MvfR, a key Pseudomonas aeruginosa pathogenicityLTTR-class regulatory protein, has dual ligands

Gaoping Xiao,1 Eric Déziel,1,2 Jianxin He,1

François Lépine,2 Biliana Lesic,1

Marie-Hélène Castonguay,2 Sylvain Milot,2

Anastasia P. Tampakaki,1† Scott E. Stachel,1

and Laurence G. Rahme1*1Department of Surgery, Harvard Medical School,Massachusetts General Hospital, and Shriner’s BurnsInstitute Boston, MA 02114, USA.2INRS-Institut Armand-Frappier, Laval, Québec, CanadaH7V 1B7, Canada.

Summary

MvfR (PqsR), a Pseudomonas aeruginosa LysR-typetranscriptional regulator, plays a critical role in thevirulence of this pathogen. MvfR modulates theexpression of multiple quorum sensing (QS)-regulatedvirulence factors; and the expression of the phnABand pqsA-E genes that encode functions mediating4-hydroxy-2-alkylquinolines (HAQs) signalling com-pounds biosynthesis, including 3,4-dihydroxy-2-heptylquinoline (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ). PQS enhances the in vitroDNA-binding affinity of MvfR to the pqsA-E promoter,to suggest it might function as the in vivo MvfR ligand.Here we identify a novel MvfR ligand, as we show thatHHQ binds to the MvfR ligand-binding-domain andpotentiates MvfR binding to the pqsA-E promoterleading to transcriptional activation of pqsA-E genes.We show that HHQ is highly produced in vivo, where itis not fully converted into PQS, and demonstrate that itis required for MvfR-dependent gene expression andpathogenicity; PQS is fully dispensable, as pqsH–

mutant cells, which produce HHQ but completely lackPQS, display normal MvfR-dependent gene expres-sion and virulence. Conversely, PQS is required for fullproduction of pyocyanin. These results uncover anovel biological role for HHQ; and provide novelinsights on MvfR activation that may aid in the devel-opment of therapies that prevent or treat P. aeruginosainfections in humans.

Introduction

The opportunistic bacterial pathogen Pseudomonasaeruginosa is a principle agent of deleterious and fatalinfections in immuno-compromised individuals, and pan-antibiotic-resistant infections in hospitals (Lyczak et al.,2000). Critical P. aeruginosa virulence factor genes havebeen identified using PA14, a highly virulent and broadhost-range human isolate (Rahme et al., 1995; 1997; Tanet al., 1999). The expression of many of these genes isclosely co-ordinated via a complex cell density-dependentregulatory circuitry (Rumbaugh et al., 2000; Smith andIglewski, 2003). This system, termed quorum sensing(QS), is mediated via diffusible intercellular signals, calledautoinducers, whose concentrations increase in concertwith increasing cell density (Fuqua et al., 2001).

The LasR and RhlR regulatory proteins, which directthe transcription of P. aeruginosa QS-controlled genes,are activated by binding to their cognate autoinducers,N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-(butanoyl)-L-homoserine lactone (C4-HSL)respectively (Juhas et al., 2005). LasR also controls theexpression of MvfR, a transcriptional regulator essentialfor full P. aeruginosa pathogenicity (Rahme et al., 1997;Mahajan-Miklos et al., 1999; Cao et al., 2001; Gallagherand Manoil, 2001; Lau et al., 2003). MvfR is required forP. aeruginosa to produce several QS-regulated virulencefactors, including pyocyanin and hydrogen cyanide(HCN) (Rahme et al., 1997; Gallagher and Manoil, 2001;Déziel et al., 2005); and directs the synthesis of a largearray of 4-hydroxy-2-alkylquinolines (HAQs). TheseHAQs comprise distinct congener families, includingderivatives of 4-hydroxy-2-heptylquinoline (HHQ), andthe corresponding dihydroxylated derivatives, such as3,4-dihydroxy-2-heptylquinoline, which is known as thePseudomonas quinolone signal (PQS), as it functions inQS cell-to-cell signalling (Gallagher et al., 2002; Dézielet al., 2004; Lépine et al., 2004; Diggle et al., 2006a).We have previously shown that exogenous HHQ mayact as a messenger molecule in a cell-cell-communication pathway (Déziel et al., 2004). The mvfRgene is contiguous and divergently transcribed withrespect to its two primary target operons, pqsABCDEand phnAB (Cao et al., 2001; Gallagher et al., 2002;Déziel et al., 2004). The conversion of HHQ into PQS ismediated by pqsH, which is regulated by LasR, and

