Skip to main content

In Vitro Reconstitution of Escherichia coli O86 O Antigen Repeating Unit

  • Protocol
  • First Online:
Functional Glycomics

Abstract

Polysaccharides constitute a major component of the bacterial cell surface. They play critical roles in the interactions between bacteria and the host environments, and consequently contribute to the virulence of pathogens. The lipopolysaccharide (LPS) found on the surface of gram-negative bacteria consists of three parts: lipid A, a core oligosaccharide, and the O antigen. The O antigen is the outermost part of LPS and contains multiple oligosaccharide repeating units. Biosynthesis of the O-repeating unit is the first committed step in LPS biosynthesis. We sequenced and characterized the O-antigen biosynthetic gene cluster of Escherichia coli serotype O86. Four glycosyltransferases encoded by the genes within the cluster were cloned and overexpressed. In vitro reconstitution of the O-repeating unit of E. coli 086 was achieved via using these enzymes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Jansson, P. E., Lindberg, B., Lindberg, A. A., and Wollin, R. (1981) Structural studies on the hexose region of the core in lipopolysaccharides from Enterobacteriaceae. Eur. J. Biochem. 115, 571–577.

    Article  CAS  PubMed  Google Scholar 

  2. Raetz, C. R. and Whitfield, C. (2002) Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700.

    Article  CAS  PubMed  Google Scholar 

  3. Amer, A. O. and Valvano, M. A. (2001) Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine. Microbiology, 147, 3015–3025.

    CAS  PubMed  Google Scholar 

  4. Amer, A. O. and Valvano, M. A. (2002) Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli. Microbiology, 148, 571–582.

    CAS  PubMed  Google Scholar 

  5. Liu, D., Cole, R. A. and Reeves, P. R. (1996) An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J. Bacteriol. 178, 2102–2107.

    CAS  PubMed  Google Scholar 

  6. Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. A. (1999) The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat. J. Biol. Chem. 274, 35129–35138.

    Article  CAS  PubMed  Google Scholar 

  7. Daniels, C., Vindurampulle, C. and Morona, R. (1998) Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy). Mol. Microbiol. 28, 1211–1222.

    Article  CAS  PubMed  Google Scholar 

  8. Franco, A. V., Liu, D. and Reeves, P. R. (1996) A Wzz (Cld) protein determines the chain length of K lipopolysaccharide in Escherichia coli O8 and O9 strains. J. Bacteriol. 178, 1903–1907.

    CAS  PubMed  Google Scholar 

  9. Heinrichs, D. E., Yethon, J. A. and Whitfield, C. (1998) Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica. Mol. Microbiol. 3, 221–232.

    Article  Google Scholar 

  10. Springer, G. F., Horton, R. E., and Forbes, M. (1959) Origin of anti-human blood group B agglutinins in white Leghorn chicks. J. Exp. Med. 110, 221–244.

    Article  CAS  PubMed  Google Scholar 

  11. Kochibe, N. and Iseki, S. (1968) Immunochemical studies on bacterial blood group substances. V. Structures of B determinant groups. Jpn. J. Microbiol. 12, 403–411.

    CAS  PubMed  Google Scholar 

  12. Feizi, T. (1985) Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens. Nature 314, 53–57.

    Article  CAS  PubMed  Google Scholar 

  13. Lemieux, R. U., Bundle, D. R. and Baker, D. A. (1975) The properties of a “synthetic” antigen related to the human blood-group Lewis a. J. Am. Chem. Soc. 97, 4076–4083.

    Article  CAS  PubMed  Google Scholar 

  14. Lemieux, R. U. and Driguez, H. (1975) The chemical synthesis of 2-O-(alpha-l-fucopyranosyl)-3-O-(alpha-d-galactopyranosyl)-d-galactose. The terminal structure of the blood-group B antigenic determinant. J. Am. Chem. Soc. 97, 4069–4075.

    Article  CAS  PubMed  Google Scholar 

  15. Seto, N. O., Palcic, M. M., Hindsgaul, O., Bundle, D. R., and Narang, S. A. (1995) Expression of a recombinant human glycosyltransferase from a synthetic gene and its utilization for synthesis of the human blood group B trisaccharide. Eur. J. Biochem. 234, 323–328.

    Article  CAS  PubMed  Google Scholar 

  16. Seto, N. O., Palcic, M. M., Compston, C. A., Li, H., Bundle, D. R., and Narang, S. A. (1997) Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes. J. Biol. Chem. 272, 14133–14138.

    Article  CAS  PubMed  Google Scholar 

  17. Sim, M. M., Kondo, H., and Wong, C. H. (1993) Synthesis of dibenzyl glycosyl phosphites using dibenzyl N,N-diethylphosphoramidite as phosphitylating reagant: An effective route to glycosyl phosphates, sugar nucleotides and glycosides. J. Am. Chem. Soc. 115, 2260–2267.

    Article  CAS  Google Scholar 

  18. Montoya-Peleaza, P. J., Rileya, J. G., Szareka, W. A., Valvano, M. A., Schutzbachb, J. S., and Brockhausenb, I. (2005) Identification of a UDP-Gal: GlcNAc-R galactosyltransferase activity in Escherichia coli VW187. Bioorg. Med. Chem. Lett. 15, 1205–1211.

    Article  Google Scholar 

  19. Wacker, M., Linton, D., Hitchen, P. G., et al. (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790–1793.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

P. G. Wang acknowledges National Cancer Institute (R01 CA118208), NSF (CHE-0616892), Bill & Melinda Gates Foundation (51946) for financial support.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Humana Press

About this protocol

Cite this protocol

Han, W. et al. (2010). In Vitro Reconstitution of Escherichia coli O86 O Antigen Repeating Unit. In: Li, J. (eds) Functional Glycomics. Methods in Molecular Biology, vol 600. Humana Press. https://doi.org/10.1007/978-1-60761-454-8_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-454-8_7

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-453-1

  • Online ISBN: 978-1-60761-454-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics