Identification, Quantification, and Determination of the Absolute Configuration of the Bacterial Quorum-Sensing Signal Autoinducer-2 by Gas Chromatography–Mass Spectrometry

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Abstract

Sensing the signal: A gas chromatography–mass spectrometry (GC–MS) method for the analysis of the quorum-sensing autoinducer-2 is described. It allows, for the first time, the direct analysis and accurate determination of this highly water soluble signaling compound, which exists in complex equilibria. The application on the caries-causing bacterium Streptococcus mutans is described.

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Autoinducer-2 (AI-2) is an important, small extracellular signaling molecule that is used by many bacteria. It is part of the AI-2 pool, a group of equilibrium-connected compounds derived from (S)-4,5-dihydroxy-2,3-pentanedione [(S)-DPD, 1]. Currently, these compounds are analyzed by indirect methods relying on the luminescence of sensor strains, the fluorescence of receptor proteins modified with fluorophores, or by isolation procedures not practical for quantitative analysis. Herein, we report a direct analytical procedure that allows for the unambiguous identification and quantification of molecular species by mass spectrometry. Phenylenediamine reacts readily and quantitatively with 1 to form the quinoxalinediol 12 under aqueous conditions. The extraction and silylation of this compound results in the formation of a silyl ether (13), which is amenable for analysis by gas chromatography–mass spectrometry. The use of an isotopically labeled variant (16) of 12 as an internal standard opens the possibility for the accurate quantification of samples containing AI-2 or its equilibrium products. The analysis of cell-free culture supernatants of Vibrio harveyi and Streptococcus mutans allowed for the accurate quantification of the AI-2 concentration above the limit of detection (0.7 ng mL−1). No compounds were detected in mutants lacking the capability to produce AI-2. In addition, the absolute configuration of 1 can be analyzed using the derivative 13 by chiral gas chromatography.

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