Pseudomonas Quinolone Signal Research Articles

Biochemical characterization of PqsD activity in alkylquinolone biosynthesis in Pseudomonas aeruginosa

Pseudomonas aeruginosa (Pa) is a versatile opportunistic pathogen. Quorum sensing (QS) is essential for Pa pathogenicity by activating virulence factor production. Among the three known QS systems in Pa, Pseudomonas Quinolone Signal (PQS) is an alkyquinolone, one of >50 quinolone compounds produced that differ in alkyl chain length and desaturation. The condensing enzyme PqsD catalyzes the branching point of quinolone synthesis, utilizing either malonyl‐CoA or 3‐oxo‐fatty acids of various chain lengths. We showed that PqsD exhibited higher activity using fatty acid substrates with 10, 12 or 14 carbons than those with 6 or 8 carbons, suggesting the fatty acid chain length is critical in positioning the carboxylic group for efficient catalysis. In addition, PqsD showed preference for malonyl‐CoA over 3‐oxo‐decanoic acid in vitro. Alanine mutants of the active site triad (C112, H257, and N287) were assayed for PqsD activity. All 3 residues were essential for condensation, whereas N287 was not required in the formation of anthraniloyl‐PqsD intermediate. Inhibitors of PqsD were identified by virtual screen and the IC50s of the top‐scoring hit compounds were determined. Our results suggest that regulation of the substrate supply in vivo may be important in determining the composition of quinolone compounds, which in turn affect the virulence factor production in Pa. (Supported by NIH P20 RR017677 and UL1 RR029882)

Interkingdom adenosine signal reduces Pseudomonas aeruginosa pathogenicity

SummaryPseudomonas aeruginosa is becoming recognized as an important pathogen in the gastrointestinal (GI) tract. Here we demonstrate that adenosine, derived from hydrolysis of ATP from the eucaryotic host, is a potent interkingdom signal in the GI tract for this pathogen. The addition of adenosine nearly abolished P. aeruginosa biofilm formation and abolished swarming by preventing production of rhamnolipids. Since the adenosine metabolite inosine did not affect biofilm formation and since a mutant unable to metabolize adenosine behaved like the wild‐type strain, adenosine metabolism is not required to reduce pathogenicity. Adenosine also reduces production of the virulence factors pyocyanin, elastase, extracellular polysaccharide, siderophores and the Pseudomonas quinolone signal which led to reduced virulence with Caenorhabditis elegans. To provide insights into how adenosine reduces the virulence of P. aeruginosa, a whole‐transcriptome analysis was conducted which revealed that adenosine addition represses genes similar to an iron‐replete condition; however, adenosine did not directly bind Fur. Therefore, adenosine decreases P. aeruginosa pathogenicity as an interkingdom signal by causing genes related to iron acquisition to be repressed.

The effect of pmpR on the type III secretion system in Pseudomonas aeruginosa

The type III secretion system (T3SS) plays important roles in Pseudomonas aeruginosa pathogenicity. Previously, we reported that the uncharacterized protein PmpR could regulate pqsR, an important regulator in the quorum-sensing system, by directly binding to its promoter region. As the T3SS is controlled by the quorum-sensing system, here, we investigated the relationship between PmpR and the T3SS. Our data showed that expression of the T3SS genes exoS, exoY, exoT, and exsD was dramatically increased in a pmpR-deletion mutant compared with that in the wild-type P. aeruginosa strain PAO1. Data from DNA mobility assays indicated that PmpR affects the T3SS indirectly. It is unlikely that PmpR controls the T3SS via the Pseudomonas quinolone signal (PQS) because the PQS negatively regulates the T3SS, while pmpR negatively regulates the PQS. The effect of PmpR on the T3SS seems to be independent of the PQS; further investigation is required to uncover the underlying regulatory pathways.

Quorum sensing modulates colony morphology through alkyl quinolones in Pseudomonas aeruginosa.

