Abstract
Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.
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Data availability
The cryo-EM volumes have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-10893 and EMD-10894, and the coordinates have been deposited in the Protein Data Bank (PDB) with accession code 6YS8. Source data are provided with the paper.
References
Guo, H. & Rubinstein, J. L. Cryo-EM of ATP synthases. Curr. Opin. Struct. Biol. 52, 71–79 (2018).
Walker, J. E. The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1–16 (2013).
Sowa, Y. & Berry, R. M. Bacterial flagellar motor. Q. Rev. Biophys. 41, 103–132 (2008).
Kojima, S. Dynamism and regulation of the stator, the energy conversion complex of the bacterial flagellar motor. Curr. Opin. Microbiol. 28, 66–71 (2015).
Deme, J. C. et al. Structures of the stator complex that drives rotation of the bacterial flagellum. Nat. Microbiol. 5, 1553–1564 (2020).
McBride, M. J. Bacteroidetes gliding motility and the type IX secretion system. Microbiol. Spectr. 7, PSIB-0002-2018 (2019).
Nakane, D., Sato, K., Wada, H., McBride, M. J. & Nakayama, K. Helical flow of surface protein required for bacterial gliding motility. Proc. Natl Acad. Sci. USA 110, 11145–11150 (2013).
Ridgway, H. F. Source of energy for gliding motility in Flexibacter polymorphus: effects of metabolic and respiratory inhibitors on gliding movement. J. Bacteriol. 131, 544–556 (1977).
Shrivastava, A., Lele, P. P. & Berg, H. C. A rotary motor drives Flavobacterium gliding. Curr. Biol. 25, 338–341 (2015).
Shrivastava, A., Roland, T. & Berg, H. C. The screw-like movement of a gliding bacterium is powered by spiral motion of cell-surface adhesins. Biophys. J. 111, 1008–1013 (2016).
Nelson, S. S., Bollampalli, S. & McBride, M. J. SprB is a cell surface component of the Flavobacterium johnsoniae gliding motility machinery. J. Bacteriol. 190, 2851–2857 (2008).
Shrivastava, A. & Berg, H. C. A molecular rack and pinion actuates a cell-surface adhesin and enables bacterial gliding motility. Sci. Adv. 6, eaay6616 (2020).
Shrivastava, A., Johnston, J. J., van Baaren, J. M. & McBride, M. J. Flavobacterium johnsoniae GldK, GldL, GldM, and SprA are required for secretion of the cell surface gliding motility adhesins SprB and RemA. J. Bacteriol. 195, 3201–3212 (2013).
Vincent, M. S. et al. Characterization of the Porphyromonas gingivalis type IX secretion trans-envelope PorKLMNP core complex. J. Biol. Chem. 292, 3252–3261 (2017).
Leone, P. et al. Type IX secretion system PorM and gliding machinery GldM form arches spanning the periplasmic space. Nat. Commun. 9, 429 (2018).
Sato, K. et al. A protein secretion system linked to bacteroidete gliding motility and pathogenesis. Proc. Natl Acad. Sci. USA 107, 276–281 (2010).
Lasica, A. M., Ksiazek, M., Madej, M. & Potempa, J. The type IX secretion system (T9SS): highlights and recent insights into its structure and function. Front. Cell Infect. Microbiol 7, 215 (2017).
de Diego, I. et al. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain. Sci. Rep. 6, 23123 (2016).
Kulkarni, S. S., Zhu, Y., Brendel, C. J. & McBride, M. J. Diverse C-terminal sequences involved in Flavobacterium johnsoniae protein secretion. J. Bacteriol. 199, e00884-16 (2017).
Lauber, F., Deme, J. C., Lea, S. M. & Berks, B. C. Type 9 secretion system structures reveal a new protein transport mechanism. Nature 564, 77–82 (2018).
Nijeholt, J. A. L. A. & Driessen, A. J. M. The bacterial Sec-translocase: structure and mechanism. Philos. Trans. R. Soc. B Biol. Sci. 367, 1016–1028 (2012).
Gorasia, D. G. et al. Structural insights into the PorK and PorN components of the Porphyromonas gingivalis type IX secretion system. PLoS Pathog. 12, e1005820 (2016).
Gorasia, D. G. et al. Structure and organisation of the type IX secretion system. Preprint at bioRxiv https://doi.org/10.1101/2020.05.13.094771 (2020).