Accepted 26 September, 2006. *For correspondence. E-mailrahme@amber.mgh.harvard.edu; Tel. (+1) 617 724 5003; Fax(+1) 617 724 8558. †Present address: Agricultural University ofAthens, Athens, Greece.

Molecular Microbiology (2006) 62(6), 1689–1699 doi:10.1111/j.1365-2958.2006.05462.xFirst published online 2 November 2006

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

encodes a putative monooxygenase (Gallagher et al.,2002; Déziel et al., 2004).

LysR-type transcriptional regulators (LTTR) are ~300amino acids in length; their N-termini include a helix-turn-helix DNA-binding domain; they are inactive prior to theirbinding to specific small molecules, termed ‘ligand’ or‘coinducer’ molecules (Schell, 1993; Tyrrell et al., 1997);and their C-termini encode a ligand binding domain (LBD)that recognizes and responds to their cognate coinducersto mediate their respective regulatory activity (Schell,1993). LTTR coinducers are typically related to theprimary function and/or target of their respective regulator,and are often a substrate or metabolite of the correspond-ing regulated pathway(s) (Schell, 1993). The MvfRC-terminal domain between amino acids 92–298 containsa predicted LBD, and as such, MvfR activation likelydepends on its binding to a specific coinducer, possiblythe product of an MvfR-regulated pathway.

PQS enhances the in vitro DNA-binding affinity ofMvfR to the pqsA-E promoter (Wade et al., 2005), tosuggest it might function as the in vivo MvfR ligand.Nonetheless, we report here that pqsH– mutant cells,which completely lack PQS production, display normalpqsA-E transcription and virulence, in contrast to mvfR–

mutant cells. This apparent incongruity suggests anothermolecule is the actual in vivo MvfR coinducer, and/or isredundant with PQS. To this end, we show both HHQand PQS function as in vivo MvfR ligands; and thesecompounds bind to the MvfR LBD, and mediate a con-formational change in MvfR that could mediate its acti-vation and transcription of target genes. That PQS isdispensable for MvfR activation and virulence suggestsHHQ and PQS have different in vivo functions. Interest-ingly, we also show PQS is significantly more potentthan HHQ for both MvfR binding and activation; andPQS, but not HHQ is required for full pyocyanin produc-tion. In addition, HHQ, but not 4-hydroxy-2-nonquinoline(HNQ) or 4-hydroxy-2-heptylquinoline N-oxide (HQNO),plays a role in P. aeruginosa virulence and MvfR-dependent gene expression.

Results

PQS is non-essential for pathogenicity andMvfR-dependent gene expression

MvfR controls HAQ biosynthesis by directly activatingpqsA-D expression (Wade et al., 2005; Xiao et al., 2006);and PQS enhances the in vitro binding of MvfR to thepqsA promoter (Wade et al., 2005). If PQS is the soleMvfR coinducer, loss of PQS synthesis should disruptboth P. aeruginosa virulence and MvfR-dependent geneexpression. To this end, we constructed a pqsH– mutant,and confirmed by LC/MS (see below) that it completelylacks PQS (Gallagher et al., 2002). Figure 1 shows pqsH–

cells display wild-type virulence in mice, and as such,have fully functional MvfR. In contrast, both mvfR– andpqsA– cells, which do not produce any HAQs, exhibitattenuated virulence (Fig. 1, Déziel et al., 2004; 2005).These results clearly demonstrate PQS is not essential forP. aeruginosa pathogenicity; and furthermore, a non-PQSHAQ is essential for MvfR function.

