Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR).

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.

A Pseudomonas aeruginosa PQS quorum-sensing system inhibitor with anti-staphylococcal activity sensitizes polymicrobial biofilms to tobramycin.

As single- and mixed-species biofilms, Staphylococcus aureus and Pseudomonas aeruginosa cause difficult-to-eradicate chronic infections. In P. aeruginosa, pseudomonas quinolone (PQS)-dependent quorum sensing regulates virulence and biofilm development that can be attenuated via antagonists targeting the transcriptional regulator PqsR (MvfR). Here, we exploited a quinazolinone (QZN) library including PqsR agonists and antagonists for their activity against S. aureus alone, when co-cultured with P. aeruginosa, and in combination with the aminoglycoside tobramycin. The PqsR inhibitor, QZN 34 killed planktonic Gram-positives but not Gram-negatives. QZN 34 prevented S. aureus biofilm formation, severely damaged established S. aureus biofilms, and perturbed P. aeruginosa biofilm development. Although P. aeruginosa protected S. aureus from tobramycin in mixed biofilms, the combination of aminoglycoside antibiotic with QZN 34 eradicated the mixed-species biofilm. The mechanism of action of QZN 34 toward Gram-positive bacteria is shown to involve membrane perturbation and dissipation of transmembrane potential.

Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia.

BACKGROUND: Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novel N-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest. RESULTS: By using a basal growth medium containing N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) as the sole source of carbon and nitrogen, the ginger rhizosphere associated bacteria were enriched for strains with AHL-degrading capabilities. Three isolates belonging to the genera Acinetobacter (GG2), Burkholderia (GG4) and Klebsiella (Se14) were identified and selected for further study. Strains GG2 and Se14 exhibited the broadest spectrum of AHL-degrading activities via lactonolysis while GG4 reduced 3-oxo-AHLs to the corresponding 3-hydroxy compounds. In GG2 and GG4, QQ was found to co-exist with AHL-dependent QS and GG2 was shown to inactivate both self-generated and exogenously supplied AHLs. GG2, GG4 and Se14 were each able to attenuate virulence factor production in both human and plant pathogens. CONCLUSIONS: Collectively our data show that ginger rhizosphere bacteria which make and degrade a wide range of AHLs are likely to play a collective role in determining the QS-dependent phenotype of a polymicrobial community.

Simultaneous quantitative profiling of N-acyl-L-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signaling molecules using LC-MS/MS.

An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-L-homoserine lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratios of AHLs and AQs produced by Pseudomonas aeruginosa and the consequences of mutating individual or multiple QSSM synthase genes (lasI, rhlI, pqsA) on AHL and AQ profiles and concentrations. The AHL profile of P. aeruginosa was dominated by N-butanoyl-L-homoserine lactone (C4-HSL) with lesser concentrations of N-hexanoyl-L-homoserine lactone (C6-HSL) and 3-oxo-substituted longer chain AHLs including N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). The AQ profile of P. aeruginosa comprised the C7 and C9 long alkyl chain AQs including 2-heptyl-4-hydroxyquinoline (HHQ), 2-nonyl-4-hydroxyquinoline, the "pseudomonas quinolone signal" (2-heptyl-3-hydroxy-4-quinolone) and the N-oxides, 2-heptyl-4-hydroxyquinoline N-oxide and 2-nonyl-4-hydroxyquinoline N-oxide. Application of the method showed significant effects of growth medium type on the ratio and the nature of the QSSMs synthesized and the dramatic effect of single, double and triple mutations in the P. aeruginosa QS synthase genes. The LC-MS/MS methodology is applicable in organisms where either or both AHL and AQ QSSMs are produced and can provide comprehensive profiles and concentrations from a single sample.

Synthesis and biotransformation of 2-alkyl-4(1H)-quinolones by recombinant Pseudomonas putida KT2440.

2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the "Pseudomonas quinolone signal" (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 µM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C3- to C13-saturated and C7:1 to C13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and ß carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C5-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in > 90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.

The Pseudomonas aeruginosa 4-quinolone signal molecules HHQ and PQS play multifunctional roles in quorum sensing and iron entrapment.

Pseudomonas aeruginosa produces 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), a quorum-sensing (QS) signal that regulates numerous virulence genes including those involved in iron scavenging. Biophysical analysis revealed that 2-alkyl-3-hydroxy-4-quinolones form complexes with iron(III) at physiological pH. The overall stability constant of 2-methyl-3-hydroxy-4-quinolone iron(III) complex was log beta(3) = 36.2 with a pFe(3+) value of 16.6 at pH 7.4. PQS was found to operate via at least three distinct signaling pathways, and its precursor, 2-heptyl-4-quinolone (HHQ), which does not form an iron complex, was discovered to function as an autoinducer molecule per se. When PQS was supplied to a P. aeruginosa mutant unable to make pyoverdine or pyochelin, PQS associated with the cell envelope and inhibited bacterial growth, a finding that reveals a secondary function for PQS in iron entrapment to facilitate siderophore-mediated iron delivery.

