Metabolic Profiling of Bacteria by Unnatural C‐terminated D‐Amino Acids

Abstract

Bacterial peptidoglycan is a mesh-like network comprised of sugars and oligopeptides. Transpeptidases cross-link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous D-amino acids onto bacterial cell surfaces with vast promiscuity for the side-chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C-terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C-terminus amidated D-amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species.

d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

“…Interestingly, conversion of the unnatural D-amino acid C terminus from a carboxylic acid to a carboxamide has been shown previously to block PBP TP on the modified stem peptide (41). Likewise, we have demonstrated previously that entirely unnatural changes to the C terminus can modulate labeling efficiency (42). It is conceivable that carboxamide modification also renders the stem peptide resistant to CP, a feature that would result in elevated labeling levels in wild-type strains that express PBP5.…”

Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens

“…27 Fitting the data according to the Michaelis-Menten mechanism yields a K M value of 23 µM (Figure S21, SI). This K M value is about an order of magnitude lower than that of a D-Lys derivative used for S. aureus labeling, 32 suggesting the D-AB3 uptake by E. coli is highly efficient. The concentration profile of D-AB3 for S. aureus staining data displays a linear relationship, suggesting the K M value is well above the highest concentration tested (125 µM, Figure S21, SI).…”

Non-Natural D-Amino Acids to Control Bacterial Virulence

“…The well-known epitope 2,4-dinitrophenylalanine (DNP) was introduced into the internal oligopeptides of B. subtilis, promoting assembly of endogenous antibodies at the cell surface [6]. The flexibility of the transpeptidase enzyme mediating this process was further employed to modify extracellular peptides at their C-termini, largely altering the topography of the bacterial cell wall [7].…”