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.

Fourteen clinical isolates of Yersinia enterocolitica serotype 0:3 and four well-documented virulent strains of serotypes 0:3, 0:8, and 0:9 were biotyped and examined for plasmid-associated autoagglutination and calcium dependency and for epithelial cell adherence. These strains were tested for the production of bacteriocin-like antagonism by using tryptone soya blood agar at room temperature and at 37°C. By using the cross-streaking method, three clinical isolates produced inhibitory substances at room temperature. These substances were active against a variety of clinical isolates and their plasmid-cured derivatives at both room temperature and 37°C. The inhibition was easier to read after incubation of the cross-streaked plate at 37°C. The inhibition patterns indicate that two of the three producer strains appear to recognize potentially virulent 0:3 strains, with or without the virulence plasmid.