Fluorescent D-amino acids


Fluorescent D-amino acids are D-amino acid derivatives whose side-chain terminal is covalently coupled with a fluorophore molecule. FDAAs incorporate into the bacterial peptidoglycan in live bacteria, resulting in strong peripheral and septal PG labeling without affecting cell growth. They are featured with their in-situ incorporation mechanisms which enable time-course tracking of new PG formation. To date, FDAAs have been employed for studying the cell wall synthesis in various bacterial species through different techniques, such as microscopy, mass spectrometry, flow cytometry.

Structures and general properties

FDAA consists of a D-amino acid and a fluorophore. The backbone is required for its incorporation into the bacterial peptidoglycan through the activity of. Once being incorporated, one can use fluorescence-detection techniques to visualize the location of new PG formation as well as the growth rate.
D-Alanine is the most well-studied D-amino acid for FDAA development because it is a naturally existing residue in bacterial peptidoglycan structures. On the other hand, various fluorophores have been employed for FDAA applications and each has its features. For example, coumarin-based FDAA is small enough to penetrate the bacterial outer membranes and thus is widely used for gram-negative bacterial studies; while TAMRA-based FDAA features its high brightness and photo/thermo-stability, which is suitable for super-resolution microscopy.

Proposed FDAA incorporation mechanisms

Peptidoglycan is a mesh-like structure containing polysaccharides cross-linked by peptide chains. Penicillin-binding proteins, in short PBPs, recognize the PG peptides and catalyze the cross-linking reactions. These enzymes are reported to have high specificity toward the chirality center of the amino acid backbone but relatively low specificity toward the side-chain structure. Therefore, when FDAAs are present, they are taken by PBPs for the cross-linking reactions, resulting in their incorporation into the PG peptide chains. At proper concentration, e.g. 1-2 mM, FDAAs labeling does not affect PG synthesis and cell growth because only 1-2% of PG peptide chains are labeled with FDAA.

Applications

Published studies utilizing FDAAs as tools include:
  • Visualizing bacterial cell wall structures.
  • Studying bacterial cell wall growth.
  • Monitoring bacterial cell wall turnover.
  • Quantifying bacterial cell wall growth activity.
  • Assaying the anti-cell wall ability of antibiotics.
  • Screening new anti-cell wall antibiotics.
  • Tracking transpeptidase activity ''in vitro.''