Functional genetic analysis reveals a 2-Alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria.

Pseudomonas aeruginosa synthesizes diverse 2-alkyl-4(1H)-quinolones (AHQs), including the signaling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), via the pqsABCDE locus. By examining the genome databases, homologs of the pqs genes were identified in other bacteria. However, apart from P. aeruginosa, only Burkholderia pseudomallei and B. thailandensis contained a complete pqsA-E operon (termed hhqA-E). By introducing the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants, we show that they are functionally conserved and restore virulence factor and PQS production. B. pseudomallei, B. thailandensis, B. cenocepacia, and P. putida each produced 2-heptyl-4(1H)-quinolone (HHQ), but not PQS. Mutation of hhqA in B. pseudomallei resulted in the loss of AHQ production, altered colony morphology, and enhanced elastase production, which was reduced to parental levels by exogenous HHQ. These data reveal a role for AHQs in bacterial cell-to-cell communication beyond that seen in P. aeruginosa.

Bacterial N-acylhomoserine lactone-induced apoptosis in breast carcinoma cells correlated with down-modulation of STAT3.

Cell growth is promoted by mitogens and survival factors, which activate intracellular signalling pathways to control cell cycle progression and cellular integrity. Proliferation signals are transmitted through Ras and Rho family small G-proteins coupled to mitogen-activated protein kinase (MAPK) cascades, while survival signals are propagated by lipid-dependent kinases such as phosphatidylinositide 3-kinases (PI3Ks) and protein kinase B (Akt/PKB). Recently, signal transducer and activator of transcription (STAT) proteins were identified as positive regulators of proliferation in a variety of cell types. Persistent activation of these pathways is associated with tumour cell growth, whereas their inhibition can halt proliferation and precipitate apoptotic cell death. The human pathogen Pseudomonas aeruginosa uses quorum-sensing signal molecules (QSSMs) to regulate virulence gene expression. QSSMs also suppress host immune responses although the mechanism of suppression is unknown. Here, we demonstrate that the QSSM N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) from P. aeruginosa blocks proliferation and induces apoptosis in human BC cell lines. Analyses of signalling events reveal that OdDHL has little or no effect on MAPK cascades, partially inhibits the Akt/PKB pathway and ablates STAT3 activity. Pharmacological inhibition of each pathway independently indicates that STAT3 activity is critical for BC cell proliferation and survival, while a constitutively active STAT3 confers resistance to OdDHL. These results support the notion of OdDHL as a bioactive molecule in eukaryotic systems and a paradigm for a novel class of antiproliferative compounds.

Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators.

Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone (3O, C(12)-HSL), is described. Twenty-four single or combination systematic alterations of the structural components of 3O, C(12)-HSL were introduced as described. Given the already defined immunological profile of the parent compound, 3O, C(12)-HSL, these compounds were assayed for their ability to inhibit murine and human leucocyte proliferation and TNF-alpha secretion by lipopolysaccharide (LPS) stimulated human leucocytes in order to provide an initial structure-activity profile. From IC(50) values obtained with a murine splenocyte proliferation assay, it is apparent that acylated l-homoserine lactones with an 11-13 C side chain containing either a 3-oxo or a 3-hydroxy group are optimal structures for immune suppressive activity. These derivatives of 3O, C(12)-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain were also potent immune suppressive agents. However, structures lacking the homoserine lactone ring, structures lacking the l-configuration at the chiral center, and those with polar substituents were essentially devoid of activity. The ability of compounds selected from the optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell proliferation and LPS-induced TNF-alpha secretion indicates the suitability of these compounds for further investigation in relation to their molecular mechanisms of action in TNF-alpha driven immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid arthritis, and type 1 (autoimmune) diabetes.

Recent progress in the design of selectin inhibitors.

The selectins are a family of cell-adhesion proteins that mediate the early stages of leukocyte recruitment from the blood stream to sites of tissue damage through recognition of the carbohydrate epitope sialyl Lewis(x) (sLe(x)). Current development of small molecule based inhibitors of this process and their clinical potential to address numerous acute and chronic diseases are explored.

Substrate-assisted O2 activation in a cofactor-independent dioxygenase.

