QUORUM SENSING AND CHROMOBACTERIUM-VIOLACEUM - EXPLOITATION OF VIOLACEIN PRODUCTION AND INHIBITION FOR THE DETECTION OF N-ACYLHOMOSERINE LACTONES

Quorum sensing relies upon the interaction of a diffusible signal mole cule with a transcriptional activator protein to couple gene expressio n with cell population density. In Cram-negative bacteria, such signal molecules are usually N-acylhomoserine lactones (AHLs) which differ i n the structure of their N-acyl side chains. Chromobacterium violaceum , a Cram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein. Previously the authors d escribed a violacein-negative, mini-Tn5 mutant of C. violaceum (CV026) in which pigment production can be restored by incubation with supern atants from the wild-type strain. To develop this mutant as a general biosensor for AHLs. the natural C. violaceun, AHL molecule was first c hemically characterized. By using solvent extraction, HPLC and mass sp ectrometry, a single AHL, N-hexanoyl-L-homoserine lactone (HHL), was i dentified in wild-type C. violaceum culture supernatants which was abs ent 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 N-acylhomocysteine thiolacton e (ART) analogues. In CV026, violacein is inducible by all the AHL and AHT compounds evaluated with N-acyl side chains from C-4 to C-8 in le ngth, with varying degrees of sensitivity. Although AHL compounds with N-acyl side chains from C-10 to C-14 are unable to induce violacein p roduction, if an activating AHL (e.g. HHL) is incorporated into the ag ar, these long-chain AHLs can be detected by their ability to inhibit violacein production. The versatility of CV026 in facilitating detecti on of AHL mixtures extracted from culture supernatants and separated b y thin-layer chromatography is also demonstrated. These simple bioassa ys employing CV026 thus greatly extend the ability to detect a wide sp ectrum of AHL signal molecules.