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In Vitro Peptidoglycan Synthesis Assay with Lipid II Substrate

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Bacterial Cell Surfaces

Part of the book series: Methods in Molecular Biology ((MIMB,volume 966))

Abstract

Bacterial cell wall peptidoglycan is synthesized from lipid II precursor by two reactions. Glycosyltransferases polymerize the glycan chains and transpeptidases form the peptide cross-links. The bifunctional class A penicillin-binding proteins catalyze both of these reactions. Here, we describe an in vitro peptidoglycan synthesis assay utilizing radiolabeled lipid II substrate to monitor simultaneously peptidoglycan glycosyltransferase and transpeptidase activities. The products of the reaction are separated by high-pressure liquid chromatography and quantified by flow-through scintillation counting.

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References

  1. Vollmer W, Blanot D, de Pedro MA (2008) Peptidoglycan structure and architecture. FEMS Microbiol Rev 32:149–167

    Article  PubMed  CAS  Google Scholar 

  2. Lovering AL, de Castro LH, Lim D, Strynadka NC (2007) Structural insight into the transglycosylation step of bacterial cell-wall biosynthesis. Science 315:1402–1405

    Article  PubMed  CAS  Google Scholar 

  3. Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P (2008) The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev 32:234–258

    Article  PubMed  CAS  Google Scholar 

  4. Vollmer W, Bertsche U (2008) Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. Biochim Biophys Acta 1778:1714–1734

    Article  PubMed  CAS  Google Scholar 

  5. Höltje J-V (1998) Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 62:181–203

    PubMed  Google Scholar 

  6. Cabeen MT, Jacobs-Wagner C (2010) The bacterial cytoskeleton. Annu Rev Genet 44:365–392

    Article  PubMed  CAS  Google Scholar 

  7. Bertsche U, Kast T, Wolf B, Fraipont C, Aarsman ME, Kannenberg K, von Rechenberg M, Nguyen-Disteche M, den Blaauwen T, Höltje J-V, Vollmer W (2006) Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli. Mol Microbiol 61:675–690

    Article  PubMed  CAS  Google Scholar 

  8. Müller P, Ewers C, Bertsche U, Anstett M, Kallis T, Breukink E, Fraipont C, Terrak M, Nguyen-Disteche M, Vollmer W (2007) The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli. J Biol Chem 282:36394–36402

    Article  PubMed  Google Scholar 

  9. Daniel RA, Errington J (2003) Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell. Cell 113:767–776

    Article  PubMed  CAS  Google Scholar 

  10. Uehara T, Park JT (2008) Growth of Escherichia coli: significance of peptidoglycan degradation during elongation and septation. J Bacteriol 190:3914–3922

    Article  PubMed  CAS  Google Scholar 

  11. van Teeffelen S, Wang S, Furchtgott L, Huang KC, Wingreen NS, Shaevitz JW, Gitai Z (2011) The bacterial actin MreB rotates, and rotation depends on cell-wall assembly. Proc Natl Acad Sci U S A 108:15822–15827

    Article  PubMed  Google Scholar 

  12. Paradis-Bleau C, Markovski M, Uehara T, Lupoli TJ, Walker S, Kahne DE, Bernhardt TG (2010) Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases. Cell 143:1110–1120

    Article  PubMed  CAS  Google Scholar 

  13. Typas A, Banzhaf M, van Saparoea VB, Verheul J, Biboy J, Nichols RJ, Zietek M, Beilharz K, Kannenberg K, von Rechenberg M, Breukink E, den Blaauwen T, Gross CA, Vollmer W (2010) Regulation of peptidoglycan synthesis by outer membrane proteins. Cell 143:1097–1109

    Article  PubMed  CAS  Google Scholar 

  14. Typas A, Banzhaf M, Gross CA, Vollmer W (2011) From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol 10:123–136

    PubMed  Google Scholar 

  15. Schwartz B, Markwalder JA, Seitz SP, Wang Y, Stein RL (2002) A kinetic characterization of the glycosyltransferase activity of Escherichia coli PBP1b and development of a continuous fluorescence assay. Biochemistry 41:12552–12561