In contrast to the majority of O2-activating enzymes, which depend on an organic cofactor or a metal ion for catalysis, a particular group of structurally unrelated oxygenases is functional without any cofactor. In this study, we characterized the mechanism of O2 activation in the reaction pathway of a cofactor-independent dioxygenase with an α/ß-hydrolase fold, which catalyzes the oxygenolytic cleavage of 2-alkyl-3-hydroxy-4(1H)-quinolones. Chemical analysis and electron paramagnetic resonance spectroscopic data revealed that O2 activation in the enzyme's active site is substrate-assisted, relying on single electron transfer from the bound substrate anion to O2 to form a radical pair, which recombines to a C2-peroxide intermediate. Thus, an oxygenase can function without a cofactor, if the organic substrate itself, after activation to a (carb)anion by an active-site base, is intrinsically reactive toward molecular oxygen.

Targeting Staphylococcus aureus quorum sensing with nonpeptidic small molecule inhibitors.

A series of 3-oxo-C12-HSL, tetramic acid, and tetronic acid analogues were synthesized to gain insights into the structural requirements for quorum sensing inhibition in Staphylococcus aureus. Compounds active against agr were noncompetitive inhibitors of the autoinducing peptide (AIP) activated AgrC receptor, by altering the activation efficacy of the cognate AIP-1. They appeared to act as negative allosteric modulators and are exemplified by 3-tetradecanoyltetronic acid 17, which reduced nasal cell colonization and arthritis in a murine infection model.

Quinolones: from antibiotics to autoinducers.

Since quinine was first isolated, animals, plants and microorganisms producing a wide variety of quinolone compounds have been discovered, several of which possess medicinally interesting properties ranging from antiallergenic and anticancer to antimicrobial activities. Over the years, these have served in the development of many synthetic drugs, including the successful fluoroquinolone antibiotics. Pseudomonas aeruginosa and related bacteria produce a number of 2-alkyl-4(1H)-quinolones, some of which exhibit antimicrobial activity. However, quinolones such as the Pseudomonas quinolone signal and 2-heptyl-4-hydroxyquinoline act as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. Here, we review selectively this extensive family of bicyclic compounds, from natural and synthetic antimicrobials to signalling molecules, with a special emphasis on the biology of P. aeruginosa. In particular, we review their nomenclature and biochemistry, their multiple properties as membrane-interacting compounds, inhibitors of the cytochrome bc(1) complex and iron chelators, as well as the regulation of their biosynthesis and their integration into the intricate quorum-sensing regulatory networks governing virulence and secondary metabolite gene expression.

Immunosuppressive but non-LasR-inducing analogues of the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone.

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (1) is involved not only in bacterial activation but also in subversion of the host immune system, and this compound might thus be used as a template to design immunosuppressive agents, provided derivatives devoid of quorum-sensing activity could be discovered. By use of a leukocyte proliferation assay and a newly developed bioluminescent P. aeruginosa reporter assay, systematic modification of 1 allowed us to delineate the bacterial LasR-induction and host immunosuppressive activities. The main determinant is replacement of the methylene group proximal to the ß-ketoamide in the acyl chain of 1 with functions containing heteroatoms, especially an NH group. This modification can be combined with replacement of the homoserine lactone system in 1 with stable cyclic groups. For example, we found the simple compound N(1)-(5-chloro-2-hydroxyphenyl)-N(3)-octylmalonamide (25d) to be over twice as potent as 1 as an immune suppressor while displaying LasR-induction antagonist activity.

Dioxygenase-mediated quenching of quinolone-dependent quorum sensing in Pseudomonas aeruginosa.

2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) is a quorum-sensing signal molecule used by Pseudomonas aeruginosa. The structural similarity between 3-hydroxy-2-methyl-4(1H)-quinolone, the natural substrate for the 2,4-dioxygenase, Hod, and PQS prompted us to investigate whether Hod quenched PQS signaling. Hod is capable of catalyzing the conversion of PQS to N-octanoylanthranilic acid and carbon monoxide. In P. aeruginosa PAO1 cultures, exogenously supplied Hod protein reduced expression of the PQS biosynthetic gene pqsA, expression of the PQS-regulated virulence determinants lectin A, pyocyanin, and rhamnolipids, and virulence in planta. However, the proteolytic cleavage of Hod by extracellular proteases, competitive inhibition by the PQS precursor 2-heptyl-4(1H)-quinolone, and PQS binding to rhamnolipids reduced the efficiency of Hod as a quorum-quenching agent. Nevertheless, these data indicate that enzyme-mediated PQS inactivation has potential as an antivirulence strategy against P. aeruginosa.

N-Acylhomoserine lactone-mediated quorum sensing: a twist in the tail and a blow for host immunity.

Communication through quorum sensing (QS) enables bacterial populations to coordinate their behavior. Recent work on N-acylhomoserine lactone-mediated QS has revealed that some soil bacteria exploit host-derived substrates to generate an alternative N-substituted homoserine lactone. New light has also been shed on the mechanism by which N-(3-oxo-dodecanoyl)-L-homoserine lactone modulates host inflammatory signaling pathways to promote bacterial survival.