BackgroundAcyl-homoserine lactone (acyl-HSL) and alkyl quinolone (AQ) based quorum-sensing (QS) systems are important for Pseudomonas aeruginosa virulence and biofilm formation. The effect of QS on biofilm formation is influenced by various genetic and environmental factors. Here, we used a colony biofilm assay to study the effect of the central acyl-HSL QS regulator, LasR, on biofilm formation and structure in the representative clinical P. aeruginosa isolate ZK2870.ResultsA lasR mutant exhibited wrinkled colony morphology at 37°C in contrast to the smooth colony morphology of the wild-type. Mutational analysis indicated that wrinkling of the lasR mutant is dependent on pel, encoding a biofilm matrix exopolysaccharide. Suppressor mutagenesis and complementation analysis implicated the AQ signaling pathway as the link between las QS and colony morphology. In this pathway, genes pqsA-D are involved in the synthesis of 4-hydroxyalkyl quinolines ("Series A congeners"), which are converted to 3,4-dihydroxyalkyl quinolines ("Series B congeners", including the well-characterized Pseudomonas Quinolone Signal, PQS) by the product of the LasR-dependent pqsH gene. Measurement of AQ in the wild-type, the lasR pqsA::Tn suppressor mutant as well as the defined lasR, pqsH, and lasR pqsH mutants showed a correlation between 4-hydroxyalkyl quinoline levels and the degree of colony wrinkling. Most importantly, the lasR pqsH double mutant displayed wrinkly morphology without producing any 3,4-dihydroxyalkyl quinolines. Constitutive expression of pqsA-D genes in a lasR pqsR::Tnmutant showed that colony wrinkling does not require the AQ receptor PqsR.ConclusionsTaken together, these results indicate that the las QS system represses Pel and modulates colony morphology through a 4-hydroxyalkyl quinoline in a PqsR-independent manner, ascribing a novel function to an AQ other than PQS in P. aeruginosa.

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

BackgroundSmall colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.Methodology/Principal FindingsOne SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10−5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.ConclusionsBy combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system.

KynR, a Lrp/AsnC-Type Transcriptional Regulator, Directly Controls the Kynurenine Pathway in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa can utilize a variety of carbon sources and produces many secondary metabolites to help survive harsh environments. P. aeruginosa is part of a small group of bacteria that use the kynurenine pathway to catabolize tryptophan. Through the kynurenine pathway, tryptophan is broken down into anthranilate, which is further degraded into tricarboxylic acid cycle intermediates or utilized to make numerous aromatic compounds, including the Pseudomonas quinolone signal (PQS). We have previously shown that the kynurenine pathway is a critical source of anthranilate for PQS synthesis and that the kynurenine pathway genes (kynA and kynBU) are upregulated in the presence of kynurenine. A putative Lrp/AsnC-type transcriptional regulator (gene PA2082, here called kynR), is divergently transcribed from the kynBU operon and is highly conserved in gram-negative bacteria that harbor the kynurenine pathway. We show that a mutation in kynR renders P. aeruginosa unable to utilize L-tryptophan as a sole carbon source and decreases PQS production. In addition, we found that the increase of kynA and kynB transcriptional activity in response to kynurenine was completely abolished in a kynR mutant, further indicating that KynR mediates the kynurenine-dependent expression of the kynurenine pathway genes. Finally, we found that purified KynR specifically bound the kynA promoter in the presence of kynurenine and bound the kynB promoter in the absence or presence of kynurenine. Taken together, our data show that KynR directly regulates the kynurenine pathway genes.

2-Heptyl-4-quinolone, a precursor of the Pseudomonas quinolone signal molecule, modulates swarming motility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen capable of group behaviors, including biofilm formation and swarming motility. These group behaviors are regulated by both the intracellular signaling molecule c-di-GMP and acylhomoserine lactone quorum-sensing systems. Here, we show that the Pseudomonas quinolone signal (PQS) system also contributes to the regulation of swarming motility. Specifically, our data indicate that 2-heptyl-4-quinolone (HHQ), a precursor of PQS, likely induces the production of the phenazine-1-carboxylic acid (PCA), which in turn acts via an as-yet-unknown downstream mechanism to repress swarming motility. We show that this HHQ- and PCA-dependent swarming repression is apparently independent of changes in global levels of c-di-GMP, suggesting complex regulation of this group behavior.

The Pseudomonas aeruginosa quinolone quorum sensing signal alters the multicellular behaviour of Pseudomonas putida KT2440

The molecule 2-heptyl-3-hydroxy-4-quinolone (referred to as the Pseudomonas quinolone signal, or PQS) is produced by Pseudomonas aeruginosa as part of its quorum sensing circuit, and has been shown to influence a variety of processes in this bacterium, including the production of siderophores and secondary metabolites, virulence determinants and biofilm development. In this report we present evidence of the effect of PQS as an interspecies signal with a negative impact on the multicellular behaviour of Pseudomonas putida KT2440. PQS reduces biofilm formation and swarming motility, and interferes with iron uptake by this bacterium. Addition of PQS also causes changes in the transcription of several P. putida genes, indicating a specific response to the signal molecule, which is not produced by strain KT2440. Among the genes with increased expression in response to PQS is PP1563, which forms part of a large prophage cluster (PP1532-PP1584); consistently, phage-mediated lysis of some cells in the population was observed in the presence of PQS. Overall, these data indicate that PQS may be used by P. aeruginosa as a chemical weapon against potential competitors.

