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Structure of Geobacter pili reveals secretory rather than nanowire behaviour

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.

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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.

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/3Source data are provided with this paper.

Code availability

No special software code was used to collect data.

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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

Affiliations

Authors

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.

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The authors declare no competing interests.

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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.

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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 (cf). 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 13.

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, bc, 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.

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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).

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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

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