Structure of the hexagonal surface layer on Caulobacter crescentus cells

Abstract

Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and Gram-negative bacteria and most archaea15. S-layers protect cells from the outside, provide mechanical stability and also play roles in pathogenicity. In situ structural information about this highly abundant class of proteins is scarce, so atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified Caulobacter crescentus' sole S-layer protein RsaA, we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. We also solved a 7.4 Å structure of the S-layer through electron cryotomography and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the electron cryotomography map, resulting in a pseudo-atomic-level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. The cellular S-layer atomic structure shows that the S-layer is porous, with a largest gap dimension of 27 Å, and is stabilized by multiple Ca2+ ions bound near the interfaces. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with electron cryotomography and sub-nanometre-resolution sub-tomogram averaging.

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Figure 1: Arrangement of the C. crescentus layer on cells and stalks.
Figure 2: The 2.7 Å X-ray structure of the outer S-layer lattice.
Figure 3: A 7.4 Å cryo-ET and sub-tomogram averaging map of the C. crescentus S-layer.
Figure 4: S-layer lattices observed in the X-ray structure and the cryo-ET map are exceptionally similar.

References

  1. 1

    Albers, S. V. & Meyer, B. H. The archaeal cell envelope. Nat. Rev. Microbiol. 9, 414–426 (2011).

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

    Glauert, A. M. The fine structure of bacteria. Br. Med. Bull. 18, 245–250 (1962).

    CAS  Article  Google Scholar 

  4. 4

    Sara, M. & Sleytr, U. B. S-layer proteins. J. Bacteriol. 182, 859–868 (2000).

    CAS  Article  Google Scholar 

  5. 5

    Zhu, C. et al. Diversity in S-layers. Prog. Biophys. Mol. Biol. 123, 1–15 (2017).

    Article  Google Scholar 

  6. 6

    Kirk, J. A., Banerji, O. & Fagan, R. P. Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics. Microb. Biotechnol. 10, 76–90 (2016).

    Article  Google Scholar 

  7. 7

    Sleytr, U. B. & Beveridge, T. J. Bacterial S-layers. Trends Microbiol. 7, 253–260 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Houwink, A. L. A macromolecular mono-layer in the cell wall of Spirillum spec. Biochim. Biophys. Acta 10, 360–366 (1953).

    CAS  Article  Google Scholar 

  9. 9

    Sleytr, U. B. & Glauert, A. M. Ultrastructure of the cell walls of two closely related Clostridia that possess different regular arrays of surface subunits. J. Bacteriol. 126, 869–882 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Baumeister, W., Wildhaber, I. & Phipps, B. M. Principles of organization in eubacterial and archaebacterial surface proteins. Can. J. Microbiol. 35, 215–227 (1989).

    CAS  Article  Google Scholar 

  11. 11

    Kessel, M., Wildhaber, I., Cohen, S. & Baumeister, W. Three-dimensional structure of the regular surface glycoprotein layer of Halobacterium volcanii from the Dead Sea. EMBO J. 7, 1549–1554 (1988).

    CAS  Article  Google Scholar 

  12. 12

    Lupas, A. et al. Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis. J. Bacteriol. 176, 1224–1233 (1994).

    CAS  Article  Google Scholar 

  13. 13

    Baranova, E. et al. SbsB structure and lattice reconstruction unveil Ca2+ triggered S-layer assembly. Nature 487, 119–122 (2012).

    CAS  Article  Google Scholar 

  14. 14

    Arbing, M. A. et al. Structure of the surface layer of the methanogenic archaean Methanosarcina acetivorans. Proc. Natl Acad. Sci. USA 109, 11812–11817 (2012).

    CAS  Article  Google Scholar 

  15. 15

    Jing, H. et al. Archaeal surface layer proteins contain beta propeller, PKD, and beta helix domains and are related to metazoan cell surface proteins. Structure 10, 1453–1464 (2002).

    CAS  Article  Google Scholar 

  16. 16

    Kern, J. et al. Structure of surface layer homology (SLH) domains from Bacillus anthracis surface array protein. J. Biol. Chem. 286, 26042–26049 (2011).

