Full color palette of fluorescent d-amino acids for in situ labeling of bacterial cell walls

Abstract

Fluorescent d-amino acids (FDAAs) enable efficient in situ labeling of peptidoglycan in diverse bacterial species. Conducted by enzymes involved in peptidoglycan biosynthesis, FDAA labeling allows specific probing of 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 studies of peptidoglycan synthesis and dynamics, which, in turn, has created a demand for the development of new FDAA probes. Here, we report the synthesis of new FDAAs, with emission wavelengths that span the entire visible spectrum. We also provide data to characterize their photochemical and physical properties, and we demonstrate their utility for visualizing peptidoglycan synthesis in Gram-negative and Gram-positive bacterial species. Finally, we show the permeability of FDAAs toward the outer-membrane of Gram-negative organisms, pinpointing the probes available for effective labeling in these species. This improved FDAA toolkit will enable numerous applications for the study of peptidoglycan biosynthesis and dynamics.

Fig. 1. PBP-facilitated FDAA incorporation mechanism and novel FDAA structures. (A) Comparison of the transpeptidation reaction performed by penicillin-binding proteins (PBPs, top) and proposed mechanism of FDAA incorporation (bottom). The peptide-PBP intermediate (acyl donor) is attacked by the free amine of lysine in another peptide chain (acyl acceptor), which leads to cross-linking. FDAAs mimic the acyl acceptor to interact with the peptide-PBP intermediate. (B) Structures of FDAAs reported in this study.

Fig. 2. Synthesis scheme and spectra of FDAAs. (A) FDAA synthesis is achieved by coupling conjugation-activated fluorophores with 3-amino-d-alanine (3-aminopropionic acid) or d-lysine. (B and C) Excitation (B) and emission (C) spectra of FDAAs in phosphate-buffer saline (1× PBS) at pH 7.4. Absorbance and fluorescence intensity were normalized to their maximum values.

Fig. 3. Virtual time-lapse FDAA labeling. (A) Streptomyces venezuelae cells were sequentially labeled with Atto₄₈₈ADA (3 h), Cy_3BADA (15 min), AF₃₅₀DL (15 min), and Atto₆₁₀ADA (15 min), and then fixed and imaged. Left: Phase-contrast; right: fluorescence. Scale bar: 5 μm. White arrows point out new branches formed at different time points (oldest to newest: (a) to (c)). (B) Lactococcus lactis cells were sequentially labeled with Atto₆₁₀ADA (8 min), HADA (5 min), YADA (8 min), sBADA (5 min), and TADA (5 min), and then fixed and imaged. Left to right: Phase channel, Atto₆₁₀ADA, HADA, YADA, sBADA, TADA, and merged image. Scale bar: 1 μm.

Fig. 4. Stochastic optical reconstruction microscopy (STORM) of E. coli imp4213 Δ6 BW25113 cells lacking all l,d-transpeptidases. Δ6 cells were labeled with Cy_3BADA (A) and TDL (B). Left: Epifluorescence microscopy; right panel: STORM (right). E. coli imp4213 Δ6 cells were treated with cephalexin for 2 h, washed, and then pulsed with 1 mM Cy_3BADA/TDL for 2 min. Insets in (A) and arrow head highlight a division septum. Scale bar: 2 μm.