Synthesis of fluorescent D-amino acids and their use for probing peptidoglycan synthesis and bacterial growth in situ

Journal name:
Nature Protocols
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Published online


Fluorescent D-amino acids (FDAAs) are efficiently incorporated into the peptidoglycans (PGs) of diverse bacterial species at the sites of PG biosynthesis, allowing specific and covalent probing of bacterial growth with minimal perturbation. Here we provide a protocol for the synthesis of four FDAAs emitting light in blue (HCC-amino-D-alanine, HADA), green (​NBD-amino-D-alanine, ​NADA, and fluorescein-D-lysine, FDL) or red (TAMRA-D-lysine, TDL) and for their use in PG labeling of live bacteria. Our modular synthesis protocol gives easy access to a library of different FDAAs made with commercially available fluorophores and diamino acid starting materials. Molecules can be synthesized in a typical chemistry laboratory in 2–3 d using standard chemical transformations. The simple labeling procedure involves the addition of the FDAAs to a bacterial sample for the desired labeling duration and stopping further label incorporation by fixing the cells with cold 70% (vol/vol) ​ethanol or by washing away excess dye. We discuss several scenarios for the use of these labels in fluorescence microscopy applications, including short or long labeling durations, and the combination of different labels in pure culture (e.g., for 'virtual time-lapse' microscopy) or in situ labeling of complex environmental samples. Depending on the experiment, FDAA labeling can take as little as 30 s for a rapidly growing species such as Escherichia coli.

At a glance


  1. Modular syntheses of FDAAs.
    Figure 1: Modular syntheses of FDAAs.

    HADA 3 and ​NADA 5 attaching commercially available fluorophores, ​7-hydroxycoumarin-3-carboxylic acid (​HCC-OH; 2) and ​4-chloro-7-nitrobenzofurazan (​NBD-Cl; 4) to the backbone ​N-Boc-D-2,3-diaminopropionic acid (i.e., ​N-α-Boc-3-amino-D-alanine; 1); and FDL 8 and TDL 10 attaching commercially available ​FITC (7) and 5 (and 6-)-carboxytetramethylrhodamine succinimidyl ester (TAMRA-OSu; 9) to the backbone ​N-α-Boc-D-lysine 6, respectively. In our experience, HADA is the FDAA probe of choice when factors regarding ease of incorporation into the PG of diverse bacterial species, brightness, photostability and price of synthesis are considered. For example, despite its inexpensive synthesis, ​NADA suffers from low photostability. In contrast, although FDL and especially TDL are brighter and more photostable than HADA, the metabolic incorporation of these larger FDAAs into cell walls is particularly limited in Gram-negative bacteria. ​DMF, ​dimethylformamide; ​THF, ​tetrahydrofuran.

  2. Virtual time-lapse microscopy with FDAAs.
    Figure 2: Virtual time-lapse microscopy with FDAAs.

    (a) A saliva sample was pulse-labeled successively with TDL (red), FDL (green) and HADA (blue) for 15 min each. The labeling patterns on each cell provide chronological account of the areas of PG synthesis during each pulse labeling, with the red and green signals representing the oldest and the newest parts of the cell wall relative to the duration of the experiment, namely 3 × 15 min. (b) Virtual time-lapse microscopy on S. venezuelae pulse-labeled with TDL (red), FDL (green) and HADA (blue) for 5 min each. Scale bars, 2 μm.

  3. Flowchart for different FDAA labeling strategies detailed in this protocol.
    Figure 3: Flowchart for different FDAA labeling strategies detailed in this protocol.

    The polarly growing Gram-positive bacterium S. venezuelae is used as an example.

  4. The quality of the FDAA labeling depends on the choice of the dye and other experimental factors.
    Figure 4: The quality of the FDAA labeling depends on the choice of the dye and other experimental factors.

