Abstract
An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-l-homoserine lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratios of AHLs and AQs produced by Pseudomonas aeruginosa and the consequences of mutating individual or multiple QSSM synthase genes (lasI, rhlI, pqsA) on AHL and AQ profiles and concentrations. The AHL profile of P. aeruginosa was dominated by N-butanoyl-l-homoserine lactone (C4-HSL) with lesser concentrations of N-hexanoyl-l-homoserine lactone (C6-HSL) and 3-oxo-substituted longer chain AHLs including N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL). The AQ profile of P. aeruginosa comprised the C7 and C9 long alkyl chain AQs including 2-heptyl-4-hydroxyquinoline (HHQ), 2-nonyl-4-hydroxyquinoline, the “pseudomonas quinolone signal” (2-heptyl-3-hydroxy-4-quinolone) and the N-oxides, 2-heptyl-4-hydroxyquinoline N-oxide and 2-nonyl-4-hydroxyquinoline N-oxide. Application of the method showed significant effects of growth medium type on the ratio and the nature of the QSSMs synthesized and the dramatic effect of single, double and triple mutations in the P. aeruginosa QS synthase genes. The LC-MS/MS methodology is applicable in organisms where either or both AHL and AQ QSSMs are produced and can provide comprehensive profiles and concentrations from a single sample.
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Acknowledgments
We thank Alex Truman for the synthesis of AHLs and AQs and Christian Pustelny for making the site-directed mutants rhlI lasI double mutant and the las I rhlI pqsA triple mutant of PAO1. This study was supported by grants from the UK Biotechnology and Biological Sciences Research Council (Grant number BB/D007038/1) and the EU QUORMETAB project.
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Ortori, C.A., Dubern, JF., Chhabra, S.R. et al. Simultaneous quantitative profiling of N-acyl-l-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signaling molecules using LC-MS/MS. Anal Bioanal Chem 399, 839–850 (2011). https://doi.org/10.1007/s00216-010-4341-0
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DOI: https://doi.org/10.1007/s00216-010-4341-0