Rhodes, R. G., Samarasam, M. N., Van Groll, E. J. & McBride, M . J. Mutations in Flavobacterium johnsoniae sprE result in defects in gliding motility and protein secretion. J. Bacteriol. 193, 5322–5327 (2011).
Sato, K., Okada, K., Nakayama, K. & Imada, K. PorM, a core component of bacterial type IX secretion system, forms a dimer with a unique kinked-rod shape. Biochem. Biophys. Res. Commun. 532, 114–119 (2020).
von Heijne, G. & Gavel, Y. Topogenic signals in integral membrane proteins. Eur. J. Biochem. 174, 671–678 (1988).
Turner, R. D., Hurd, A. F., Cadby, A., Hobbs, J. K. & Foster, S. J. Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture. Nat. Commun. 4, 1496 (2013).
Turner, R. D., Mesnage, S., Hobbs, J. K. & Foster, S. J. Molecular imaging of glycan chains couples cell-wall polysaccharide architecture to bacterial cell morphology. Nat. Commun. 9, 1263 (2018).
Koraimann, G. Lytic transglycosylases in macromolecular transport systems of Gram-negative bacteria. Cell. Mol. Life Sci. 60, 2371–2388 (2003).
Scheurwater, E. M. & Burrows, L. L. Maintaining network security: how macromolecular structures cross the peptidoglycan layer. FEMS Microbiol. Lett. 318, 1–9 (2011).
Vincent, M. S., Durand, E. & Cascales, E. The PorX response regulator of the Porphyromonas gingivalis PorXY two-component system does not directly regulate the type IX secretion genes but binds the PorL subunit. Front. Cell. Infect. Microbiol. 6, 96 (2016).
Celia, H. et al. Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry. Commun. Biol. 2, 358 (2019).
McBride, M. J. & Kempf, M. J. Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae. J. Bacteriol. 178, 583–590 (1996).
Liu, J., McBride, M. J. & Subramaniam, S. Cell surface filaments of the gliding bacterium Flavobacterium johnsoniae revealed by cryo-electron tomography. J. Bacteriol. 189, 7503–7506 (2007).
Agarwal, S., Hunnicutt, D. W. & McBride, M. J. Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA. Proc. Natl Acad. Sci. USA 94, 12139–12144 (1997).
Dietsche, T. et al. Structural and functional characterization of the bacterial type III secretion export apparatus. PLoS Pathog. 12, e1006071 (2016).
Drew, D., Lerch, M., Kunji, E., Slotboom, D. J. & de Gier, J. W. Optimization of membrane protein overexpression and purification using GFP fusions. Nat. Methods 3, 303–313 (2006).
Zhu, Y. et al. Genetic analyses unravel the crucial role of a horizontally acquired alginate lyase for brown algal biomass degradation by Zobellia galactanivorans. Environ. Microbiol. 19, 2164–2181 (2017).
Jacobus, A. P. & Gross, J. Optimal cloning of PCR fragments by homologous recombination in Escherichia coli. PLoS ONE 10, e0119221 (2015).
Thanbichler, M., Iniesta, A. A. & Shapiro, L. A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus. Nucleic Acids Res. 35, e137 (2007).
Simon, R., Priefer, U. & Puhler, A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Nat. Biotechnol. 1, 784–791 (1983).
Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor, 1972).
Tartof, K. D. A. & Hobbs, C. A. Improved media for growing plasmid and cosmid clones. Bethesda Res. Lab. Focus 9, 12 (1987).
Baumgarten, T. et al. Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3). Sci. Rep. 7, 45089 (2017).
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).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Reboul, C. F., Eager, M., Elmlund, D. & Elmlund, H. Single-particle cryo-EM-improved ab initio 3D reconstruction with SIMPLE/PRIME. Protein Sci. 27, 51–61 (2018).
Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–W350 (2016).
Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
Grimm, J. B., Brown, T. A., English, B. P., Lionnet, T. & Lavis, L. D. Synthesis of Janelia Fluor HaloTag and SNAP-tag ligands and their use in cellular imaging experiments. Methods Mol. Biol. 1663, 179–188 (2017).
Grimm, J. B. et al. Bright photoactivatable fluorophores for single-molecule imaging. Nat. Methods 13, 985–988 (2016).
Chang, L. Y. E. & Pate, J. L. Nutritional-requirements of Cytophaga johnsonae and some of its auxotrophic mutants. Curr. Microbiol. 5, 235–240 (1981).
The R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2017).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Kamisetty, H., Ovchinnikov, S. & Baker, D. Assessing the utility of coevolution-based residue–residue contact predictions in a sequence- and structure-rich era. Proc. Natl Acad. Sci. USA 110, 15674–15679 (2013).
