Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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

Nature Protocols volume 10, pages 33–52 (2015)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Modular syntheses of FDAAs.
Figure 2: Virtual time-lapse microscopy with FDAAs.
Figure 3: Flowchart for different FDAA labeling strategies detailed in this protocol.
Figure 4: The quality of the FDAA labeling depends on the choice of the dye and other experimental factors.

Similar content being viewed by others

References

  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).

    Article  CAS  Google Scholar 

  2. Gould, I.M. Coping with antibiotic resistance: the impending crisis. Int. J. Antimicrob. Agents 36, S1–S2 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Neu, H.C. The crisis in antibiotic resistance. Science 257, 1064–1073 (1992).

    Article  CAS  PubMed  Google Scholar 

  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, 617–624 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. van der Donk, W.A. Lighting up the nascent cell wall. ACS Chem. Biol. 1, 425–428 (2006).

    Article  CAS  PubMed  Google Scholar 

  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, 12519–12523 (2012).

    Article  CAS  Google Scholar 

  7. Pilhofer, M. et al. Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ. Nat. Commun. 4, 2856 (2013).

    Article  PubMed  CAS  Google Scholar 

  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, 601–614 (2013).

    Article  CAS  PubMed  Google Scholar 

  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, 2452–2462 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tocheva, E.I. et al. Peptidoglycan transformations during Bacillus subtilis sporulation. Mol. Microbiol. 88, 673–686 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Daniel, R.A. & Errington, J. Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell. Cell 113, 767–776 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Tiyanont, K. et al. Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics. Proc. Natl. Acad. Sci. USA 103, 11033–11038 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. de Pedro, M.A., Quintela, J., Holtje, J. & Schwarz, H. Murein segregation in Escherichia coli. J. Bacteriol. 179, 2823–2834 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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, 1124–1133 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Sadamoto, R., Niikura, K., Monde, K. & Nishimura, S.-I. Cell wall engineering of living bacteria through biosynthesis. Methods Enzymol. 362, 273–286 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Sadamoto, R. et al. Cell-wall engineering of living bacteria. J. Am. Chem. Soc. 124, 9018–9019 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Sadamoto, R. et al. Control of bacterial adhesion by cell-wall engineering. J. Am. Chem. Soc. 126, 3755–3761 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Liechti, G.W. et al. A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis. Nature 506, 507–510 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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, 484–491 (2006).

    Article  CAS  PubMed  Google Scholar 

  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, 1147–1152 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Brown, P.J.B. et al. Polar growth in the alphaproteobacterial order rhizobiales. Proc. Natl. Acad. Sci. USA 109, 1697–1701 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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, 817–831 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Lam, H. et al. d-Amino acids govern stationary phase cell wall remodeling in bacteria. Science 325, 1552–1555 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lupoli, T.J. et al. Transpeptidase-mediated incorporation of D-amino acids into bacterial peptidoglycan. J. Am. Chem. Soc. 133, 10748–10751 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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, 3442–3453 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Siegrist, M.S. et al. d-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen. ACS Chem. Biol. 8, 500–505 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Zapun, A. et al. In vitro reconstitution of peptidoglycan assembly from the Gram-positive pathogen Streptococcus pneumoniae. ACS Chem. Biol. 8, 2688–2696 (2013).

    Article  CAS  PubMed  Google Scholar 

  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, 199–215 (1992).

    Article  CAS  PubMed  Google Scholar 

  29. Höltje, J.-V. Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol. Mol. Biol. Rev. 62, 181–203 (1998).

    Article  PubMed  PubMed Central  Google Scholar 

  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, 4782–4785 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Vollmer, W., Blanot, D. & de Pedro, M.A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008).

    Article  CAS  PubMed  Google Scholar 

  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, 2004–2021 (2001).

    Article  CAS  Google Scholar 

  33. Prescher, J.A. & Bertozzi, C.R. Chemistry in living systems. Nat. Chem. Biol. 1, 13–20 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Fura, J.M., Sabulski, M.J. & Pires, M.M. Amino acid mediated recruitment of endogenous antibodies to bacterial surfaces. ACS Chem. Biol. 9, 1480–1489 (2014).

    Article  CAS  PubMed  Google Scholar 

  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, 1286–1296 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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, E1025–E1034 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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, 489–493 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Eun, Y.J. et al. Divin: a small molecule inhibitor of bacterial divisome assembly. J. Am. Chem. Soc. 135, 9768–9776 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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, 1953–1965 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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, 731–737 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lupoli, T.J. et al. Lipoprotein activators stimulate Escherichia coli penicillin-binding proteins by different mechanisms. J. Am. Chem. Soc. 136, 52–55 (2014).

    Article  CAS  PubMed  Google Scholar 

  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, 5456–5461 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Takacs, C.N. et al. Growth medium-dependent glycine incorporation into the peptidoglycan of Caulobacter crescentus. PLoS ONE 8, e57579 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Institutes of Health (AI 059327 to M.S.V. and GM051986 to Y.V.B.). We thank E. Garner, K.C. Huang, P. Brown, V. Hughes, A. Ducret, S. Carmody and B. Turner for their helpful discussions.

Author information

Authors and Affiliations

  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

Authors
  1. Erkin Kuru
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Srinivas Tekkam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Edward Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yves V Brun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael S Van Nieuwenhze
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Yves V Brun or Michael S Van Nieuwenhze.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kuru, E., Tekkam, S., Hall, E. et al. Synthesis of fluorescent D-amino acids and their use for probing peptidoglycan synthesis and bacterial growth in situ. Nat Protoc 10, 33–52 (2015). https://doi.org/10.1038/nprot.2014.197

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2014.197

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology