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.

Spent culture supernatants from both Aeromonas hydrophila and Aeromonas salmonicida activate a range of biosensors responsive to N-acylhomoserine lactones (AHLs). The genes for a quorum sensing signal generator and a response regulator were cloned from each Aeromonas species and termed ahyRI and asaRI, respectively. Protein sequence homology analysis places the gene products within the growing family of LuxRI homologs. ahyR and asaR are transcribed divergently from ahyI and asaI, respectively, and in both Aeromonas species, the genes downstream have been identified by DNA sequence and PCR analysis. Downstream of both ahyI and asaI is a gene with close homology to iciA, an inhibitor of chromosome replication in Escherichia coli, a finding which implies that in Aeromonas, cell division may be linked to quorum sensing. The major signal molecule synthesized via both AhyI and AsaI was purified from spent culture supernatants and identified as N-(butanoyl)-Lhomoserine lactone (BHL) by thin-layer chromatography, high-pressure liquid chromatography analysis, and mass spectrometry. In addition, a second, minor AHL, N-hexanoyl-L-homoserine lactone, was identified. Transcriptional reporter studies with ahyI::luxCDABE fusions indicate that AhyR and BHL are both required for ahyI transcription. For A. salmonicida, although the addition of exogenous BHL gives only a small stimulation of the production of serine protease with comparison to the control culture, the incorporation of a longer-chain AHL, N-(3-oxodecanoyl)-L-homoserine lactone, reduced the final level (by approximately 50%) and delayed the appearance (from an A 650 of 0.9 in the control to an A 650 of 1.2 in the test) of protease in the culture supernatant. These data add A. hydrophila and A. salmonicida to the growing family of gram-negative bacteria now known to control gene expression through quorum sensing.Quorum sensing (19) is a mechanism for controlling gene expression in response to an expanding bacterial population. In many gram-negative bacteria, the diffusible quorum sensing signal molecule is a member of the N-acylhomoserine lactone (AHL) family (18,45,51). Accumulation of this molecule above a threshold concentration, through the activity of a signal generator protein, indicates that the population has reached a minimum population size, and the appropriate target gene(s) is activated via the action of a member of the LuxR family of transcriptional activators (18,45,51). The LuxR protein appears to consist of two domains containing an AHL binding site within the N-terminal domain and a helix-turn-helix DNA binding motif located within the C-terminal domain (10,22).In general, the signal generator proteins responsible for the synthesis of AHLs belong to the LuxI family, the archetypal member of which was originally identified within the Vibrio (Photobacterium) fischeri lux operon as the gene product responsible for the synthesis of N-(3-oxohexanoyl)homoserine lactone (13,14,16). However, AHL synthesis is not always directed by a LuxI homolog. In both V. ...

Plasmid reporter vectors have been constructed which respond to activation of LuxR and its homologues LasR and RhlR (VsmR) by N-acyl homoserine lactones (AHLs). The expression of luxCDABE from transcriptional fusions to PluxI, PlasI and PrhlI respectively, occurs in the presence of activating AHLs. A profile of structure/activity relationships is seen where the natural ligand is most potent. The characterisation of individual LuxR homologue/AHL combinations allows a comprehensive evaluation of quorum sensing signals from a test organism.

In Aeromonas hydrophila, the ahyI gene encodes a protein responsible for the synthesis of the quorum sensing signal N-butanoyl-l-homoserine lactone (C4-HSL). Inactivation of the ahyI gene on the A. hydrophila chromosome abolishes C4-HSL production. The exoprotease activity of A. hydrophila consists of both serine protease and metalloprotease activities; in theahyI-negative strain, both are substantially reduced but can be restored by the addition of exogenous C4-HSL. In contrast, mutation of the LuxR homolog AhyR results in the loss of both exoprotease activities, which cannot be restored by exogenous C4-HSL. Furthermore, a substantial reduction in the production of exoprotease by the ahyI + parent strain is obtained by the addition of N-acylhomoserine lactone analogs that have acyl side chains of 10, 12, or 14 carbons. The inclusion ofN-(3-oxododecanoyl)-l-homoserine lactone orN-(3-oxotetradecanoyl)-l-homoserine lactone at 10 μM in overnight cultures of A. hydrophila abolishes exoprotease production in azocasein assays and reduces the activity of all the exoprotease species seen in zymograms.