    CAS  Article  Google Scholar 

  17. 17

    Jiang, C., Brown, P. J., Ducret, A. & Brun, Y. V. Sequential evolution of bacterial morphology by co-option of a developmental regulator. Nature 506, 489–493 (2014).

    CAS  Article  Google Scholar 

  18. 18

    Wagner, J. K. & Brun, Y. V. Out on a limb: how the Caulobacter stalk can boost the study of bacterial cell shape. Mol. Microbiol. 64, 28–33 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Amat, F. et al. Analysis of the intact surface layer of Caulobacter crescentus by cryo-electron tomography. J. Bacteriol. 192, 5855–5865 (2010).

    CAS  Article  Google Scholar 

  20. 20

    Smit, J., Engelhardt, H., Volker, S., Smith, S. H. & Baumeister, W. The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy. J. Bacteriol. 174, 6527–6538 (1992).

    CAS  Article  Google Scholar 

  21. 21

    Ford, M. J., Nomellini, J. F. & Smit, J. S-layer anchoring and localization of an S-layer-associated protease in Caulobacter crescentus. J. Bacteriol. 189, 2226–2237 (2007).

    CAS  Article  Google Scholar 

  22. 22

    Nomellini, J. F., Kupcu, S., Sleytr, U. B. & Smit, J. Factors controlling in vitro recrystallization of the Caulobacter crescentus paracrystalline S-layer. J. Bacteriol. 179, 6349–6354 (1997).

    CAS  Article  Google Scholar 

  23. 23

    Garnham, C. P., Campbell, R. L. & Davies, P. L. Anchored clathrate waters bind antifreeze proteins to ice. Proc. Natl Acad. Sci. USA 108, 7363–7367 (2011).

    CAS  Article  Google Scholar 

  24. 24

    Ireland, M. M., Karty, J. A., Quardokus, E. M., Reilly, J. P. & Brun, Y. V. Proteomic analysis of the Caulobacter crescentus stalk indicates competence for nutrient uptake. Mol. Microbiol. 45, 1029–1041 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Bharat, T. A. M. & Scheres, S. H. W. Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION. Nat. Protoc. 11, 2054–2065 (2016).

    CAS  Article  Google Scholar 

  26. 26

    Schur, F. K. M. et al. An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation. Science 353, 506–508 (2016).

    CAS  Article  Google Scholar 

  27. 27

    Howorka, S. Rationally engineering natural protein assemblies in nanobiotechnology. Curr. Opin. Biotechnol. 22, 485–491 (2011).

    CAS  Article  Google Scholar 

  28. 28

    Mark, S. S. et al. Bionanofabrication of metallic and semiconductor nanoparticle arrays using S-layer protein lattices with different lateral spacings and geometries. Langmuir 22, 3763–3774 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Chang, Y.-W. et al. Architecture of the type IVa pilus machine. Science 351, aad2001 (2016).

    Article  Google Scholar 

  30. 30

    Poindexter, J. S. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28, 231–295 (1964).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Stock, D., Perisic, O. & Löwe, J. Robotic nanolitre protein crystallisation at the MRC Laboratory of Molecular Biology. Prog. Biophys. Mol. Biol. 88, 311–327 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011).

    CAS  Article  Google Scholar 

  34. 34

    Sheldrick, G. M. in Direct Methods for Solving Macromolecular Structures (ed. Fortier, S. ) 401–411 (Springer, 1998).

    Google Scholar 

  35. 35

    McCoy, A. J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D 63, 32–41 (2007).

    CAS  Article  Google Scholar 

  36. 36

    Cowtan, K. Recent developments in classical density modification. Acta Crystallogr. D 66, 470–478 (2010).

    CAS  Article  Google Scholar 

  37. 37

    Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006).

    Article  Google Scholar 

  38. 38

    Turk, D. MAIN software for density averaging, model building, structure refinement and validation. Acta Crystallogr. D 69, 1342–1357 (2013).

    CAS  Article  Google Scholar 

  39. 39

    Adams, P. D. et al. PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  40. 40

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    CAS  Article  Google Scholar 

  41. 41

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  Google Scholar 

  42. 42

    Hagen, W. J. H., Wan, W. & Briggs, J. A. G. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191–198 (2016).

