Quorum sensing relies upon the interaction of a diffusible signal molecule with a transcriptional activator protein to couple gene expression with cell population density. In Gram-negative bacteria, such signal molecules are usually -acylhomoserine lactones (AHLs) which differ in the structure of their -acyl side chains. a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigmen violacein. Previously the authors described a violacein-negative, mini-Tn5 mutant of (CV026) in which pigment production can be restored by incubation with supernatants from the wild-type strain. To develop this mutant as a general biosensor for AHLs, the natural AHL molecule was first chemically characterized. By using solvent extraction, HPLC and mass spectrometry, a single AHL, -hexanoyl--homoserine lactone (HHL), was identified in wild-type culture supernatants which was absent from CV026. Since the production of violacein constitutes a simple assay for the detection of AHLs, we explored the ability of CV026 to respond to a series of synthetic AHL and -acylhomocysteine thiolactone (AHT) analogues. In CV026, violacein is inducible by ail the AHL and AHT compounds evaluated with -acyl side chains from C to C in length, with varying degrees of sensitivity. Although AHL compounds with -acyl side chains from C to C are unable to induce violacein production, if an activating AHL (e.g. HHL) is incorporated into the agar, these long-chain AHLs can be detected by their ability to inhibit violacein production. The versatility of CV026 in facilitating detection of AHL mixtures extracted from culture supernatants and separated by thin-layer chromatography is also demonstrated. These simple bioassays employing CV026 thus greatly extend the ability to detect a wide spectrum of AHL signa molecules.


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