Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones

Abstract

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 N-acylhomoserine lactones (AHLs) which differ in the structure of their N-acyl side chains. Chromobacterium violaceurn, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein. Previously the authors described a violacein-negative, mini-Tn5 mutant of C. violaceurn (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 C. violaceurn AHL molecule was first chemically characterized. By using solvent extraction, HPLC and mass spectrometry, a single AHL, N-hexanoyl-L-homoserine lactone (HHL), was identified in wild-type C. violaceurn 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 N-acylhomocysteine thiolactone (AHT) analogues. In CV026, violacein is inducible by all the AHL and AHT compounds evaluated with N-acyl side chains from C, to C, in length, with varying degrees of sensitivity. Although AHL compounds with N-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 signal molecules.

Inhibition of quorum sensing in Chromobacterium violaceum by Syzygium cumini L. and Pimenta dioica L.

“…Inhibition of QS regulated violacein production in C. violaceum is commonly used for anti-QS screening studies. The AHL synthesized by different strains of C. violaceum vary in their acyl chain length or substitution, and the two wild type strains used in this study though produce two different major AHL molecules [11] , the tested plant extracts were able to inhibit the violacein synthesis in both. Other reports have also found natural plant extracts, such as that of Myristica cinnamomea, showing anti-QS activity at 3 mg/mL against C. violaceum CV026 [14] .…”

“…Biosensor bioassay for anti-QS activity was carried out according to the methods described previously [11] . Briefly, plant extracts re-suspended in ethanol were loaded onto 6 mm sterile discs (Himedia, India) at different concentrations (100 µg-3.0 mg/disc).…”

“…Further, the whole genome studies on C. violaceum showed that violacein production is regulated by vioD, vioC, vioB and vioA genes arranged in an operon through a QS system mediated by AHL [10] . A biosensor strain, C. violaceum CV026 (mini-Tn5 mutant of wild type strain), deficient in autoinducer synthase which requires exogenous addition of AHL to produce violacein was developed for studying various QS mechanisms, and it offers a convenient tool for the biological assay of screening QS inhibitors [11] . This study investigated into the anti-QS activity of S. cumini and P. dioica using C. violaceum strains.…”

Inhibition ofSteptococcus mutansbiofilm formation by extracts ofTenacibaculumsp. 20J, a bacterium with wide-spectrum quorum quenching activity

“…The AHL degradation activity of the different extracts was tested by incubating 500 μl of each extract with N -hexanoyl-L-homoserine lactone (C6-HSL) 10 μM for 24 h at 22°C 200 rpm and the remaining AHL was detected using the biosensor strain Chromobacterium violaceum CV026 [39,40]. After 24 h of degradation, 100 μl of the mixture were placed in wells made on an LB plate overlaid with a 1/100 dilution of an overnight culture of C. violaceum CV026 in soft LB (8% agar).…”

Quorum Sensing Signaling Molecules Involved in the Production of Violacein byPseudoalteromonas

“…Previous research has shown that C6-HSL also acts as an AHL and induces violacein production in the wild-type strain of Chromobacterium violaceum. 7) The physiological function of C14-HSL is unknown at present, but it might play an important role in the quorum sensing regulation of other bacterial behaviors. Strain 520P1 (1 ml) was incubated in the wells of a 24-well plate with 62 mM 3-oxo-C8-HSL standard, 48 mM C14-HSL standard, or 100 ml of the AHL sample extracted from the spot corresponding to 3-oxo-C8-HSL on a TLC plate (A).…”