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 β 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 quinolone signal (PQS), and its precursor HHQ, modulate interspecies and interkingdom behaviour

The Pseudomonas quinolone signal (PQS), and its precursor 2-heptyl-4-quinolone (HHQ), play a key role in coordinating virulence in the important cystic fibrosis pathogen Pseudomonas aeruginosa. The discovery of HHQ analogues in Burkholderia and other microorganisms led us to investigate the possibility that these compounds can influence interspecies behaviour. We found that surface-associated phenotypes were repressed in Gram-positive and Gram-negative bacteria as well as in pathogenic yeast in response to PQS and HHQ. Motility was repressed in a broad range of bacteria, while biofilm formation in Bacillus subtilis and Candida albicans was repressed in the presence of HHQ, though initial adhesion was unaffected. Furthermore, HHQ exhibited potent bacteriostatic activity against several Gram-negative bacteria, including pathogenic Vibrio vulnificus. Structure-function analysis using synthetic analogues provided an insight into the molecular properties that underpin the ability of these compounds to influence microbial behaviour, revealing the alkyl chain to be fundamental. Defining the influence of these molecules on microbial-eukaryotic-host interactions will facilitate future therapeutic strategies which seek to combat microorganisms that are recalcitrant to conventional antimicrobial agents.

Small RNAs as regulators of primary and secondary metabolism in Pseudomonas species

Small RNAs (sRNAs) exert important functions in pseudomonads. Classical sRNAs comprise the 4.5S, 6S, 10Sa and 10Sb RNAs, which are known in enteric bacteria as part of the signal recognition particle, a regulatory component of RNA polymerase, transfer-messenger RNA (tmRNA) and the RNA component of RNase P, respectively. Their homologues in pseudomonads are presumed to have analogous functions. Other sRNAs of pseudomonads generally have little or no sequence similarity with sRNAs of enteric bacteria. Numerous sRNAs repress or activate the translation of target mRNAs by a base-pairing mechanism. Examples of this group in Pseudomonas aeruginosa are the iron-repressible PrrF1 and PrrF2 sRNAs, which repress the translation of genes encoding iron-containing proteins, and PhrS, an anaerobically inducible sRNA, which activates the expression of PqsR, a regulator of the Pseudomonas quinolone signal. Other sRNAs sequester RNA-binding proteins that act as translational repressors. Examples of this group in P. aeruginosa include RsmY and RsmZ, which are central regulatory elements in the GacS/GacA signal transduction pathway, and CrcZ, which is a key regulator in the CbrA/CbrB signal transduction pathway. These pathways largely control the extracellular activities (including virulence traits) and the selection of the energetically most favourable carbon sources, respectively, in pseudomonads.

Growth phase-differential quorum sensing regulation of anthranilate metabolism in Pseudomonas aeruginosa.

Pseudomonas quinolone signal (PQS) plays a role in the regulation of virulence genes and it is intertwined in the las/rhl quorum sensing (QS) circuits of Pseudomonas aeruginosa. PQS is synthesized from anthranilate by pqsA-D and pqsH whose expression is influenced by the las/rhl systems. Since anthranilate can be degraded by functions of antABC and catBCA, PQS synthesis might be regulated by the balance between the expression of the pqsA-D/phnAB, pqsH, antABC, and catBCA gene loci. antA and catA are repressed by LasR during log phase and activated by RhlR in late stationary phase, whereas pqsA-E/phnAB is activated by LasR in log phase and repressed by RhlR. QscR represses both but each repression occurs in a different growth phase. This growth phase-differential regulation appears to be accomplished by the antagonistic interplay of LasR, RhlR, and QscR, mediated by two intermediate regulators, AntR and PqsR, and their cofactors, anthranilate and PQS, where the expressions of antR and pqsR and the production of anthranilate and PQS are growth phase-differentially regulated by QS systems. Especially, the anthranilate level increases in an RhlR-dependent manner at late stationary phase. From these results, we suggest that RhlR and LasR regulate the anthranilate metabolism in a mutually antagonistic and growth phase-differential manner by affecting both the expressions and activities of AntR and PqsR, and that QscR also phase-differentially represses both LasR and RhlR functions in this regulation.

