title: Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis.
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.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University , Cambridge, Massachusetts 02138, United States.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Lebar
authors.forename: Janine M
authors.initials: JM
authors.lastname: May
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Meeske
authors.forename: Sara A
authors.initials: SA
authors.lastname: Leiman
authors.forename: Tania J
authors.initials: TJ
authors.lastname: Lupoli
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tsukamoto
authors.forename: Richard
authors.initials: R
authors.lastname: Losick
authors.forename: David Z
authors.initials: DZ
authors.lastname: Rudner
authors.forename: Suzanne
authors.initials: S
authors.lastname: Walker
authors.forename: Daniel
authors.initials: D
authors.lastname: Kahne
mesh_terms: D001412:Bacillus subtilis / Q000166:cytology / Q000201:enzymology; D002473:Cell Wall / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000378:metabolism; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; D010458:Peptidyl Transferases