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...

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 pathogen that causes both acute and chronic infections in immunocompromised individuals. This gram-negative bacterium produces a battery of virulence factors that allow it to infect and survive in many different hostile environments. The control of many of these virulence factors falls under the influence of one of three P. aeruginosa cell-to-cell signaling systems. The focus of this study, the quinolone signaling system, functions through the Pseudomonas quinolone signal (PQS), previously identified as 2-heptyl-3-hydroxy-4-quinolone. This signal binds to and activates the LysR-type transcriptional regulator PqsR (also known as MvfR), which in turn induces the expression of the pqsABCDE operon. The first four genes of this operon are required for PQS synthesis, but the fifth gene, pqsE, is not. The function of the pqsE gene is not known, but it is required for the production of multiple PQS-controlled virulence factors and for virulence in multiple models of infection. In this report, we show that PqsE can activate PQS-controlled genes in the absence of PqsR and PQS. Our data also suggest that the regulatory activity of PqsE requires RhlR and indicate that a pqsE mutant can be complemented for pyocyanin production by a large excess of exogenous N-butyryl homoserine lactone (C 4 -HSL). Finally, we show that PqsE enhances the ability of Escherichia coli expressing RhlR to respond to C 4 -HSL. Overall, our data lead us to conclude that PqsE functions as a regulator that is independent of PqsR and PQS but dependent on the rhl quorum-sensing system.Pseudomonas aeruginosa is a serious opportunistic pathogen that can cause infections in a diverse group of organisms from the animal, plant, and insect kingdoms (9, 47, 53). In humans, P. aeruginosa can cause multiple types of infections that are generally difficult to treat due to the high level of antibiotic resistance exhibited by this microbe (15). These infections are complex and generally involve numerous virulence determinants, with some factors playing a larger role than others in different types of infections (16). One aspect of P. aeruginosa virulence that seems to be important in most infections is the ability of the bacteria to communicate via intercellular signals (11). P. aeruginosa uses at least three cell-to-cell signaling systems to control the expression of assorted virulence factors. There are two classical acyl-homoserine lactone (HSL)-based quorum-sensing systems, the las and rhl systems (see reference 43 for a review of P. aeruginosa quorum sensing). The las and rhl quorum-sensing systems function via the signals N-(3-oxododecanoyl) HSL (3-oxo-C 12 -HSL) and N-butyryl HSL (C 4 -HSL), respectively (40, 41). When at a threshold concentration, 3-oxo-C 12 -HSL and C 4 -HSL serve as coinducers for the transcriptional activators LasR and RhlR, respectively (21,35,36,39). The other cell-to-cell signaling system of P. aeruginosa functions through the quinolone compound 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas ...

Bacterial bile salt hydrolases catalyze the degradation of conjugated bile acids in the mammalian gut. The crystal structures of conjugated bile acid hydrolase (CBAH) from Clostridium perfringens as apoenzyme and in complex with taurodeoxycholate that was hydrolyzed to the reaction products taurine and deoxycholate are described here at 2.1 and 1.7 A resolution, respectively. The crystal structures reveal close relationship between CBAH and penicillin V acylase from Bacillus sphaericus. This similarity together with the N-terminal cysteine classifies CBAH as a member of the N-terminal nucleophile (Ntn) hydrolase superfamily. Both crystal structures show an identical homotetrameric organization with dihedral (D(2) or 222) point group symmetry. The structure analysis of C. perfringens CBAH identifies critical residues in catalysis, substrate recognition, and tetramer formation which may serve in further biochemical characterization of bile acid hydrolases.