    (a,b) Although cells grown with HADA, ​NADA or TDL (500 μM, several generations for E. coli and 20 min for B. subtilis) show similar labeling patterns, the labeling quality, i.e., the SNR, can differ substantially. Under these experimental and imaging conditions, SNRs of HADA, ​NADA and TDL are 6.3, 1.9 and 1.07 (i.e., the signal is 7% above background) for E. coli and 2.69, 1.55 and 2.91 for B. subtilis. In E. coli, the low SNR of TDL is due to its poor outer-membrane permeability. The lack of labeling on the poles of B. subtilis is typical in briefly pulsed rod-shaped bacteria, and it represents the inertness of the polar cell walls. (c,d) SNR depends on other experimental factors such as effective washes. Without washes, the signal from HADA-labeled E. coli cells (500 μM, several generations) is obscured by the background fluorescence and therefore SNR is 1 (c, left, and d, left and middle) and the image is saturated and completely white. Each wash improves the SNR (SNRHADA,wash×1 =1.5; SNRHADA,wash×3 = 3.03). After the washes, the L-isomer (HALA)-treated cells (SNRHALA,wash×3 = 1.02) show ∼100 times less normalized signal relative to the HADA-labeled cells. Display ranges within c or d were kept constant for visual comparison. Scale bars, 4 μm.


  1. Schneider, T. & Sahl, H.-G. An oldie but a goodie–cell wall biosynthesis as an antibiotic target pathway. Int. J. Med. Microbiol. 300, 2010 (2010).
  2. Gould, I.M. Coping with antibiotic resistance: the impending crisis. Int. J. Antimicrob. Agents 36, S1S2 (2010).
  3. Neu, H.C. The crisis in antibiotic resistance. Science 257, 10641073 (1992).
  4. van Dam, V., Olrichs, N. & Breukink, E. Specific labeling of peptidoglycan precursors as a tool for bacterial cell wall studies. Chem. Bio. Chem. 10, 617624 (2009).
  5. van der Donk, W.A. Lighting up the nascent cell wall. ACS Chem. Biol. 1, 425428 (2006).
  6. Kuru, E. et al. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angew. Chem. Int. Ed. 51, 1251912523 (2012).
  7. Pilhofer, M. et al. Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ. Nat. Commun. 4, 2856 (2013).
  8. Pinho, M.G., Kjos, M. & Veening, J.-V. How to get (a)round: mechanisms controlling growth and division of coccoid bacteria. Nat. Rev. Microbiol. 11, 601614 (2013).
  9. Ranjit, D.K. & Young, K.D. The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli. J. Bacteriol. 195, 24522462 (2013).
  10. Tocheva, E.I. et al. Peptidoglycan transformations during Bacillus subtilis sporulation. Mol. Microbiol. 88, 673686 (2013).
  11. Daniel, R.A. & Errington, J. Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell. Cell 113, 767776 (2003).
  12. Tiyanont, K. et al. Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics. Proc. Natl. Acad. Sci. USA 103, 1103311038 (2006).
  13. de Pedro, M.A., Quintela, J., Holtje, J. & Schwarz, H. Murein segregation in Escherichia coli. J. Bacteriol. 179, 28232834 (1997).
  14. Olrichs, N.K. et al. A novel in vivo cell-wall labeling approach sheds new light on peptidoglycan synthesis in Escherichia coli. Chembiochem 12, 11241133 (2011).
  15. Sadamoto, R., Niikura, K., Monde, K. & Nishimura, S.-I. Cell wall engineering of living bacteria through biosynthesis. Methods Enzymol. 362, 273286 (2003).
  16. Sadamoto, R. et al. Cell-wall engineering of living bacteria. J. Am. Chem. Soc. 124, 90189019 (2002).
  17. Sadamoto, R. et al. Control of bacterial adhesion by cell-wall engineering. J. Am. Chem. Soc. 126, 37553761 (2004).
  18. Liechti, G.W. et al. A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis. Nature 506, 507510 (2013).
  19. Schouten, J.A. et al. Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5. Mol. BioSyst. 2, 484491 (2006).
  20. de Pedro, M.A., Young, K.D., Höltje, J.-V. & Schwarz, H. Branching of Eschereichia coli cells arises from multiple sites of inert peptidoglycan. J. Bacteriol. 185, 11471152 (2003).
  21. Brown, P.J.B. et al. Polar growth in the alphaproteobacterial order rhizobiales. Proc. Natl. Acad. Sci. USA 109, 16971701 (2012).
  22. Cava, F., Lam, H., de Pedro, M.A. & Waldor, M.K. Emerging knowledge of regulatory roles of D-amino acids in bacteria. Cell. Mol. Life Sci. 68, 817831 (2011).
  23. Lam, H. et al. d-Amino acids govern stationary phase cell wall remodeling in bacteria. Science 325, 15521555 (2009).
  24. Lupoli, T.J. et al. Transpeptidase-mediated incorporation of D-amino acids into bacterial peptidoglycan. J. Am. Chem. Soc. 133, 1074810751 (2011).
  25. Cava, F., de Pedro, M.A., Lam, H., Davis, B.M. & Waldor, M.K. Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. EMBO J. 30, 34423453 (2011).
  26. Siegrist, M.S. et al. d-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen. ACS Chem. Biol. 8, 500505 (2013).
  27. Zapun, A. et al. In vitro reconstitution of peptidoglycan assembly from the Gram-positive pathogen Streptococcus pneumoniae. ACS Chem. Biol. 8, 26882696 (2013).
  28. Bugg, T.D.H. & Walsh, C.T. lntracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Nat. Prod. Rep. 9, 199215 (1992).
  29. Höltje, J.-V. Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol. Mol. Biol. Rev. 62, 181203 (1998).
  30. Magnet, S., Dubost, L., Marie, A., Arthur, M. & Guttmann, L. Identification of the L,D-transpeptidases for peptidoglycan cross-linking in Escherichia coli. J. Bacteriol. 190, 47824785 (2008).
  31. Vollmer, W., Blanot, D. & de Pedro, M.A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149167 (2008).
  32. Kolb, H.C., Finn, M.G. & Sharpless, K.B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 20042021 (2001).
  33. Prescher, J.A. & Bertozzi, C.R. Chemistry in living systems. Nat. Chem. Biol. 1, 1320 (2005).
  34. Fura, J.M., Sabulski, M.J. & Pires, M.M. Amino acid mediated recruitment of endogenous antibodies to bacterial surfaces. ACS Chem. Biol. 9, 14801489 (2014).
  35. Kerr, C.H., Culham, D.E., Marom, D. & Wood, J.M. Salinity-dependent impacts of ProQ, Prc, and Spr deficiencies on Escherichia coli cell structure. J. Bacteriol. 196, 12861296 (2014).
  36. Ursell, T.S. et al. Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization. Proc. Natl. Acad. Sci. USA 111, E1025E1034 (2014).
  37. Jiang, C., Brown, P.J., Ducret, A. & Brun, Y.V. Sequential evolution of bacterial morphology by co-option of a developmental regulator. Nature 506, 489493 (2014).
  38. Eun, Y.J. et al. Divin: a small molecule inhibitor of bacterial divisome assembly. J. Am. Chem. Soc. 135, 97689776 (2013).
  39. Fenton, A.K. & Gerdes, K. Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli. EMBO J. 32, 19531965 (2013).
  40. Cava, F., Kuru, E., Brun, Y.V. & de Pedro, M.A. Modes of cell wall growth differentiation in rod-shaped bacteria. Curr. Opin. Microbiol. 16, 731737 (2013).
  41. Lupoli, T.J. et al. Lipoprotein activators stimulate Escherichia coli penicillin-binding proteins by different mechanisms. J. Am. Chem. Soc. 136, 5255 (2014).
  42. Shieh, P., Siegrist, M.S., Cullen, A.J. & Bertozzi, C.R. Imaging bacterial peptidoglycan with near-infrared fluorogenic azide probes. Proc. Natl. Acad. Sci. USA 111, 54565461 (2014).
  43. Takacs, C.N. et al. Growth medium-dependent glycine incorporation into the peptidoglycan of Caulobacter crescentus. PLoS ONE 8, e57579 (2013).
  44. Fleurie, A. et al. Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division. PLoS Genet. 10, e1004275 (2014).

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  1. Interdisciplinary Biochemistry Program, Indiana University, Bloomington, Indiana, USA.

    • Erkin Kuru
  2. Department of Chemistry, Indiana University, Bloomington, Indiana, USA.

    • Srinivas Tekkam,
    • Edward Hall &
    • Michael S Van Nieuwenhze
  3. Department of Biology, Indiana University, Bloomington, Indiana, USA.

    • Yves V Brun


E.K., M.S.V. and Y.V.B. designed the study; E.K. designed, and S.T. and E.H. synthesized FDAAs; E.K. designed and conducted experiments involving microscopy; and E.K., S.T., E.H., Y.V.B. and M.S.V. wrote the manuscript.

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