To ask if PQS is dispensable for mvfR-dependentgene expression, we compared the whole-genomeexpression profiles of PA14 versus pqsH– mutant cells,minus and plus 10 mg l-1 PQS, with that of mvfR– cells,which we previously analysed to show that pqs, phn,mex and phz expression are most greatly altered bymvfR loss (Déziel et al., 2005). Table 1 demonstratesPQS is fully dispensable for QS-regulated MvfR-dependent gene expression: the expression of MvfR-regulated genes, with the exception of phzA1, isunaffected by PQS deficiency, nor by PQS addition topqsH– cells. These results clearly demonstrate that MvfRactivity is PQS-independent. Nonetheless, this activitydoes depend on the HAQ biosynthesis pathway,because MvfR is not activated in pqsA– cells, whichcompletely lack HAQs (see below). Also, pqsH expres-sion is MvfR-independent (Déziel et al., 2005).

Figure 2 shows PQS is completely dispensable for bothpqsA-E transcription and HAQ production, as pqsA–lacZexpression (Fig. 2A), and HAQ production (Fig. 2B), are

Fig. 1. PQS is fully dispensable forP. aeruginosa pathogenicity. Thermally injuredmice were infected with 105 PA14, mvfR–,pqsA– or pqsH– cells. Fifteen mice were usedfor each experiment. Each experiment wasperformed twice. Note that the mutant strainsare isogenic with PA14.

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essentially the same in pqsH– versus PA14 cells. Also,while HHQ concentration is higher in the pqsH– cells, totalmolar HHQ + PQS in the PA14 cells is essentially equal tomolar HHQ in the mutant cells (Fig. 2B), to confirm thatPA14 cells completely convert HHQ into PQS.

A non-PQS HAQ functions as an in vivo MvfR ligand

Because MvfR controls HAQ biosynthesis, and LTTRsoften mediate the synthesis of their ligands, one or moreHAQs might function as an MvfR co-ligand to activatepqsA-E expression. Indeed, PQS enhances the in vitrobinding of MvfR to the pqsA-E promoter, suggesting it isan MvfR co-ligand (Wade et al., 2005). Nonetheless, ourpqsH– results strongly suggest a PqsABCDE pathway

product other than PQS functions as the in vivo co-ligand.To this end, Fig. 2A demonstrates that pqsA-E expres-sion, which is PQS-independent, nonetheless depends onthe HAQ pathway: while pqsA– cells express wild-typelevels of mvfR and completely lack HAQs (data notshown; (Déziel et al., 2004)), the pqsA mutation largelyabolishes pqsA–lacZ expression (Fig. 2A). Thus, the pqsoperon encodes functions essential both for MvfR regu-latory activity, and for its own expression.

That non-PQS HAQs activate MvfR function is furtherconfirmed by LC/MS analysis using a heterologousEscherichia coli system that synthesizes HAQs. Table 2presents the concentrations of four different HAQs pro-duced by E. coli cells that harbour both pDN18-mvfR,which constitutively expresses MvfR, and pLG12, which

Table 1. Fold change of MvfR-regulated pqs, phn, phz and mex expression in pqsH– and wild-type cells, minus and plus PQS.

