Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis

Matthew D. Lebar, Janine M. May, Alexander J. Meeske, Sara A. Leiman, Tania Lupoli, Hirokazu Tsukamoto, Richard Losick, David Z. Rudner, Suzanne Walker, Daniel Kahne

Research output: Contribution to journalArticle

Abstract

The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both d-amino acids and d-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged d-amino acids. We exploited these observations to design a fluorescent d-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents.

Original languageEnglish (US)
Pages (from-to)10874-10877
Number of pages4
JournalJournal of the American Chemical Society
Volume136
Issue number31
DOIs
StatePublished - Aug 6 2014

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Peptidoglycan
Bacillus subtilis
Fluorescent Dyes
Cell Wall
Lipids
Cells
Escherichia coli
Enzymes
Amino acids
Labels
Bacteria
Penicillin-Binding Proteins
Amino Acids
Bacilli
Peptides
Polysaccharides
muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol
Gram-Positive Bacteria
Gram-Negative Bacteria

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis. / Lebar, Matthew D.; May, Janine M.; Meeske, Alexander J.; Leiman, Sara A.; Lupoli, Tania; Tsukamoto, Hirokazu; Losick, Richard; Rudner, David Z.; Walker, Suzanne; Kahne, Daniel.

In: Journal of the American Chemical Society, Vol. 136, No. 31, 06.08.2014, p. 10874-10877.

Research output: Contribution to journalArticle

Lebar, MD, May, JM, Meeske, AJ, Leiman, SA, Lupoli, T, Tsukamoto, H, Losick, R, Rudner, DZ, Walker, S & Kahne, D 2014, 'Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis', Journal of the American Chemical Society, vol. 136, no. 31, pp. 10874-10877. https://doi.org/10.1021/ja505668f
Lebar, Matthew D. ; May, Janine M. ; Meeske, Alexander J. ; Leiman, Sara A. ; Lupoli, Tania ; Tsukamoto, Hirokazu ; Losick, Richard ; Rudner, David Z. ; Walker, Suzanne ; Kahne, Daniel. / Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis. In: Journal of the American Chemical Society. 2014 ; Vol. 136, No. 31. pp. 10874-10877.
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