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.

Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens

“…In comparison, the diazaborine formation is faster than the azide-alkyne click chemistry or the alkyne-nitrone cycloaddition by 2–3 orders of magnitude. Although the tetrazine ligation can be fast, its reaction rate is quite sensitive to how the tetrazine moiety is linked to its biological target 36 and typically bulky diaryl tetrazine derivatives are needed to afford fast kinetics, 37 which are less ideal due to the outer membrane barrier.…”

Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Escherichia coli Strain DC2

“…The specific tasks of each PBP have been difficult to discern, largely due to the redundant functions of these enzymes in most organisms (7). However, recent work has illuminated much about the substrate specificity (8)(9)(10)(11), activators (12), and association of several PBPs with cell elongation or septal synthesis (13)(14)(15). Because the PBPs are crucial for cell wall biosynthesis, cell division, growth, and resistance to ␤-lactams, study of the roles of individual family members may be critical to devising ways to overcome and/or evade bacterial resistance.…”

A biocatalytic approach to capuramycin analogues by exploiting a substrate permissive N-transacylase CapW

“…18 Two distinct and mechanistically comparable penicillin binding proteins have recently been demonstrated to catalyze an intermolecular transamidation that was exploited to introduce alternative amines including fluorescent probes in place of the terminal d -Ala of peptidoglycan. 19,20 Similarly, all of the substrates tested here with CapW were incorporated in place of the naturally occurring l -ACL, and it is hypothesized that this thermodynamically driven reaction proceeds through the reversible formation of an acyl-enzyme intermediate. The ability of CapW to catalyze direct amine exchange is particularly significant when considering that 1 or 3 are the congeners with the highest yield from the respective wild-type stains, thus making them desirable semisynthetic precursors.…”