Identifiera mvfR–b pqsH–c pqsH–+ PQSc Probable functiond

PA0996_pqsA 16.0 < 2 < 2 Probable coenzyme A ligasePA0997_pqsB 109.0 < 2 < 2 Hypothetical proteinPA0998_pqsC 158.0 < 2 < 2 Hypothetical proteinPA0999_pqsD 28.0 < 2 < 2 3-oxoacyl-(acyl-carrier-protein) synthase IIIPA1000_pqsE 80.0 < 2 < 2 Hypothetical proteinPA1001_phnA 45.0 < 2 < 2 Anthranilate synthase component IPA1002_phnB 13.0 < 2 < 2 Anthranilate synthase component IIPA2300_chiC 3.8 < 2 < 2 ChitinasePA2570_lecA 15.6 < 2 < 2 Galactose binding PA-I lectinPA3361_lecB 6.8 < 2 < 2 Fucose-binding PA-II lectinPA4205_mexG 29.0 < 2 < 2 Hypothetical proteinPA4206_mexH 8.0 < 2 < 2 Probable RND efflux membrane fusion protein precursorPA4207_mexI 18.0 < 2 < 2 Probable RND efflux transporterPA4208_ompD 33.0 < 2 < 2 Probable outer membrane protein precursorPA4209_phzM 7.0 < 2 < 2 Probable O-methyltransferasePA4210_phzA1 4.0 5 < 2 Probable phenazine biosynthesis protein PhzAPA4211_phzB1 11.0 < 2 < 2 Probable phenazine biosynthesis protein PhzBPA4212_phzC1 9.0 < 2 < 2 Phenazine biosynthesis protein PhzCPA4213_phzD1 7.0 < 2 < 2 Phenazine biosynthesis protein PhzDPA4214_phzE1 12.0 < 2 < 2 Phenazine biosynthesis protein PhzEPA4215_phzF1 10.0 < 2 < 2 Phenazine biosynthesis protein PhzFPA4216_phzG1 17.0 < 2 < 2 Phenazine biosynthesis protein PhzGPA4217_phzS 11.0 < 2 < 2 FAD-dependent monooxygenase

a. The Affymetrix P. aeruginosa microarray contains probe sets for only one copy of each phzA-G gene, labelled PA4210 (phzA1), PA4211(phzB1)and PA1901 through PA1905 (phzC2D2E2F2G2). PA1901-PA1905 have been reassigned to PA4212-PA4216.b. Fold change ratios in mvfR– are from Déziel et al. (2005), except for PA4207_mexI, PA4208_ompD and PA4210_phzA1, which are from anindependent unpublished experiment.c. Fold change ratios represent expression levels in PA14 versus pqsH– or pqsH– cells + 10 mg l-1 PQS at OD600 = 3; and were calculated fromthe average of three data sets using the Genespring algorithm after inverse log transformation.d. PA numbers and annotation are from http://www.vz.pseudomonas.com.

Table 2. pqsA-D genes encode enzymes that mediate HHQ and HNQ biosynthesis in E. coli in response to MvfR regulation.

Plasmid HHQ (mg l-1) HNQ (mg l-1) HQNO (mg l-1) PQS (mg l-1)

No 0 0 0 0PpqsA -pqsA-D 0 0 0 0Plac-MvfR 0 0 0 0Plac-MvfR + PpqsA -pqsA-D 8.04 � 0.84 4.40 � 0.58 0 0

Plac-MvfR and PpqsA -pqsA-D correspond to pDN18-mvfR and pLG12 respectively. Cells were grown at 37°C in LB and supplemented with 10 mg l-1

anthranilic acid.

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carries the pqsA-D operon under the control of its ownpromoter; versus control cells that harbour pDN18-mvfRalone, pLG12 alone or no plasmid. These data show bothHHQ, and its HNQ analogue, but neither the N-oxideHQNO, nor PQS, are produced by the cells that carry bothplasmids, but not by any of the control cells. These resultsclearly demonstrate the MvfR protein and the pqsA-Dgene products are necessary and sufficient to reconstitutepqs regulation and HAQ biosyntheses. In addition, thatMvfR is activated in E. coli to induce pqsA-D mediatedHHQ and HNQ production further confirms these genes

encode activities that catalyse HHQ congener synthesis,including an MvfR co-inducer(s). Note these cells still failto produce HQNO or PQS due to the absence of pqsL(Lépine et al., 2004) and pqsH in E. coli.

Both HHQ and PQS are in vivo MvfR ligands of pqsgene expression

To determine if HHQ and/or HNQ activate MvfR function inP. aeruginosa, pqsA–lacZ expression was assayed in aPA14 pqsA–/pqsH– double mutant, which completely lacksendogenous HAQs and cannot convert HHQ into PQS.Figure 3 shows exogenous HHQ and PQS, and to alesser degree HNQ, stimulate MvfR-dependent pqsA–lacZ expression, while only basal activity is seen minuscompound, or plus anthranilic acid (AA) or HQNO. Theseresults show both HHQ and PQS have significant in vivocoinducer activity; and suggest HHQ functions as an invivo MvfR ligand.

