Article

Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ

  • Nature Communications 4, Article number: 2856 (2013)
  • doi:10.1038/ncomms3856
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Abstract

Chlamydiae are important pathogens and symbionts with unique cell biological features. They lack the cell-division protein FtsZ, and the existence of peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG together function in orchestrating cell division and maintaining cell shape in almost all other bacteria. Using electron cryotomography, mass spectrometry and fluorescent labelling dyes, here we show that some environmental chlamydiae have cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential.

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Change history

  • Updated online 03 December 2013

    The HTML version of this Article previously published did not acknowledge Grant J. Jensen as a corresponding author. This has now been corrected in the HTML; the PDF version of the paper was correct from the time of publication.

References

  1. 1.

    & The Planctomycetes, Verrucomicrobia, Chlamydiae and sister phyla comprise a superphylum with biotechnological and medical relevance. Curr. Opin. Biotechnol. 17, 241–249 (2006).

  2. 2.

    & Evolution. Intermediate steps. Science 330, 1187–1188 (2010).

  3. 3.

    & Transitional forms between the three domains of life and evolutionary implications. Proc. Biol. Sci. 278, 3321–3328 (2011).

  4. 4.

    , & FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one. Microbiol. Mol. Biol. Rev. 74, 504–528 (2010).

  5. 5.

    et al. Characterization and evolution of cell division and cell wall synthesis genes in the bacterial phyla Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes and phylogenetic comparison with rRNA genes. J. Bacteriol. 190, 3192–3202 (2008).

  6. 6.

    , & Cell division in a minimal bacterium in the absence of ftsZ. Mol. Microbiol. 78, 278–289 (2010).

  7. 7.

    , , , & Life without a wall or division machine in Bacillus subtilis. Nature 457, 849–853 (2009).

  8. 8.

    & Preparation and chemical composition of the cell walls of mature infectious dense forms of meningopneumonitis organisms. J. Bacteriol. 94, 1178–1183 (1967).

  9. 9.

    & Preparation and chemical composition of the cell membranes of developmental reticulate forms of meningopneumonitis organisms. J. Bacteriol. 94, 1184–1188 (1967).

  10. 10.

    , & Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31, 1161–1176 (1981).

  11. 11.

    et al. Muramic acid is not detectable in Chlamydia psittaci or Chlamydia trachomatis by gas chromatography-mass spectrometry. Infect. Immun. 58, 835–837 (1990).

  12. 12.

    , , , & Chlamydia trachomatis has penicillin-binding proteins but not detectable muramic acid. J. Bacteriol. 151, 420–428 (1982).

  13. 13.

    & Building the invisible wall: updating the chlamydial peptidoglycan anomaly. Trends. Microbiol. 14, 70–77 (2006).

  14. 14.

    & Cell-wall constituents of Rickettsiae and Psittacosis-Lymphogranuloma organisms. J. Gen. Microbiol. 30, 469–480 (1963).

  15. 15.

    , & A search for the bacterial mucopeptide component, muramic acid, in Chlamydia. J. Gen. Microbiol. 80, 315–318 (1974).

  16. 16.

    Why is Chlamydia sensitive to penicillin in the absence of peptidoglycan? Infect. Agents. Dis. 2, 87–99 (1993).

  17. 17.

    Recent progress of electron microscopy in microbiology and its development in future: from a study of the obligate intracellular parasites, chlamydia organisms. J. Electron. Microsc. 28, 57–64 (1979).

  18. 18.

    , , & Electron microscopic observations on the structure of the envelopes of mature elementary bodies and developmental reticulate forms of Chlamydia psittaci. J. Bacteriol. 105, 355–360 (1971).

  19. 19.

    et al. Cryo-electron tomography of Chlamydia trachomatis gives a clue to the mechanism of outer membrane changes. J. Electron. Microsc. 59, 237–241 (2010).

  20. 20.

    & Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity. Mol. Microbiol. 57, 41–52 (2005).

  21. 21.

    , & In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance. J. Bacteriol. 185, 1218–1228 (2003).

  22. 22.

    et al. Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity. J. Bacteriol. 185, 6507–6512 (2003).

  23. 23.

    , , , & Functional and biochemical analysis of the Chlamydia trachomatis ligase MurE. J. Bacteriol. 191, 7430–7435 (2009).

  24. 24.

    , , , & Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem. Arch. Microbiol. 194, 505–512 (2012).

  25. 25.

    et al. Functional conservation of the lipid II biosynthesis pathway in the cell wall-less bacteria Chlamydia and Wolbachia: why is lipid II needed? Mol. Microbiol. 73, 913–923 (2009).

  26. 26.

    Chlamydiae as symbionts in eukaryotes. Annu. Rev. Microbiol. 62, 113–131 (2008).

  27. 27.

    , & Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure. Proc. Natl Acad. Sci. USA 109, 8959–8964 (2012).

  28. 28.

    , & Electron cryotomography. Cold Spring Harb. Perspect. Biol. 2, a003442 (2010).

  29. 29.

    et al. Cellular architecture of Treponema pallidum: novel flagellum, periplasmic cone, and cell envelope as revealed by cryo electron tomography. J. Mol. Biol. 403, 546–561 (2010).

  30. 30.

    et al. Peptidoglycan remodeling and conversion of an inner membrane into an outer membrane during sporulation. Cell 146, 799–812 (2011).

  31. 31.

    Disulfide cross-linked envelope proteins: the functional equivalent of peptidoglycan in chlamydiae? J. Bacteriol. 178, 1–5 (1996).

  32. 32.

    Separation and quantification of muropeptides with high-performance liquid chromatography. Anal. Biochem. 172, 451–464 (1988).

  33. 33.

