Fluorescent D-amino acids
Fluorescent D-amino acids (FDAAs) enable efficient in situ labeling of bacterial peptidoglycan, the main component of the cell wall, in diverse bacterial species. Conducted by enzymes involved in peptidoglycan biosynthesis, FDAA labeling allows specific probing of the cell wall formation/remodeling activity, bacterial growth, and cell morphology. Their broad application and high biocompatibility have made FDAAs an important and effective tool for studying peptidoglycan synthesis and dynamics.
FDAAs were first developed and reported by the VanNueuwenhze and Brun laboratories at Indiana University in 2012. [1] To date, more than twenty fluorophores have been investigated for FDAA development and nine FDAAs that exhibit outstanding peptidoglycan labeling ability have been reported. The FDAA toolset covers the entire range of visible spectrum and possesses high compatibility with most fluorescent protein fusions and macromolecule staining. [2]
Mechanism of FDAA labeling
FDAAs are known as metabolic probes for in situ labeling of peptidoglycan, a process conducted by the enzymes involved in the peptidoglycan biosynthesis. Bacterial peptidoglycan is a mesh-like structure composed of glycan strands cross-linked by polypeptides. The polypeptide cross-linkages are constructed by an enzyme-directed reaction called transpeptidation. Studies have shown that transpeptidases have high specificity toward the chirality of substrates, the D-amino acids in the peptidoglycan polypeptides, but not toward their side chain structures. As a result, D-amino acids with various side chain structures can be incorporated into peptidoglycan structures by transpeptidase's activity. Exploiting this enzyme promiscuity, FDAAs are designed and synthesized for studying transpeptidase activities in vivo. Their incorporation into peptidoglycan reflects the synthesis activity of the cell wall, which is supported by mass spectrometry analyses. [1] In short, FDAAs are indicators of transpeptidase activity and can be applied for visualizing peptidoglycan formation and remodeling using microscopy and spectrophotometric analyses.
Proposed mechanism of FDAA incorporation
FDAA applications
FDAAs have been used for visualizing peptidoglycan structures in various bacterial species. Compared to other existing peptidoglycan labeling tools, such as fluorescent wheat germ agglutinin (FWGA) and fluorescent antibiotics, FDAAs have significantly improved membrane permeability and biocompatibility to bacterial cells. This ensures efficient peptidoglycan labeling with minimized artificial effects caused by the probe toxicity.
Most importantly, FDAAs are the first tools that directly visualize transpeptidase activity in live bacterial cells. Since transpeptidase activity is required for new peptidoglycan formation and its remodeling, FDAAs have been employed for studying peptidoglycan growth pattern in many bacterial species as well as the dynamics/mechanism of the enzyme activity. Check out our application section for more details!
Sequential labeling using four different FDAAS in Streptomyces venezuelae
Sequential labeling using four different FDAAS in Streptomyces venezuelae
Sequential labeling using five different FDAAS in Lactococcus lactis
FDAA spectra and microscopy settings
The reported FDAA toolsets have covered the entire range of visible light. These differently colored FDAA can be distinguished by microscopy when appropriate excitation and emission filters are used. The FDAA toolsets not only enable virtual time-lapse tracking of peptidoglycan synthesis (by sequential labeling as shown above) but also the study of interactions between newly made peptidoglycan and fluorescently labeled synthases, such as penicillin-binding proteins.
Filter selection for visualizing FDAA using microscopy
References
[1] Kuru et al. In Situ Probing of Newly Synthesized Peptidoglycan in Live Bacteria with Fluorescent D-Amino Acids. Angew. Chem. 2012, 124, 12687-12691.
[2] Hsu et al. Full-color palette of fluorescent D-amino acids for in situ labeling of bacterial cell walls. Chem. Sci. 2017, 8, 6313-6321.