FluorescentD-amino acids (FDAAs) areD-amino acid derivatives whose side-chain terminal is covalently coupled with afluorophore molecule.[1] FDAAs incorporate into the bacterialpeptidoglycan (PG) in live bacteria, resulting in strong peripheral and septal PG labeling without affecting cell growth. They are featured with theirin-situ incorporation mechanisms which enable time-course tracking of new PG formation.[2] To date, FDAAs have been employed for studying the cell wall synthesis in various bacterial species (bothgram-positives andgram-negatives) through different techniques, such asmicroscopy,mass spectrometry,flow cytometry.
Collection of reported fluorescentD-amino acids and their structures.
FDAA consists of aD-amino acid and a fluorophore (coupled through the amino acid side chain). TheD-amino acid backbone is required for its incorporation into the bacterial peptidoglycan through the activity ofDD-transpeptidases.[3] Once being incorporated, one can use fluorescence-detection techniques to visualize the location of new PG formation as well as the growth rate.[4]
D-Alanine is the most well-studiedD-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.[5] For example, coumarin-based FDAA (HADA) is small enough to penetrate the bacterial outer membranes and thus is widely used for gram-negative bacterial studies; while TAMRA-based FDAA (TADA) features its high brightness and photo/thermo-stability, which is suitable for super-resolution microscopy (strong excitation light is used).[5]
Proposed mechanism of FDAA incorporation into bacterial peptidoglycan.[1]
Peptidoglycan (PG) is a mesh-like structure containing polysaccharides cross-linked by peptide chains.[6]Penicillin-binding proteins (DD-transpeptidases), in short PBPs, recognize the PG peptides and catalyze the cross-linking reactions.[7] These enzymes are reported to have high specificity toward the chirality center of the amino acid backbone (D-chiral center) 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.[2]
Sequential labeling of FDAAs revealed the growth pattern of peptidoglycan inStreptomyces venezuelae.