HHQ enhances MvfR binding to the pqsA promoterin vitro

Both HHQ and PQS potentiate the DNA-binding activity ofthe MvfR protein, and as such, function as MvfR ligands.Figure 4 shows that when a 174 bp radiolabelled DNAfragment containing the pqsA promoter region, includingthe MvfR binding site (Xiao et al., 2006), is mixed withlysate from E. coli cells overexpressing full-length MvfR,both HHQ and PQS stimulate MvfR-pqsA promoterbinding, although significantly weaker potentiation is seenwith HHQ.

Fig. 2. PQS is fully dispensable for MvfR activation of pqsA-Etranscription.A. b-Galactosidase activity expressed from pqsA–lacZ at differenttime points in PA14, mvfR–, pqsA– or pqsH–cells.B. Accumulation kinetics of HHQ, PQS and HQNO in PA14 andpqsH– cells, and their total production.

Fig. 3. Both HHQ and PQS function as co-ligands to activate MvfRfunction for pqsA-E transcriptional activation. b-Galactosidaseactivity expressed from pqsA–lacZ at different time points in PA14,pqsA– or pqsA–/pqsH–cells, minus and plus 10 mg l-1 anthranilicacid (AA), HQNO, HNQ, HHQ or PQS.

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Both HHQ and PQS induce conformational change ofthe MvfR protein

MvfR harbours a predicted LTTR LBD within its C-terminalregion that HHQ and PQS might interact with to activateMvfR function that then mediates target gene activation.To this end, we asked if either HHQ or PQS bind to MvfRprotein. Figure 5 shows addition of either of these com-pounds to a solution containing a truncated C-terminalHis-tagged MvfR (MvfRC87), which starts at residue 87 andcontains the MvfR LBD, converts the soluble MvfRC87 intoa precipitable form that is removed from the mixture bycentrifugation. These results further suggest both HHQand PQS function as MvfR ligands. PQS quickly andefficiently precipitates MvfRC87, suggesting it mediates aspecific conformational change within the protein frag-ment: MvfR precipitation occurs by 1 min after ligand addi-tion, with ~50% of the putative protein-ligand complex

precipitating within 4 min (data not shown). HHQ alsoprecipitates MvfR, but to a lesser extent than does PQS,correlating with its lower activation of pqsA-E transcriptionin pqsA–/pqsH– cells (Fig. 3), and its lower potentiation ofMvfR binding to the pqsA-E promoter (Fig. 4). Figure 5also shows that neither PQS nor HHQ lead to any MvfRdegradation, as the total amount of MvfRC87 is essentiallythe same in the uncentrifuged protein-ligand mixturesminus or plus ligand.

PQS is 100-fold more potent than HHQ in activatingpqsA-E transcription

Figure 6 shows the relative potency of PQS is 100-foldgreater than that of HHQ in mediating pqsA-E induction:addition of 0.05 mg l-1 PQS to pqsA–/pqsH– cells carryinga pqsA–lacZ reporter gene induces the same level ofpqsA–lacZ expression as addition of 5 mg l-1 HHQ.

PQS is required to induce full phzA1-G1 transcriptionand pyocyanin production

Table 1 shows that while expression of pqsA-E and otherMvfR-regulated genes are normal in pqsH– cells, phzAtranscription is fivefold less than in PA14. Accordingly,pqsH– cells produce less pyocyanin than wild-type cells[(Gallagher et al., 2002); Fig. 7A], even while producinghigher HHQ levels (Fig. 2B). Figure 7 also shows PQSaddition to pqsH– cells restores both phzA1 expressionand pyocyanin production to wild-type levels. Theseresults suggest PQS, but not HHQ, is essential for fullpyocyanin production, and further differentiate betweenthe respective biological activities of these two MvfRligands.

