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A Pseudomonas putida bioreporter for the detection of enzymes active on 2-alkyl-4(1H)-quinolone signalling molecules

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Abstract

The quorum sensing signalling molecules 2-heptyl-3-hydroxy-4(1H)-quinolone, termed the “Pseudomonas quinolone signal” (PQS), and 2-heptyl-4(1H)-quinolone (HHQ) play an important role in the control of virulence gene expression in Pseudomonas aeruginosa. To construct a bioreporter for the specific and sensitive detection of these compounds, a plasmid with the pqsR gene encoding the PQS- and HHQ-responsive transcriptional regulator PqsR, and with the PqsR-controlled pqsA promoter fused to the lacZ gene, was established in Pseudomonas putida KT2440. The bioreporter responds to HHQ and PQS at concentrations in the range of 0.1–10 and 0.01–5 μM, respectively, with EC50 values of 1.50 ± 0.25 μM for HHQ and 0.15 ± 0.02 μM for PQS. 2,4-Dihydroxyquinoline, a metabolite produced abundantly by P. aeruginosa, did not elicit an increase in reporter enzyme activity. To test whether the bioreporter can be used for the detection of enzymes active on AQ signalling molecules, the hodC gene coding for 2-methyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase was expressed in the reporter strain. This dioxygenase catalyses the cleavage of PQS, albeit with very low activity. The response of the bioreporter to PQS was significantly quenched by co-expression of the hodC gene, and HPLC analysis of culture extracts verified that the PQS levels decreased during cultivation. The bioreporter can be applied to screen for AQ-converting enzymes, which will be useful tools to interfere with quorum sensing and thus virulence in P. aeruginosa.

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Acknowledgments

We thank Prof. Dr. Paul Williams and Dr. Stephan Heeb, University of Nottingham, UK, for providing the pME6032 vector, and Prof. Dr. Svein Valla, Norwegian University of Science and Technology, Trondheim, Norway, for the gift of plasmid pPR9TT. We also thank Olha Schneider (Münster) for help in β-galactosidase experiments, Sven Thierbach (Münster) for help in determination of kinetic parameters of HodC and data analysis, and Heiko Niewerth (Münster) for devising the HPLC protocol and for critical reading of the manuscript. This work was supported, in part, by the German Research Foundation (DFG, grant FE 383/16-1 to S.F.).

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  1. Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstraße 3, 48149, Münster, Germany

    Christine Müller & Susanne Fetzner

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  1. Christine Müller
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Correspondence to Susanne Fetzner.

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Müller, C., Fetzner, S. A Pseudomonas putida bioreporter for the detection of enzymes active on 2-alkyl-4(1H)-quinolone signalling molecules. Appl Microbiol Biotechnol 97, 751–760 (2013). https://doi.org/10.1007/s00253-012-4236-4

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