Acknowledgements
We thank M. McBride and Y. Zhu for providing reagents for the genetic manipulation of F. johnsoniae. We thank L. Lavis for supplying the Janelia Fluor ligands, S. Hickman for advice on fluorescence imaging and A. Wainman for assistance with phase contrast imaging. We thank P. Guy for his involvement in producing the pulse-chase mCherry–CTD fusion construct and R. Berry for discussions. We acknowledge the use of the Central Oxford Structural Microscopy and Imaging Centre (COSMIC) and the Oxford Micron Advanced Imaging Facility. We thank K. Foster for providing additional imaging facilities. This work was supported by Wellcome Trust studentships to R.H.J. and A.S. (grant no. 1009136), a Biotechnology and Biological Sciences Research Council studentship to A.K. and Wellcome Trust Investigator Awards (grant nos. 107929/Z/15/Z and 100298/Z/12/Z). COSMIC was supported by a Wellcome Trust Collaborative Award (grant no. 201536/Z/16/Z), the Wolfson Foundation, a Royal Society Wolfson Refurbishment Grant, the John Fell Fund, and the EPA and Cephalosporin Trusts.
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Contributions
R.H.J. carried out all genetic and biochemical work except as credited otherwise. J.C.D. and S.M.L. collected electron microscopy data and determined the structures. A.K. developed and carried out the SprB tracking experiments including strain construction. F.A. carried out the pulse chase analysis of protein export. A.S. assayed cellular ATP levels. F.L. constructed the ∆gldL strain and produced the GldL and GldM antibodies. B.C.B. and S.M.L. conceived the project. All authors interpreted data and wrote the manuscript.
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Extended data
Extended Data Fig. 1 Purification of the GldLM’ and PorLM complexes.
Coomassie Blue-stained SDS-PAGE gels and size-exclusion column traces of complexes used for cryoEM structure determination. In each case the size exclusion column fractions that were pooled to make the cryoEM grids are denoted by an arrow and correspond to the fractions analysed on the gels. A280 is the absorption at 280 nm in arbitrary units. a, GldLM225(produced from pRHJ007). b, GldLM232 (produced from pRHJ008). c, PorLM (produced from pRHJ006).
Extended Data Fig. 2 Cryo-EM workflow for GldLM′.
a, Exemplar micrograph collected at a defocus value of approximately −2.5 μm, Scale bar is 200 Å. b, Image processing workflow for GldLM’. Scale bar is 100 Å. c, Directional Fourier Shell Correlation (FSC) plot for GldLM′ calculated using the 3DFSC server52. d, Particle Orientation Distributions from final refined volume. Rotation (phi) is plotted along the x axis and tilt (theta) on the y axis. The area of the dot at the angular position is proportional to the number of particles assigned to that view.
Extended Data Fig. 3 Cryo-EM workflow for PorLM.
a, Image processing workflow for PorLM. Scale bar is 100 Å. b, Fourier Shell Correlation (FSC) plot for PorLM Red, phase randomised; Green, unmasked; Blue, masked; Black, corrected. c, Local resolution histogram of PorLM volume. d, Particle Orientation Distributions from final refined volume. Rotation (phi) is plotted along the x axis and tilt (theta) on the y axis. The area of the dot at the angular position is proportional to the number of particles assigned to that view.
Extended Data Fig. 4 Cryo-EM map quality and resolution estimates.
a, Fourier Shell Correlation (FSC) plot for the GldLM′ structure. The resolution at the gold-standard cutoff (FSC = 0.143) is indicated by the dashed line. Curves: Red, phase-randomized; Green, unmasked; blue, masked; black, MTF-corrected. b, Local resolution estimates (in Å) for the sharpened GldLM′ map. c, Representative modelled densities.
Extended Data Fig. 5
Cryo-EM data collection, refinement and validation statistics.
Extended Data Fig. 6 Contacts between the periplasmic loops of GldL and GldM.
The PDBePISA server (http://www.ebi.ac.uk/pdbe/pisart.html)60 was used to calculate the sizes of the interfaces between the periplasmic loops of each copy of GldL and the periplasmic domains of GldM. The periplasmic loops of GldL were taken to include residues 29–41.
Extended Data Fig. 7 Conservation analysis of the GldLM′ complex. a-c, Sequence conservation assessed using Consurf57.
a, The whole complex in cartoon representation. The chains shown individually in b and c are indicated with black arrows. b,c, GldM (chain B) and GldL (chain F) in surface representation. The left hand panels show the chains in the same orientation as the upper panel of a. d, Co-varying residue pairs in the first periplasmic domain of GldM identified using the Gremlin server61. The highest-scoring pairs (score cut-off 1.63) are shown on the structure of the GldLM′ complex. Contacts with a minimum atom-atom distance of <5 Å are shown in green, and 5–10 Å in yellow. Note, that no high-scoring GldM-GldM intermolecular pairs were observed for D1. Conversely dimerization of the other periplasmic domains is supported by co-variance with the following strongly co-varying pairs identified in the other domains D2:Val253-Ile284, Phe248-Glu-293, Tyr236-Arg261; D3: Thr327-Ala349, Asp331-Lys371; D4: Ala456-Asp483.
Extended Data Fig. 8 The full length PorLM volume is compatible with the shape of GldLM′ but not with the domain arrangements of earlier crystallographic structures of GldM (6ey4,) or PorM (6ey5) fragments.
The PorLM volume is contoured at 2.5 s and the upper and lower panels are related by a 90o rotation about the y axis. The approximate domain boundaries are indicated by the dashed lines and domains in these regions are labelled on the right hand side of the figure. The cryoEM structure of GldLM′ is positioned by docking the GldM′ TMHs onto the PorM TMHs as described in the text and Fig. 3. The D1 dimer of GldM 6ey415 is docked into the D1 portion of the volume and shows (1) poor fit within D1 because the centre of the closed crystallographic dimer protrudes from the volume and (2) poor fit in the D2-D4 region when no bend is introduced between D1-D2. PorM 6ey515 is positioned by placement of D2 into its portion of the volume and shows a poor fit for D3-4 which protrude from the volume. The hybrid model presented in Fig. 3 is shown for comparison, with GldM coloured as in other panels and GldL chains coloured individually.
Extended Data Fig. 9 Gliding motility and T9SS phenotypes of mutant strains.
Footnotes for table:1 Data from Fig. 4a.2 Activity of the major T9SS substrate chitinase in the extracellular medium. Includes data shown in Fig. 4b.3+ ++, spreads as well as the parental strain; ++ spreads less well than the parental strain; + residual spreading; - does not spread. Includes data shown in Fig. 4c. 4 Includes data from Supplementary Movie 1. Note that only strains possessing cell surface adhesins are able to adhere to glass slides, and that adhesin export to the cell surface requires the T9SS which in turn depends on functional GldLM. 5Data from Fig. 4d.
Extended Data Fig. 10 Closeup views of density around side chains discussed in manuscript.
Cryo-EM volumes contoured at 3 sigma. Key side chains are shown for both copies of GldM and for the GldL chain involved in the putative GldLE49 – GldMR9 salt-bridge.
Supplementary information
Supplementary Information
Supplementary Fig. 1, Supplementary Tables 1–3 and a list of Supplementary Videos 1–8.
Supplementary Video 1
Mutant strains gliding on glass. Wild-type, gldLY13A, gldLY13F, gldLK27A, gldLH30A, gldLE49D, gldMR9K, gldMY17A and gldMY17F cells are shown.
Supplementary Video 2
Fluorophore-labelled SprB adhesin moving on the surface of a wild-type cell.
Supplementary Video 3
Fluorophore-labelled SprB adhesin moving on the surface of a gldLY13A cell.
Supplementary Video 4
Fluorophore-labelled SprB adhesin moving on the surface of a gldLK27A cell.
Supplementary Video 5
Fluorophore-labelled SprB adhesin moving on the surface of a gldLH30A cell.
Supplementary Video 6
Fluorophore-labelled SprB adhesin moving on the surface of a gldMR9K cell.
Supplementary Video 7
Fluorophore-labelled SprB adhesin moving on the surface of a gldMY17F cell.
Supplementary Video 8
Animation of a rotary model of GldLM mechanism.
Source data
Source Data Fig. 1
Uncropped gels for Fig. 1.
Source Data Fig. 4
Uncropped gels for Fig. 4.
Source Data Fig. 4
Uncropped motility plates for Fig. 4.
Source Data Fig. 5
Uncropped gels for Fig. 5.
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Hennell James, R., Deme, J.C., Kjӕr, A. et al. Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility. Nat Microbiol 6, 221–233 (2021). https://doi.org/10.1038/s41564-020-00823-6
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DOI: https://doi.org/10.1038/s41564-020-00823-6
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