C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa

Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during infection natriuretic peptides, particularly CNP, could enhance the virulence of PAO1. This activity is relayed by Vfr and PtxR activation, and a general diagram of the virulence activation cascade involving AHL, HCN and exotoxin A is proposed.

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 bc1 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.

Detection of the Pseudomonas Quinolone Signal (PQS) by cyclic voltammetry and amperometry using a boron doped diamond electrode

2-Heptyl-3-hydroxy-4-quinolone, known as the Pseudomonas Quinolone Signal, is a key regulator of bacterial cooperative behaviour known as quorum sensing. A simple electrochemical strategy was employed for its sensitive detection using a bare boron-doped diamond electrode by cyclic voltammetry and amperometry. PQS (and potentially other quinolones) was then detected in cultures of P. aeruginosa pqsL(-) mutant strains.

Microwave and flow syntheses of Pseudomonasquinolone signal (PQS) and analogues

Expedient syntheses of Pseudomonas quinolone signal (PQS) and related structural analogues using microwave and flow methods are reported.

Full Virulence of Pseudomonas aeruginosa Requires OprF

OprF is a general outer membrane porin of Pseudomonas aeruginosa, a well-known human opportunistic pathogen associated with severe hospital-acquired sepsis and chronic lung infections of cystic fibrosis patients. A multiphenotypic approach, based on the comparative study of a wild-type strain of P. aeruginosa, its isogenic oprF mutant, and an oprF-complemented strain, showed that OprF is required for P. aeruginosa virulence. The absence of OprF results in impaired adhesion to animal cells, secretion of ExoT and ExoS toxins through the type III secretion system (T3SS), and production of the quorum-sensing-dependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. Accordingly, in the oprF mutant, production of the signal molecules N-(3-oxododecanoyl)-l-homoserine lactone and N-butanoyl-l-homoserine lactone was found to be reduced and delayed, respectively. Pseudomonas quinolone signal (PQS) production was decreased, while its precursor, 4-hydroxy-2-heptylquinoline (HHQ), accumulated in the cells. Taken together, these results show the involvement of OprF in P. aeruginosa virulence, at least partly through modulation of the quorum-sensing network. This is the first study showing a link between OprF, PQS synthesis, T3SS, and virulence factor production, providing novel insights into virulence expression.

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 of 3-halo-analogues of HHQ, subsequent cross-coupling and first crystal structure of Pseudomonas quinolone signal (PQS)

2-Aryl- and 2-alkyl-quinolin-4-ones and their N-substituted derivatives have several important biological functions such as the Pseudomonas quinolone signal (PQS) molecule participation in quorum sensing. Herein, we report the synthesis of its biological precursor, 2-heptyl-4-hydroxy-quinoline (HHQ) and possible isosteres of PQS; the C-3 Cl, Br and I analogues. N-Methylation of the iodide was also feasible and the usefulness of this compound showcased in Pd-catalysed cross-coupling reactions, thus allowing access to a diverse set of biologically important molecules. The first crystal structure of PQS is also included.

Oxygen levels rapidly modulate Pseudomonas aeruginosa social behaviours via substrate limitation of PqsH

Many bacteria use extracellular signals to coordinate group behaviours, a process referred to as quorum sensing (QS). The bacterium Pseudomonas aeruginosa utilizes a complex QS system to control expression of over 300 genes, including many involved in host colonization and disease. The Pseudomonas quinolone signal (PQS) is a component of P. aeruginosa QS, and although it contributes to virulence in some models of infection, the PQS biosynthetic pathway is not fully elucidated. Here, we show that PqsH catalyses the terminal step in PQS production, synthesizing PQS in vitro using the substrates 2-heptyl-4-quinolone (HHQ), NADH and oxygen. Structure function studies reveal that the alkyl side-chain of HHQ is critical for PqsH activity with the highest activity observed for alkyl chain lengths of 7 and 9 carbons. Due to the PqsH requirement for oxygen, PQS and PQS-controlled virulence factors are not produced by anaerobic P. aeruginosa. Interestingly, anaerobic P. aeruginosa produced PQS in the absence of de novo protein synthesis upon introduction of oxygen, indicating that oxygen is the sole limiting substrate during anaerobic growth. We propose a model in which PqsH poises anaerobic P. aeruginosa to activate PQS-controlled factors immediately upon exposure to molecular oxygen.