    Article  PubMed  CAS  Google Scholar 

  16. Offant J, Terrak M, Derouaux A, Breukink E, Nguyen-Disteche M, Zapun A, Vernet T (2010) Optimization of conditions for the glycosyltransferase activity of penicillin-binding protein 1a from Thermotoga maritima. FEBS J 277:4290–4298

    Article  PubMed  CAS  Google Scholar 

  17. Barrett D, Wang TS, Yuan Y, Zhang Y, Kahne D, Walker S (2007) Analysis of glycan polymers produced by peptidoglycan glycosyltransferases. J Biol Chem 282:31964–31971

    Article  PubMed  CAS  Google Scholar 

  18. Tamura T, Suzuki H, Nishimura Y, Mizoguchi J, Hirota Y (1980) On the process of cellular division in Escherichia coli: isolation and characterization of penicillin-binding proteins 1a, 1b, and 3. Proc Natl Acad Sci U S A 77:4499–4503

    Article  PubMed  CAS  Google Scholar 

  19. Tomioka S, Ishino F, Tamaki S, Matsuhashi M (1982) Formation of hyper-crosslinked peptidoglycan with multiple crosslinkages by a penicillin-binding protein, 1A, of Escherichia coli. Biochem Biophys Res Commun 106:1175–1182

    Article  PubMed  CAS  Google Scholar 

  20. van Heijenoort Y, Gomez M, Derrien M, Ayala J, van Heijenoort J (1992) Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3. J Bacteriol 174:3549–3557

    PubMed  Google Scholar 

  21. Breukink E, van Heusden HE, Vollmerhaus PJ, Swiezewska E, Brunner L, Walker S, Heck AJ, de Kruijff B (2003) Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes. J Biol Chem 278:19898–19903

    Article  PubMed  CAS  Google Scholar 

  22. Schwartz B, Markwalder JA, Wang Y (2001) Lipid II: total synthesis of the bacterial cell wall precursor and utilization as a substrate for glycosyltransfer and transpeptidation by penicillin binding protein (PBP) 1b of Escherichia coli. J Am Chem Soc 123:11638–11643

    Article  PubMed  CAS  Google Scholar 

  23. Born P, Breukink E, Vollmer W (2006) In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli. J Biol Chem 281:26985–26993

    Article  PubMed  CAS  Google Scholar 

  24. Terrak M, Ghosh TK, van Heijenoort J, Van Beeumen J, Lampilas M, Aszodi J, Ayala JA, Ghuysen JM, Nguyen-Disteche M (1999) The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli. Mol Microbiol 34:350–364

    Article  PubMed  CAS  Google Scholar 

  25. Bertsche U, Breukink E, Kast T, Vollmer W (2005) In vitro murein peptidoglycan synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli. J Biol Chem 280:38096–38101

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  27. Sung MT, Lai YT, Huang CY, Chou LY, Shih HW, Cheng WC, Wong CH, Ma C (2009) Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli. Proc Natl Acad Sci U S A 106:8824–8829

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Carolin Ewers for the SDS-PAGE analysis shown in Fig. 1b. This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and the EU Commission within the DIVINOCELL and EUR-INTAFAR projects.

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Authors and Affiliations

  1. Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell Biology, Newcastle University, Newcastle upon Tyne, UK

    Jacob Biboy, Nhat Khai Bui & Waldemar Vollmer

Authors
  1. Jacob Biboy
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  2. Nhat Khai Bui
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  3. Waldemar Vollmer
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Corresponding author

Correspondence to Waldemar Vollmer .

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Editors and Affiliations

  1. , Dept. of Biology and Biochemistry, University of Houston, Calhoun Road 4800, Houston, 77004, Texas, USA

    Anne H. Delcour

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Biboy, J., Bui, N.K., Vollmer, W. (2013). In Vitro Peptidoglycan Synthesis Assay with Lipid II Substrate. In: Delcour, A. (eds) Bacterial Cell Surfaces. Methods in Molecular Biology, vol 966. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-245-2_17

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  • DOI: https://doi.org/10.1007/978-1-62703-245-2_17

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-244-5

  • Online ISBN: 978-1-62703-245-2

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