    Article  Google Scholar 

  43. 43

    Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    CAS  Article  Google Scholar 

  44. 44

    Bharat, T. A. M. et al. Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells. PLoS Biol. 9, e1001196 (2011).

    CAS  Article  Google Scholar 

  45. 45

    Bharat, T. A. M., Russo, C. J., Löwe, J., Passmore, L. A. & Scheres, S. H. W. Advances in single-particle electron cryomicroscopy structure determination applied to sub-tomogram averaging. Structure 23, 1743–1753 (2015).

    CAS  Article  Google Scholar 

  46. 46

    Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  Google Scholar 

  47. 47

    Briggs, J. A. et al. Structure and assembly of immature HIV. Proc. Natl Acad. Sci. USA 106, 11090–11095 (2009).

    CAS  Article  Google Scholar 

  48. 48

    Förster, F., Medalia, O., Zauberman, N., Baumeister, W. & Fass, D. Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography. Proc. Natl Acad. Sci. USA 102, 4729–4734 (2005).

    Article  Google Scholar 

  49. 49

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  Article  Google Scholar 

  50. 50

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

    CAS  Article  Google Scholar 

  51. 51

    Söding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005).

    Article  Google Scholar 

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Acknowledgements

The authors thank M. Skehel and F. Begum (MRC Laboratory of Molecular Biology, LMB) for mass-spectrometric identification of proteins, M. Yu (LMB) for help with X-ray data collection, C. Savva (LMB) for help with cryo-EM data collection, S. Scheres (LMB) for help with RELION software, F. Schur and W. Wan (European Molecular Biology Laboratory, EMBL) for providing high-resolution image-processing code and scripts before publication and for advice on their implementation, F. van den Ent (LMB) for advice on protein purification and T. Darling and J. Grimmett (LMB) for help with high-performance computing. The authors also thank Y. Modis (Cambridge University) for pointing out the similarity to anti-freeze proteins. Part of this work was funded by the European Molecular Biology Organization (aALTF 778-2015 to T.A.M.B.), the Medical Research Council (U105184326 to J.L.), the Wellcome Trust (095514/Z/11/Z to J.L.) and the National Institutes of Health (GM51986 to Y.V.B.). This work was supported by iNEXT, project no. 1482, funded by the Horizon 2020 programme of the European Union.

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Authors

Contributions

T.A.M.B., Y.V.B. and J.L. designed the research. T.A.M.B., D.K.-C., G.G.H., E.W.Y., J.M.D., W.J.H.H. and J.L. performed experiments. W.J.H.H. and J.A.G.B. supported high-resolution cryo-ET and image processing. TA.M.B., D.K.-C., J.M.D., E.W.Y. and J.L. analysed the data. T.A.M.B. and J.L. wrote the manuscript with support from all authors.

Corresponding authors

Correspondence to Tanmay A. M. Bharat or Jan Löwe.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2; Supplementary Figures 1–7; legends for Supplementary Videos (PDF 2780 kb)

Supplementary Video 1

Whole cell cryo-ET of a C. crescentus cell (related to Figure 1). (AVI 21817 kb)

Supplementary Video 2

Cryo-ET of reconstituted 2D sheets of RsaA (related to Figure 2). (AVI 18787 kb)

Supplementary Video 3

The X-ray structure and map of a RsaA monomer (related to Figure 2). (MOV 35083 kb)

Supplementary Video 4

X-ray crystallography structure of the outer S-layer lattice (related to Figure 2). (MOV 35508 kb)

Supplementary Video 5

Cryo-ET of a C. crescentus stalk (related to Figure 3). (AVI 5543 kb)

Supplementary Video 6

Fit of the X-ray structure of the RsaA hexamer into the sub-tomogram averaging map (related to Figures 3,4). (AVI 28218 kb)

Supplementary Video 7

The structure and arrangement of the S-layer on cells (related to Figure 4). (AVI 46055 kb)

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Bharat, T., Kureisaite-Ciziene, D., Hardy, G. et al. Structure of the hexagonal surface layer on Caulobacter crescentus cells. Nat Microbiol 2, 17059 (2017). https://doi.org/10.1038/nmicrobiol.2017.59

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