Comprehensive profiling of N-acylhomoserine lactones produced by Yersinia pseudotuberculosis using liquid chromatography coupled to hybrid quadrupole-linear ion trap mass spectrometry.

A method for the comprehensive profiling of the N-acylhomoserine lactone (AHL) family of bacterial quorum-sensing molecules is presented using liquid chromatography (LC) coupled to hybrid quadrupole-linear ion trap (QqQLIT) mass spectrometry. Information-dependent acquisition (IDA), using triggered combinations of triple-quadrupole and linear ion trap modes in the same LC-MS/MS run, was used to simultaneously screen, quantify and identify multiple AHLs in a single sample. This MS method uses common AHL fragment ions attributed to the homoserine moiety and the 3-oxo-, 3-hydroxy- or unsubstituted acyl side chains, to identify unknown AHLs in cell-free culture supernatants in an unbiased manner. This LC-MS technique was applied to determine the relative molar ratios of AHLs produced by Yersinia pseudotuberculosis and the consequences of inactivating by mutation either or both of the AHL synthase genes (ypsI and ytbI) on AHL profile and concentration. The Y. pseudotuberculosis wild type but not the ypsI ytbI double mutant produced at least 24 different AHLs with acyl chains ranging from C4 to C15 with or without 3-oxo or 3-hydroxy substituents. YtbI, in contrast to YpsI, could direct the synthesis of all of the AHLs identified. The most abundant and hence most biologically relevant Y. pseudotuberculosis AHLs were found to be the 3-oxo-substituted C6, C7 and C8 AHLs and the unsubstituted C6 and C8 compounds. The LC-QqQLIT methodology is broadly applicable to quorum-sensing signal molecule analysis and can provide comprehensive AHL profiles and concentrations from a single sample and simultaneously collect confirmatory spectra for each AHL identified.

The immunomodulatory Pseudomonas aeruginosa signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone enters mammalian cells in an unregulated fashion.

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.

N-acyl homoserine lactones are degraded via an amidolytic activity in Comamonas sp. strain D1.

Comamonas strain D1 enzymatically inactivates quorum-sensing (QS) signal molecules of the N-acyl homoserine lactone (N-AHSL) family, and exhibits the broadest inactivation range of known bacteria. It degrades N-AHSL with acyl-side chains ranging from 4 to 16 carbons, with or without 3-oxo or 3-hydroxy substitutions. N-AHSL degradation yields HSL but not N-acyl homoserine: strain D1 therefore harbors an amidohydrolase activity. Strain D1 is the fifth bacterium species in which an N-AHSL amidohydrolase is described. Consistent with its N-AHSL degradation ability, strain D1 efficiently quenches various QS-dependent functions in other bacteria, such as violacein production by Chromobacterium violaceum and pathogenicity and antibiotic production in Pectobacterium.

A dual biosensor for 2-alkyl-4-quinolone quorum-sensing signal molecules.

Pseudomonas, Burkholderia and Alteromonas species produce diverse 2-alkyl-4-quinolones (AHQs) which inhibit the growth of bacteria, algae and phytoplankton, chelate iron, modulate mammalian host immune defences and act as quorum-sensing (QS) signal molecules. To facilitate the detection, identification and quantification of the major Pseudomonas aeruginosa AHQs 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) we developed two different AHQ biosensors. These were constructed by introducing either a lecA::luxCDABE or a pqsA::luxCDABE reporter gene fusion into a P. aeruginosa pqsA mutant which cannot synthesize AHQs. While both biosensors responded similarly to PQS (EC(50) 18 +/- 4 microM), the pqsA::luxCDABE biosensor was most sensitively activated by HHQ (EC(50) 0.44 +/- 0.1 microM). This biosensor was also activated albeit less sensitively by (i) PQS analogues with alkyl chains varying from C1 to C11, (ii) HHQ analogues with C9 and C11 alkyl chains and (iii) 2-heptyl-4-hydroxyquinoline-N-oxide (HHQNO). The AHQ biosensor also responded differentially to the AHQs present in cell free culture supernatants prepared from PAO1 and isogenic strains carrying mutations in genes (pqsA, pqsH, lasR, lasI, rhlR, rhlI) known to influence AHQ production. The AHQ profiles of P. aeruginosa strains was also evaluated by overlaying thin layer chromatogram (TLC) plates with the pqsA::luxCDABE biosensor. In PAO1, three major bioluminescent spots were observed which correspond to PQS, HHQ and a mixture of 2 nonyl-4-quinolone and HHQNO. We also noted that on TLC plates the biosensor not only produced bioluminescence in response to AHQs but also the green pigment, pyocyanin which offers an alternative visual indicator for AHQ production.