Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic d-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of d,d- and l,d-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.
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The authors thank K. C. Huang for providing strain E. coli imp4213, S. Walker for providing S. aureus PBP4 plasmid, D. Kysela for help with image processing and analysis and J. Rittichier for providing the enzyme substrates used in the in vitro assays and his advice on RfDAA synthesis. This study is supported by NIH grants 5R01GM113172 to M.S.V. and Y.V.B. and R35GM122556 to Y.V.B., and by a Canada 150 research Chair in Bacterial Cell Biology to Y.V.B.
The authors declare no competing interests.
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The protocol for RfDAA synthesis, characterization data and bacteria labelling and imaging experiments. The file also includes supplementary discussions, figures, tables and NMR spectra of the probes.
The time-lapse microscopy of PG synthesis labelled by Rf470DL in S. venezuelae.
The time-lapse microscopy of PG synthesis labelled by Rf470DL in B. subtilis.
The time-lapse microscopy of PG synthesis labelled by Rf470DL in B. subtilis. The effect of RfDAA labeling in dead cells was highlighted.
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Hsu, Y., Hall, E., Booher, G. et al. Fluorogenic d-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays. Nat. Chem. 11, 335–341 (2019). https://doi.org/10.1038/s41557-019-0217-x
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