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Extraction and Purification of Wall-Bound Polymers of Gram-Positive Bacteria

  • Alice Chateau
  • Olaf Schneewind
  • Dominique MissiakasEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1954)

Abstract

The envelope of gram-positive bacteria encompasses the cell wall, a rigid exoskeleton comprised of peptidoglycan that provides protection against lysis and governs bacterial cell shapes. Peptidoglycan also serves as the site of attachment for proteins and nonproteinaceous polymers that interact with the bacterial environment. Nonproteinaceous molecules include teichoic acids, capsular polysaccharides, and secondary cell wall polysaccharides (SCWP). Treatment of gram-positive bacterial cells with proteases, nucleases, and detergents results in the isolation of “murein sacculi” (i.e., peptidoglycan with bound carbohydrate polymers). Incubation of sacculi with acid or base releases carbohydrate polymers that can be purified for further biochemical characterization. This protocol describes the hydrofluoric acid extraction and purification of the secondary cell wall polymer of Bacillus anthracis that is also found in the envelope of the other members of the Bacillus cereus sensu lato group of bacteria.

Key words

Secondary cell wall polysaccharide Bacillus cereus Murein sacculus Peptidoglycan Hydrofluoric acid Phosphodiester bond 

Notes

Acknowledgments

We thank current and past members of the laboratory who helped develop and refine this protocol. Research in our laboratory is supported by grant number AI069227 from the National Institute of Allergy and Infectious Diseases, Infectious Diseases Branch.

References

  1. 1.
    Navarre WW, Schneewind O (1999) Surface proteins of gram-positive bacteria and the mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev 63:174–229PubMedPubMedCentralGoogle Scholar
  2. 2.
    Schneewind O, Fowler A, Faull KF (1995) Structure of the cell wall anchor of surface proteins in Staphylococcus aureus. Science 268:103–106CrossRefGoogle Scholar
  3. 3.
    Munoz E, Ghuysen J-M, Heymann H (1967) Cell walls of Streptococcus pyogenes type 14. C polysaccharide-peptidoglycan and G polysaccharide-peptidoglycan complexes. Biochemistry 6:3659–3670CrossRefGoogle Scholar
  4. 4.
    Chan YG, Kim HK, Schneewind O et al (2014) The capsular polysaccharide of Staphylococcus aureus is attached to peptidoglycan by the LytR-CpsA-Psr (LCP) family of enzymes. J Biol Chem 289:15680–15690CrossRefGoogle Scholar
  5. 5.
    Coley J, Archibald AR, Baddiley J (1976) A linkage unit joining peptidoglycan to teichoic acid in Staphylococcus aureus H. FEBS Lett 61:240–242CrossRefGoogle Scholar
  6. 6.
    Ries W, Hotzy C, Schocher I et al (1997) Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer. J Bacteriol 179:3892–3898CrossRefGoogle Scholar
  7. 7.
    Schneewind O, Missiakas D (2014) Sec-secretion and sortase-mediated anchoring of proteins in gram-positive bacteria. Biochim Biophys Acta 1843:1687–1697CrossRefGoogle Scholar
  8. 8.
    Kawai Y, Marles-Wright J, Cleverley RM et al (2011) A widespread family of bacterial cell wall assembly proteins. EMBO J 30:4931–4941CrossRefGoogle Scholar
  9. 9.
    Eberhardt A, Hoyland CN, Vollmer D et al (2012) Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae. Microb Drug Resist 18:240–255CrossRefGoogle Scholar
  10. 10.
    Chan YGY, Frankel MB, Dengler V et al (2013) Staphylococcus aureus mutants lacking the LytR-CpsA-Psr (LCP) family of enzymes release wall teichoic acids into the extracellular medium. J Bacteriol 195:4650–4659CrossRefGoogle Scholar
  11. 11.
    Liszewski Zilla M, Chan YG, Lunderberg JM et al (2015) LytR-CpsA-Psr enzymes as determinants of Bacillus anthracis secondary cell wall polysaccharide assembly. J Bacteriol 197:343–353CrossRefGoogle Scholar
  12. 12.
    Kojima N, Arakai Y, Ito E (1985) Structure of the linkage units between ribitol teichoic acids and peptidoglycan. J Bacteriol 161:299–306PubMedPubMedCentralGoogle Scholar
  13. 13.
    de Jonge BL, Chang YS, Gage D et al (1992) Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A. J Biol Chem 267(16):11248–11254PubMedGoogle Scholar
  14. 14.
    Choudhury B, Leoff C, Saile E et al (2006) The structure of the major cell wall polysaccharide of Bacillus anthracis is species specific. J Biol Chem 281:27932–27941CrossRefGoogle Scholar
  15. 15.
    Mesnage S, Fontaine T, Mignot T et al (2000) Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation. EMBO J 19:4473–4484CrossRefGoogle Scholar
  16. 16.
    Missiakas D, Schneewind O (2017) Assembly and function of the Bacillus anthracis S-layer. Annu Rev Microbiol 71:79–98CrossRefGoogle Scholar
  17. 17.
    Molnár J, Prágai B (1971) Attempts to detect the presence of teichoic acid in Bacillus anthracis. Acta Microbiol Acad Sci Hung 18:105–108PubMedGoogle Scholar
  18. 18.
    Leoff C, Saile E, Sue D et al (2008) Cell wall carbohydrate compositions of strains from Bacillus cereus group of species correlate with phylogenetic relatedness. J Bacteriol 190:112–121CrossRefGoogle Scholar
  19. 19.
    Forsberg LS, Choudhury B, Leoff C et al (2011) Secondary cell wall polysaccharides from Bacillus cereus strains G9241, 03BB87 and 03BB102 causing fatal pneumonia share similar glycosyl structures with the polysaccharides from Bacillus anthracis. Glycobiology 21:934–948CrossRefGoogle Scholar
  20. 20.
    Forsberg LS, Abshire TG, Friedlander A et al (2012) Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose deficient wall polysaccharide from avirulent B. anthracis CDC 684. Glycobiology 22:1103–1117CrossRefGoogle Scholar
  21. 21.
    Chateau A, Lunderberg JM, Oh SY et al (2018) Galactosylation of the secondary cell wall polysaccharide of Bacillus anthracis and its contribution to anthrax pathogenesis. J Bacteriol 200(5).  https://doi.org/10.1128/JB.00562-17
  22. 22.
    Mo KF, Li X, Li H et al (2012) Endolysins of Bacillus anthracis bacteriophages recognize unique carbohydrate epitopes of vegetative cell wall polysaccharides with high affinity and selectivity. J Am Chem Soc 134:15556–15562CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Alice Chateau
    • 1
    • 2
  • Olaf Schneewind
    • 1
    • 2
  • Dominique Missiakas
    • 1
    • 2
    Email author
  1. 1.Howard Taylor Ricketts LaboratoryArgonne National LaboratoryLemontUSA
  2. 2.Department of MicrobiologyUniversity of ChicagoChicagoUSA

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