Numerous species of bacteria use an elegant regulatory mechanism known as quorum sensing to control the expression of specific genes in a cell-density dependent manner. In Gram-negative bacteria, quorum sensing systems function through a cell-to-cell signal molecule (autoinducer) that consists of a homoserine lactone with a fatty acid side chain. Such is the case in the opportunistic human pathogen Pseudomonas aeruginosa, which contains two quorum sensing systems (las and rhl) that operate via the autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-Lhomoserine lactone. The study of these signal molecules has shown that they bind to and activate transcriptional activator proteins that specifically induce numerous P. aeruginosa virulence genes. We report here that P. aeruginosa produces another signal molecule, 2-heptyl-3-hydroxy-4-quinolone, which has been designated as the Pseudomonas quinolone signal. It was found that this unique cell-to-cell signal controlled the expression of lasB, which encodes for the major virulence factor, LasB elastase. We also show that the synthesis and bioactivity of Pseudomonas quinolone signal were mediated by the P. aeruginosa las and rhl quorum sensing systems, respectively. The demonstration that 2-heptyl-3-hydroxy-4-quinolone can function as an intercellular signal sheds light on the role of secondary metabolites and shows that P. aeruginosa cell-to-cell signaling is not restricted to acyl-homoserine lactones.

A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of Pseudomonas aeruginosa PAO1. The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects. The largest set of pleiotropic mutations blocked the production of the extracellular Pseudomonas quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes. Most of these pqs mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented. Two of the genes required for PQS synthesis (phnA and phnB) had previously been assumed to encode phenazine biosynthetic functions. The transcription of one of the genes required for PQS synthesis (PA2587/pqsH) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation. Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice.A complex network of regulatory factors governs the production of secondary metabolites and other virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. This network regulates gene expression in response to stimuli such as growth phase, culture density, and oxygen and iron availability (12,26,28,37). Central components of the network are the las and rhl quorum-sensing systems, which activate gene expression in response to culture density (13). Each system is made up of two genes, one encoding an enzyme which produces a specific acylated homoserine lactone autoinducer (lasI/rhlI), and a second encoding a transcriptional activator that binds the corresponding autoinducer (lasR/rhlR). The las system directs expression of virulence factors such as elastases A and B and alkaline protease (16,25). The rhl system directs expression of rhamnolipid biosynthesis enzymes, pyocyanin biosynthesis enzymes, and hydrogen cyanide synthase (3,24,28). In addition, LasI/R regulates expression of both itself and rhlI (1, 26). The las and rhl systems together have been shown to influence the expression of over two hundred genes (36).Recently, a third signaling system based on 2-heptyl-3-hydroxy-4-quinolone, designated the Pseudomonas quinolone signal (PQS), has been shown to be a part of the quorumsensing regulatory network in P. aeruginosa (27). The production of PQS depends on lasR (27), and exogenous PQS strongly induces expression of elastase B and rhlI in a lasR mutant background (22). These results place PQS between the las and rhl quorum-sensing systems in the quorum-sensing regulatory network (22).We have described a process ("paralytic killing") in which P. aeruginosa PAO1 rapidly kills the nematode Caenorhabditis elegans by cyanide poisoning (8, 14). Previous studies of a different P....

The opportunistic pathogen Pseudomonas aeruginosa uses intercellular signals to control the density-dependent expression of many virulence factors. The las and rhl quorum-sensing systems function, respectively, through the autoinducers N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone (C 4 -HSL), which are known to positively regulate the transcription of the elastase-encoding gene, lasB. Recently, we reported that a second type of intercellular signal is involved in lasB induction. This signal was identified as 2-heptyl-3-hydroxy-4-quinolone and designated the Pseudomonas quinolone signal (PQS). PQS was determined to be part of the quorum-sensing hierarchy since its production and bioactivity depended on the las and rhl quorum-sensing systems, respectively. In order to define the role of PQS in the P. aeruginosa quorumsensing cascade, lacZ gene fusions were used to determine the effect of PQS on the transcription of the quorum-sensing system genes lasR, lasI, rhlR, and rhlI. We found that in P. aeruginosa, PQS caused a major induction of rhlI-lacZ and had lesser effects on the transcription of lasR-lacZ and rhlR-lacZ. We also observed that the transcription of both rhlI-lacZ and lasB-lacZ was cooperatively effected by C 4 -HSL and PQS. Additionally, we present data indicating that PQS was not produced maximally until cultures reached the late stationary phase of growth. Taken together, our results imply that PQS acts as a link between the las and rhl quorum-sensing systems and that this signal is not involved in sensing cell density.Pseudomonas aeruginosa is a ubiquitous environmental organism capable of infecting a wide variety of animals, plants, and insects. As a human pathogen, this bacterium is a major source of opportunistic infections in both immunocompromised individuals and cystic fibrosis patients. P. aeruginosa is now the leading source of gram-negative nosocomial infections (25) and causes chronic lung infections in approximately 90% of cystic fibrosis patients (7). The ability of this organism to cause devastating infections stems from the production of an arsenal of virulence factors, several of which are controlled according to cell density through an elegant mechanism known as quorum sensing. In gram-negative bacteria, most quorumsensing systems consist of a LuxR-type transcriptional activator (R protein) and an acylated homoserine lactone signal molecule (autoinducer) (see reference 4 for a review). When a bacterial culture is at a low cell density, basal levels of autoinducer and R protein are produced. As a population grows, the concentration of autoinducer increases with cell density until it reaches a threshold concentration where it binds to and thereby activates an R protein. Activated R protein then activates specific genes, causing cell density-dependent gene expression.In P. aeruginosa, there are at least two quorum-sensing systems, las and rhl, which control the expression of numerous genes (see reference 21 for a review). The las quorum-sensing system consis...