow (hydrophobic) to blue (hydrophilic). c, Interactions between PilA-N determines the structure as 1-start, 3-start and 4-start helix. d, Interactions between PilA-N and PilA-C, which is consistent with the studies on monomers that mutating E39 or E60 could disrupt the interactions while mutating E48 showed no disruption.

Extended Data Fig. 6 Post-translational modifications in PilA-N–C filament.

a, Lack of PTM in Y32 of PilA-N. Mass spectra of PilA-N did not show any modified peptide in AYNSAASSDLR with expected mass of 1.154 kDa. Inset, MS/MS spectra showed no modification on Y32 in PilA-N. b, Mass spectra showed methylated peptide FTLIELLVVAIIGILAAIAIPQQFSAYR with mass 3.044 kDa. Inset, MS/MS spectra showed N-terminal methylation. c, Cryo-EM map for PilA-C showing an extra density on Ser94, suggesting a post-translational modification. d, Gel of purified filaments showing glycosylated PilA-C (left lane) and positive control using horseradish peroxidase (right lane).

Extended Data Fig. 7 Geobacter sulfurreducens PilA-N–C pilus is structurally similar to a T2SS pseudopilus and does not show structure or functions of T4aP.

a, The globular domain of the PilA-N–C pilin protomer lacks hallmarks of T4P (PDB code 5XVY): disulfide bridge (green), four β-strands motif (blue) and D-region (magenta), consistent with pseudopili (PDB code 5O2Y). b, Hydrophobic interactions are the main interactions between PilA-N chains, similar to pseudopili, whereas T4P are additionally stabilized via intersubunit electrostatic interactions between F1 and E5. c, d, Comparison of bacterial adhesion to glass (c) and twitching motility (d). Error bars, s.d. (n = 3). e, f, Core aromatic residues in the theoretical model of PilA-N filament24 (e) and cryo-EM structure of PilA-N–C filament (f). g, AFM image of a PilA-N–C filament (red) bridging gold electrodes and corresponding height profile at location shown by a black line. Scale bar, 200 nm. h, i, Current–voltage curve (h) and corresponding conductivity comparison (i) for individual PilA-N–C filament versus OmcS nanowire3. Error bars, s.d. (n = 4 biological replicates).

Source data.

Extended Data Fig. 8 Thermal stability comparison between filaments of G. sulfurreducens PilA-N–C with P. aeruginosa T4P.

a, b, Circular dichroism spectra for P. aeruginosa T4P (a) and G. sulfurreducens PilA-N–C filaments (b) showing thermal denaturation. c, d, TEM images of P. aeruginosa T4P (c) and G. sulfurreducens PilA-N–C filaments (d) treated at the temperatures indicated for 5 min. Scale bars, 200 nm.

Extended Data Fig. 9 Cryo-EM reconstruction suggests that the previously claimed ‘PilA-N-alone’ filament could be DNA.

a, Cryo-EM image of filaments claimed to be PilA-N alone3. b, Cryo-EM images showed similar filaments in our OmcS filament preparations. Scale bars, 20 nm (a, b). Black arrows represent OmcS filaments in a, b. c, Two-dimensional average showed no helical features consistent with T4P. Scale bar, 5 nm. d, e, Cryo-EM density map (d) and docking with DNA molecule (e) (PDB code 1BNA), suggested the identity of the filaments to be DNA.

Extended Data Fig. 10 Expression of PilA-N–C filaments restores the secretion of OmcS and OmcZ nanowires in ΔpilA-N cells.

a, AFM images of ΔpilA-N/pilA-N-C cell showed pili on the surface of the cell. b, Zoomed-in image of ΔpilA-N/pilA-N-C cell. c, Height analysis of the pili consistent with PilA-N–C filament. d, e, ΔpilA-N/pilA-N-C cell showed the secretion of OmcS (d) and OmcZ (d) nanowires. Scale bars, 1 μm (a), 300 nm (b), 100 nm (d, e). f, Height analysis of filaments at locations shown in e showed the diameter consistent with OmcS and OmcZ filaments. g, Immunoblotting with OmcS antibody showing the restoration of secretion defect in OmcS nanowires in ΔpilA-N/pilA-N-C cells. FP, filament preparation; PP, periplasmic fraction; M, marker. For gel source data, see Supplementary Fig. 16. h, i, TEM image of filament preparation from ΔpilA-N showing no filament (h) and OmcS filaments from ΔpilA-N/pilA-N-C (i). Scale bars, 200 nm (h, i). ΔpilA-N/pilA-N-C: in-trans expression of an episomal copy of wild-type pilA-N and pilA-C in ΔpilA-N.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-16 and Supplementary Tables 1- 4.

Reporting Summary

Peer Review File

Video 1

Heterodimeric assembly of Geobacter pilin subunit. PilA-N (gold) and PilA-C (cyan) form a heterodimeric pilin in contrast to the model proposed since 2005 that Geobacter type 4 pili composed solely of PilA-N protein. Cryo-EM structure reveals that the C-terminal residues of PilA-N stabilize its copolymerization with PilA-C via electrostatic and hydrophobic interactions that position PilA-C along the outer surface of the filament. PilA-N is composed of two α-helices, linked by a short coil (Fig. 2d). A staggered helical array of PilA-N subunits forms the core of the PilA-N-C filament (Fig. 2e). PilA-C consists of four anti-parallel β-strands surrounded by a web of loops (Fig. 2d).

41586_2021_3857_MOESM5_ESM.mp4

Video 2 Geobacter PilA-N binds to PilA-C to form heterodimers. PilA-N’s C terminus (gold) protrudes into a socket in PilA-C (cyan). PilA-C binds PilA-N via electrostatic and hydrophobic interactions that stabilizes the PilA-N/PilA-C heterodimer. In addition, the C-terminal 5-residues of PilA-N protrude and are held between two "flaps" of PilA-C (Extended Data Fig. 4). The N-terminus of PilA-C (Ala1) interacts with the C-terminus of PilA-N (Ser61) via hydrogen bonding or possibly a salt bridge (Fig. 2f). As the N-terminal of PilA-N is primarily hydrophobic, binding to PilA-C prevents the exposure of the hydrophobic side chains to the aqueous environment that could increase filament stability (Fig. 2e, Extended Data Fig. 5b).

Video 3

Assembly of Geobacter pseudopilus filament. A heterodimer of PilA-N (gold) and PilA-C (cyan) polymerizes into a filament. The overall structural features of the filament are similar to T4P, with a helical core and globular head domain arranged within a right-handed helix (Fig. 2b). The electrostatic and hydrophobic interactions (Fig. 2g) appear critical for filament stability. The filament is mainly organized via the interactions between adjacent PilA-N subunits (Extended Data Fig.5a,c), with little direct interaction between PilA-C within the filament. The filament shows N-terminal methylation of PilA-N (Extended Data Fig. 6) and extra density around S94 for PilA-C for an O-linked glycosylation (Extended Data Fig. 6b-d).

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gu, Y., Srikanth, V., Salazar-Morales, A.I. et al. Structure of Geobacter pili reveals secretory rather than nanowire behaviour. Nature 597, 430–434 (2021). https://doi.org/10.1038/s41586-021-03857-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-021-03857-w

This article is cited by

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.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing