High-Throughput Screening to Identify Chemoreceptor Ligands

  • Matilde Fernández
  • Álvaro Ortega
  • Miriam Rico-Jiménez
  • David Martín-Mora
  • Abdelali Daddaoua
  • Miguel A. Matilla
  • Tino Krell
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)


The majority of bacterial chemoreceptors remain functionally un-annotated. The knowledge of chemoreceptor function, however, is indispensable to understanding the evolution of the chemotaxis system in bacteria with different lifestyles. Significant progress in the annotation of chemoreceptor function has been made using experimental strategies that are based on the individual, genetically engineered ligand binding domain (LBD) of chemoreceptors. There is now evidence that all major classes of LBDs can be produced as individual domains that retain their ligand binding activity. Here, we provide a protocol for the combined use of high-throughput ligand screening using Differential Scanning Fluorimetry followed by Isothermal Titration Calorimetry to identify and characterize ligands that bind to recombinant chemoreceptor LBDs. This approach has been shown to be very efficient for determining the function of novel chemoreceptors.


Chemoreceptor Ligand binding domain Thermal-shift assays Isothermal titration calorimetry Differential scanning fluorimetry 



We acknowledge financial support from FEDER funds and Fondo Social Europeo through grants from the Junta de Andalucía (grant CVI-7335) and the Spanish Ministry for Economy and Competitiveness (grants BIO2013-42297 and BIO2016-76779-P).


  1. 1.
    Wuichet K, Zhulin IB (2010) Origins and diversification of a complex signal transduction system in prokaryotes. Sci Signal 3:ra50CrossRefGoogle Scholar
  2. 2.
    Parkinson JS, Hazelbauer GL, Falke JJ (2015) Signaling and sensory adaptation in Escherichia coli chemoreceptors: 2015 update. Trends Microbiol 23:257–266CrossRefGoogle Scholar
  3. 3.
    Lacal J, Garcia-Fontana C, Munoz-Martinez F, Ramos JL, Krell T (2010) Sensing of environmental signals: classification of chemoreceptors according to the size of their ligand binding regions. Environ Microbiol 12:2873–2884CrossRefGoogle Scholar
  4. 4.
    Kaneko T, Minamisawa K, Isawa T, Nakatsukasa H, Mitsui H et al (2010) Complete genomic structure of the cultivated rice endophyte Azospirillum sp. B510. DNA Res 17:37–50CrossRefGoogle Scholar
  5. 5.
    Parales RE, Luu RA, Chen GY, Liu X, Wu V et al (2013) Pseudomonas putida F1 has multiple chemoreceptors with overlapping specificity for organic acids. Microbiology 159:1086–1096CrossRefGoogle Scholar
  6. 6.
    Alvarez-Ortega C, Harwood CS (2007) Identification of a malate chemoreceptor in Pseudomonas aeruginosa by screening for chemotaxis defects in an energy taxis-deficient mutant. Appl Environ Microbiol 73:7793–7795CrossRefGoogle Scholar
  7. 7.
    Milligan DL, Koshland DE Jr (1993) Purification and characterization of the periplasmic domain of the aspartate chemoreceptor. J Biol Chem 268:19991–19997PubMedGoogle Scholar
  8. 8.
    Ni B, Huang Z, Wu YF, W, Fan Z, Jiang CY et al (2015) A novel chemoreceptor MCP2983 from Comamonas testosteroni specifically binds to cis-aconitate and triggers chemotaxis towards diverse organic compounds. Appl Microbiol Biotechnol 99:2773–2781CrossRefGoogle Scholar
  9. 9.
    Garcia V, Reyes-Darias JA, Martin-Mora D, Morel B, Matilla MA et al (2015) Identification of a chemoreceptor for C2 and C3 carboxylic acids. Appl Environ Microbiol 81:5449–5457CrossRefGoogle Scholar
  10. 10.
    Goers Sweeney E, Henderson JN, Goers J, Wreden C, Hicks KG et al (2012) Structure and proposed mechanism for the pH-sensing Helicobacter pylori chemoreceptor TlpB. Structure 20:1177–1188CrossRefGoogle Scholar
  11. 11.
    Glekas GD, Mulhern BJ, Kroc A, Duelfer KA, Lei V et al (2012) The Bacillus subtilis chemoreceptor McpC senses multiple ligands using two discrete mechanisms. J Biol Chem 287:39412–39418CrossRefGoogle Scholar
  12. 12.
    Nishiyama S, Takahashi Y, Yamamoto K, Suzuki D, Itoh Y et al (2016) Identification of a Vibrio cholerae chemoreceptor that senses taurine and amino acids as attractants. Sci Rep 6:20866CrossRefGoogle Scholar
  13. 13.
    Lacal J, Alfonso C, Liu X, Parales R, Morel B et al (2010) Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands. J Biol Chem 285:23126–23136CrossRefGoogle Scholar
  14. 14.
    Rico-Jimenez M, Reyes-Darias JA, Ortega A, Diez Pena AI, Morel B et al (2016) Two different mechanisms mediate chemotaxis to inorganic phosphate in Pseudomonas aeruginosa. Sci Rep 6:28967CrossRefGoogle Scholar
  15. 15.
    Krell T (2008) Microcalorimetry: a response to challenges in modern biotechnology. Microb Biotechnol 1:126–136CrossRefGoogle Scholar
  16. 16.
    McKellar JL, Minnell JJ, Gerth ML (2015) A high-throughput screen for ligand binding reveals the specificities of three amino acid chemoreceptors from Pseudomonas syringae pv. actinidiae. Mol Microbiol 96:694–707CrossRefGoogle Scholar
  17. 17.
    Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221CrossRefGoogle Scholar
  18. 18.
    Krell T (2015) Tackling the bottleneck in bacterial signal transduction research: high-throughput identification of signal molecules. Mol Microbiol 96:685–688CrossRefGoogle Scholar
  19. 19.
    Fernandez M, Morel B, Corral-Lugo A, Krell T (2016) Identification of a chemoreceptor that specifically mediates chemotaxis toward metabolizable purine derivatives. Mol Microbiol 99:34–42CrossRefGoogle Scholar
  20. 20.
    Corral-Lugo A, de la Torre J, Matilla MA, Fernandez M, Morel B et al (2016) Assessment of the contribution of chemoreceptor-based signaling to biofilm formation. Environ Microbiol 18:3355–3372CrossRefGoogle Scholar
  21. 21.
    Martín-Mora D, Ortega A, Reyes-Darias JA, García V, López-Farfán D et al (2016) Identification of a chemoreceptor in Pseudomonas aeruginosa that specifically mediates chemotaxis towards alpha-ketoglutarate. Front Microbiol 7:1937CrossRefGoogle Scholar
  22. 22.
    Martin-Mora D, Reyes-Darias JA, Ortega A, Corral-Lugo A, Matilla MA et al (2016) McpQ is a specific citrate chemoreceptor that responds preferentially to citrate/metal ion complexes. Environ Microbiol 18:3284–3295CrossRefGoogle Scholar
  23. 23.
    Fernández M, Morel B, Corral-Lugo A, Rico-Jiménez M, Martín-Mora D et al (2016) Identification of ligands for bacterial sensor proteins. Curr Genet 62:143–147CrossRefGoogle Scholar
  24. 24.
    Krell T, Lacal J, Garcia-Fontana C, Silva-Jimenez H, Rico-Jimenez M et al (2014) Characterization of molecular interactions using isothermal titration calorimetry. Methods Mol Biol 1149:193–203CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Matilde Fernández
    • 1
  • Álvaro Ortega
    • 1
  • Miriam Rico-Jiménez
    • 1
  • David Martín-Mora
    • 1
  • Abdelali Daddaoua
    • 1
  • Miguel A. Matilla
    • 1
  • Tino Krell
    • 1
  1. 1.Department of Environmental Protection, Estación Experimental del ZaidínConsejo Superior de Investigaciones CientíficasGranadaSpain

Personalised recommendations