Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P. aeruginosa between survival of the many and persistence of the few.Pseudomonas aeruginosa, although readily defined taxonomically (69), shows no evidence of being constrained to a particular bacterial lifestyle (72). Indeed, this opportunistic pathogen proliferates within hosts as varied as plants, insects, nematodes, and mammals and interacts with amoebae and yeasts as well (13,16,31,44,58). The broad host range of P. aeruginosa can be used both to uncover the role of specific virulence factors and to characterize new regulatory systems controlling virulence (10,12,13,16,21,44,58). One study identified transposon insertion mutants of P. aeruginosa strain PAO1 that were less virulent in the fruit fly model host (16). Most such strains had an insertion in a chemotaxis-like regulatory locus (PA0408 to PA0417) required for surface spreading by twitching motility and controlling unknown factors required for efficient fly killing. Based only on their distinctive compact colony morphology, a large collection of these mutants was assembled for testing in the fly model.In this same study (16), two even more striking colony morphologies were noted, and strains of each type were saved. One set of mutants formed wrinkled colonies and had additional properties described for autoaggregating cells of Pseudomonas fluorescens (59,68) and Salmonella enterica serovar Typhimurium (61,62,83). The second set of mutants formed colonies with visible lysis, a characteristic noted in some of the earliest descriptions of P. aeruginosa isolates (5, 6, 26, 32), but for which the molecular basis has been elusive. Since autoaggregation and autolysis are likely to reflect fundamental aspects of the biology of P. aeruginosa, the genetic basis of the two mutant colony morphologies was explored. Autoaggregat...

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and ␤-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium that can infect insects, plants, and animals. As an opportunistic pathogen of humans, P. aeruginosa causes acute infections in immunocompromised individuals and chronic lung infections in cystic fibrosis patients. Such infections are made possible through the production of an arsenal of virulence factors, many of which are regulated by cell-to-cell signals (see reference 36 for a review). P. aeruginosa produces at least three small compounds that function as intercellular communication signals. The acyl homoserine lactone signals, N-(3-oxododecanoyl) homoserine lactone (3-oxo-C 12 -HSL) and Nbutyryl homoserine lactone (C 4 -HSL), have been well studied and function in combination with the LuxR homologs LasR and RhlR, respectively (16,30,31,33,34). Together, these quorum-sensing signals control 6 to 11% of the P. aeruginosa genome (44, 46, 48). The third P. aeruginosa intercellular signal is a quinolone compound that was identified as 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal [PQS]) (37). This signal controls multiple virulence factors and is intertwined in the quorum-sensing cascade, where it appears to be a regulatory link between the las and rhl quorum-sensing systems (14,27). PQS is produced in the lungs of cystic fibrosis patients infected with P. aeruginosa (9) and is required for v...

SummaryFarnesol is a sesquiterpene produced by many organisms, including the fungus Candida albicans. Here, we report that the addition of farnesol to cultures of Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, leads to decreased production of the Pseudomonas quinolone signal (PQS) and the PQS-controlled virulence factor, pyocyanin. Within 15 min of farnesol addition, decreased transcript levels of pqsA, the first gene in the PQS biosynthetic operon, were observed. Transcript levels of pqsR (mvfR), which encodes the transcription factor that positively regulates pqsA, were unaffected. An Escherichia coli strain producing PqsR and containing the pqsA promoter fused to lacZ similarly showed that farnesol inhibited PQS-stimulated transcription. Electrophoretic mobility shift assays showed that, like PQS, farnesol stimulated PqsR binding to the pqsA promoter at a previously characterized LysR binding site, suggesting that farnesol promoted a non-productive interaction between PqsR and the pqsA promoter. Growth with C. albicans leads to decreased production of PQS and pyocyanin by P. aeruginosa, suggesting that the amount of farnesol produced by the fungus is sufficient to impact P. aeruginosa PQS signalling. Related isoprenoid compounds, but not other long-chain alcohols, also inhibited PQS production at micromolar concentrations, suggesting that related compounds may participate in interspecies interactions with P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that controls numerous virulence factors through intercellular signals. This bacterium has two quorum-sensing systems (las and rhl), which act through the intercellular signals N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12-HSL) and N-butyryl-L-homoserine lactone (C 4-HSL), respectively. P. aeruginosa also produces a third intercellular signal that is involved in virulence factor regulation. This signal, 2-heptyl-3-hydroxy-4-quinolone [referred to as the Pseudomonas quinolone signal (PQS)], is a secondary metabolite that is part of the P. aeruginosa quorum-sensing hierarchy. PQS can induce both lasB (encodes LasB elastase) and rhlI (encodes the C 4-HSL synthase) in P. aeruginosa and is produced maximally during the late stationary phase of growth. Because PQS is an intercellular signal that is part of the quorum-sensing hierarchy and controls multiple virulence factors, we began basic studies designed to elucidate its biosynthetic pathway. First, we present data that strongly suggest that anthranilate is a precursor for PQS. P. aeruginosa converted radiolabeled anthranilate into radioactive PQS, which was bioactive. We also found that an anthranilate analog (methyl anthranilate) would inhibit the production of PQS. This analog was then shown to have a major negative effect on elastase production by P. aeruginosa. These data provide evidence that precursors of intercellular signals may provide viable targets for the development of therapeutic treatments that will reduce P. aeruginosa virulence.

Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro-and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.Pseudomonas aeruginosa is a common environmental organism and also an opportunistic human pathogen. A large number of the virulence factors which it produces during infection are regulated by low-molecular-weight molecules which are part of the cell-to-cell signaling systems of this organism. One class of signals, which are common in gram-negative bacteria, is the acyl homoserine lactone molecules. In the case of P. aeruginosa, the molecules in this class are N-(3-oxo-dodecanoyl)-L-homoserine lactone, which is the signal of the las quorum-sensing system, and N-butanoyl-homoserine lactone, which is the signal of the rhl quorum-sensing system (28,40,(43)(44)(45). The second signal class, the members of which thus far appear to be synthesized only by P. aeruginosa and certain Burkholderia species (22), is based on the 2-alkyl-4-quinolone structure. The compound 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS), has been shown to control the expression of multiple virulence factors in P. aeruginosa (20,37,47). PQS has been shown to act as a ligand for PqsR, the transcriptional activator which controls the pqsABCDE operon (57). The biosynthesis of PQS appears to involve the condensation of anthranilate with a 10-carbon fatty acid and requires the expression of multiple genetic operons (Fig. 1) (10,12,19,27). Based on protein similarities, the pqsABCDE operon is thought to code for the activating and condensation enzymes (except for pqsE, which is not required for bi...