    & Abnormal peptidoglycan produced in a methicillin-resistant strain of Staphylococcus aureus grown in the presence of methicillin: functional role for penicillin-binding protein 2A in cell wall synthesis. Antimicrob. Agents. Chemother. 37, 342–346 (1993).

  34. 34.

    et al. The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development. J. Bacteriol. 191, 494–505 (2009).

  35. 35.

    et al. In Situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angew. Chem. 51, 12519–12523 (2012).

  36. 36.

    & Electron microscopic observations on the effects of penicillin on the morphology of Chlamydia psittaci. J. Bacteriol. 101, 278–285 (1970).

  37. 37.

    et al. Penicillin induced persistence in Chlamydia trachomatis: high quality time lapse video analysis of the developmental cycle. PLoS One 4, e7723 (2009).

  38. 38.

    , & Persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis. Microbiol. Rev. 58, 686–699 (1994).

  39. 39.

    , & Antibiotic susceptibilities of Parachlamydia acanthamoeba in amoebae. Antimicrob. Agents Chemother. 46, 3065–3067 (2002).

  40. 40.

    , , , & Description and partial characterization of a new chlamydia-like microorganism. FEMS. Microbiol. Lett. 109, 329–334 (1993).

  41. 41.

    & Identification of an antigen localized to an apparent septum within dividing chlamydiae. Infect. Immun. 68, 708–715 (2000).

  42. 42.

    et al. Illuminating the evolutionary history of chlamydiae. Science 304, 728–730 (2004).

  43. 43.

    et al. Unity in variety—the Pan-genome of the Chlamydiae. Mol. Biol. Evol. 28, 3253–3270 (2011).

  44. 44.

    & Peptidoglycan synthesis in the absence of Class A penicillin-binding proteins in Bacillus subtilis. J. Bacteriol. 185, 1423–1431 (2003).

  45. 45.

    et al. Role of class A penicillin-binding proteins in PBP5-mediated β-lactam resistance in Enterococcus faecalis. J. Bacteriol. 186, 1221–1228 (2004).

  46. 46.

    , & Phylogeny of Prosthecobacter, the fusiform caulobacters: members of a recently discovered division of the Bacteria. Int. J. Syst. Bacteriol. 46, 960–966 (1996).

  47. 47.

    et al. Diversity of bacterial endosymbionts of environmental Acanthamoeba isolates. Appl. Environ. Microbiol. 74, 5822–5831 (2008).

  48. 48.

    et al. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919–1925 (1990).

  49. 49.

    An Illustrated Key to Freshwater and Soil Amoebae Freshwater Biological Association (1976).

  50. 50.

    , & An improved cryogen for plunge freezing. Microsc. Microanal. 14, 375–379 (2008).

  51. 51.

    et al. Electron cryotomography sample preparation using the Vitrobot. Nat. Protocols 1, 2813–2819 (2006).

  52. 52.

    et al. UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction. J. Struct. Biol. 157, 138–147 (2007).

  53. 53.

    , & Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 1996/01/01, 71–76 (1996).

  54. 54.

    et al. Markov random field based automatic image alignment for electron tomography. J. Struct. Biol. 161, 260–275 (2008).

  55. 55.

    et al. Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens. PLoS One 8, e55010 (2013).

  56. 56.

    et al. Peptidoglycan-modifying enzyme Pgp1 is required for helical cell shape and pathogenicity traits in Campylobacter jejuni. PLoS. Pathog. 8, e1002602 (2012).

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Acknowledgements

This work was funded by the Austrian Science Fund FWF (Y277-B03 to M.H.), the European Research Council (ERC StG ‘EvoChlamy’ to M.H.), NIH grant GM094800B (to G.J.J.), the Caltech Center for Environmental Microbiology Interactions (to G.J.J., M.P.), a gift from the Gordon and Betty Moore Foundation to Caltech, the BBSRC (BB/I020012/1 to W.V.), NIH grant AI059327 (to M.S.V.) and NIH grant GM051986 (to Y.V.B.). We thank Elitza Tocheva for discussions on the preparation of sacculi.

Author information

Author notes

    • Martin Pilhofer
    •  & Karin Aistleitner

    These authors contributed equally to this work

Affiliations

  1. Division of Biology, California Institute of Technology, Pasadena, California 91125, USA

    • Martin Pilhofer
    •  & Grant J. Jensen
  2. Howard Hughes Medical Institute, Pasadena, California 91125, USA

    • Martin Pilhofer
    •  & Grant J. Jensen
  3. Division of Microbial Ecology, University of Vienna, Vienna, A-1090, Austria

    • Karin Aistleitner
    •  & Matthias Horn
  4. Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell Biology, Newcastle University, Newcastle upon Tyne, NE2 4AX, UK

    • Jacob Biboy
    •  & Waldemar Vollmer
  5. Institute for Cell and Molecular Biosciences, Pinnacle Laboratory, Newcastle University, Newcastle upon Tyne, NE2 4AX, UK

    • Joe Gray
  6. Indiana University, Bloomington, Indiana 47405, USA

    • Erkin Kuru
    • , Edward Hall
    • , Yves V. Brun
    •  & Michael S. VanNieuwenhze

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Contributions

M.P. initiated the study. M.P. and K.A. performed all experiments except HPLC/MS analyses of sacculi, which were done by J.B., J.G. and W.V. E.K., E.H., Y.V.B. and M.S.V. provided the FDAA dyes and advice on the FDAA labelling experiments. M.P., K.A., W.V., M.H. and G.J.J. designed the experiments and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Matthias Horn or Grant J. Jensen.

Supplementary information

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    Supplementary Information

    Supplementary Figures S1-S4 and Supplementary Table S1

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