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 violaceum, 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 C. violaceum (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. violaceum 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. violaceum 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 ail the AHL and AHT compounds evaluated with N-acyl side chains from C₄ to C₈ in length, with varying degrees of sensitivity. Although AHL compounds with N-acyl side chains from C₁₀ to C₁₄ 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.

For many years bacteria were considered primarily as autonomous unicellular organisms with little capacity for collective behaviour. However, we now appreciate that bacterial cells are in fact, highly communicative. The generic term ‘quorum sensing’ has been adopted to describe the bacterial cell-to-cell communication mechanisms which co-ordinate gene expression usually, but not always, when the population has reached a high cell density. Quorum sensing depends on the synthesis of small molecules (often referred to as pheromones or autoinducers) that diffuse in and out of bacterial cells. As the bacterial population density increases, so does the synthesis of quorum sensing signal molecules, and consequently, their concentration in the external environment rises. Once a critical threshold concentration has been reached, a target sensor kinase or response regulator is activated (or repressed) so facilitating the expression of quorum sensing-dependent genes. Quorum sensing enables a bacterial population to mount a co-operative response that improves access to nutrients or specific environmental niches, promotes collective defence against other competitor prokaryotes or eukaryotic defence mechanisms and facilitates survival through differentiation into morphological forms better able to combat environmental threats. Quorum sensing also crosses the prokaryotic–eukaryotic boundary since quorum sensing-dependent signalling can be exploited or inactivated by both plants and mammals.

Summary: In Pseudomonas aeruginosa, diverse exoproduct virulence determinants are regulated via N‐acylhomoserine lactone‐dependent quorum sensing. Here we show that 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) is also an integral component of the quorum sensing circuitry and is required for the production of rhl‐dependent exoproducts at the onset of stationary phase. Analysis of spent P. aeruginosa culture supernatants revealed that PQS is produced at the end of exponential phase in the parent strain and in the late stationary phase of a lasR mutant. Mutants defective in both PQS production (pqsR‐) and response (pqsE‐) produced substantially reduced levels of exoproducts but retained wild‐type N‐butanoyl homoserine lactone (C4‐HSL) levels. In the wild type, provision of exogenous PQS at the time of inoculation significantly increased PA‐IL lectin, pyocyanin and elastase production during early stationary phase and promoted biofilm formation. Exogenous PQS but not PQS derivatives lacking the 3‐hydroxy group overcame the cell density but not growth phase‐dependent production of exoproducts. PQS also overcame the transcriptional and post‐transcriptional repression of lecA (which codes for the PA‐IL lectin) mediated via the negative regulators MvaT and RsmA respectively. Increased expression of lecA in the presence of exogenous PQS can be explained partially by increases in RhlR, RpoS and C4‐HSL levels. A refined model for quorum sensing in P. aeruginosa is presented.

In gram-negative bacterial pathogens, such asPseudomonas aeruginosaandYersinia pseudotuberculosis, cell-to-cell communication via theN-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures ofY. pseudotuberculosisandP. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the majorP. aeruginosa[N-butanoylhomoserine lactone (C4-HSL) andN-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] andY. pseudotuberculosis[N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) andN-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL),N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as theN-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, andN-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37°C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require anN-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.

Although unicellular, bacteria are highly interactive and employ a range of cell-to-cell communication or ‘quorum sensing (QS)’ systems for promoting collective behaviour within a population. QS is generally considered to facilitate gene expression only when the population has reached a sufficient cell density and depends on the synthesis of small molecules that diffuse in and out of bacterial cells. As the bacterial population density increases, so does the synthesis of QS signal molecules and consequently, their concentration in the external environment increases. Once a critical threshold concentration is reached, a target sensor kinase or response regulator is activated, so facilitating the expression of QS-dependent target genes. Several chemically distinct families of QS signal molecules have been described, of which the N-acylhomoserine lactone (AHL) family in Gram-negative bacteria have been the most intensively investigated. QS contributes to environmental adaptation by facilitating the elaboration of virulence determinants in pathogenic species and plant biocontrol characteristics in beneficial species as well as directing biofilm formation and colony escape. QS also crosses the prokaryotic–eukaryotic boundary in that QS signal molecules influence the behaviour of eukaryotic organisms in both the plant and mammalian worlds such that QS signal molecules may directly facilitate bacterial survival by promoting an advantageous lifestyle within a given environmental niche.