HHQ is highly produced in vivo and it is never fullyconverted into PQS

In agreement with the result that PQS is not essential forP. aeruginosa pathogenicity, Fig. 8 shows that HHQ is themost prominent HAQ produced by PA14 cells in vivo,versus other principal HAQs, PQS and HQNO. Fig. 8further shows that HHQ levels increase over time, ratherthan decrease as the result of conversion to PQS. HHQconcentration remains 10 times higher than PQS at 35 hpost burn and infection, by which time the infected miceare moribund. These findings further indicate the impor-tance of HHQ in P. aeruginosa pathogenesis, presumablyvia its role as an in vivo MvfR co-ligand.

Discussion

This study presents new insights into MvfR activation andidentifies a novel MvfR autoinducer. Expanding on a

Fig. 4. Both HHQ and PQS increase the affinity of the MvfRprotein for the pqsA-E promoter.A. A 32P-labelled 174 bp pqsA promoter region DNA fragment wasmixed with E. coli cell lysate containing MvfR protein minus ligand(lanes 2 and 3); plus 10 mg l-1 HHQ (lanes 4 and 5); and plus10 mg l-1 PQS (lanes 6 and 7). Protein per reaction are 0 ng ml-1

(lanes 1); 30 ng ml-1 (lane 2, 4 and 6); and 60 ng ml-1 (lane 3, 5 and7). Mixtures were electrophoresed on 5% non-denaturatedpolyacrylamide gels.B. Densitometric relative intensity of DNA band shift signals.

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recent report (Wade et al., 2005), our work demonstratesthat both HHQ and PQS function as MvfR ligands toinduce transcription of target genes with PQS showinghigher affinity in MvfR binding than HHQ. Several lines ofevidence support this conclusion. First, in the PA14 pqsH–

strain, in which PQS is absent, MvfR protein is functionalin the QS MvfR-controlled gene transcriptional regulation

and virulence. Second, MvfR activates the production ofHHQ and HNQ in the presence of the pqsA-D genes inE. coli, demonstrating the production of an active MvfRligand, which can not be PQS as PQS is neither producedin nor added to the E. coli cells. Third, adding HHQ, anend product of PqsA-D enzymatic activity, results in acti-vation of the MvfR function in pqsA–/pqsH– double mutant

Fig. 5. Both HHQ and PQS induce a conformational change in the MvfR protein.A. Both HHQ and PQS precipitate MvfRC87, which contains the MvfR LBD. HHQ, PQS and HQNO were each dissolved in ethyl acetate, anddried under a nitrogen stream. A His-MvfRC87 solution was added to each pellet to achieve maximal HAQ concentration; the mixtures wereincubated at room temperature for 30 min; and the supernatants were separated on SDS-PAGE minus (–) or plus (+) centrifugation. HQNOserved as the control, because it does not enhance MvfR binding to the pqsA promoter (Fig. 3; data not shown). MW, molecular weight; kDa,kilodalton.B. Densitometric relative intensity of the His-MvfRC87 bands.

Fig. 6. PQS is 100-fold more potent thanHHQ for activation of the MvfR regulatoryfunction. b-Galactosidase activity expressedfrom pqsA–lacZ in PA14 or pqsA–/pqsH– cells,minus and plus different concentrations ofHHQ or PQS, at OD600 = 3.0.

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cells as assessed by pqsA-E transcriptional activation.Fourth, like PQS, HHQ enhances MvfR binding to thepqsA-E promoter; and induces a conformational changein the MvfR protein. Fifth, in contrast to our in vitro results,HHQ is not fully converted into PQS in vivo, as its levelscontinue to increase over time, and furthermore remainhigher than the corresponding PQS levels.

That PQS is dispensable for MvfR-controlled gene acti-vation and virulence suggests HHQ and PQS may havedifferent in vivo functions. At least two specific PQS-dependent functions support this hypothesis. Vesicle for-mation has been shown to require PQS but not HHQ(Mashburn and Whiteley, 2005), and our results demon-strate that both full phzA1 gene expression and pyocyaninproduction, are PQS dependent, suggesting PQS func-tions in pyocyanin regulation. However, because additionof either HHQ or PQS in the mvfR– cells does not induce

phzA–lacZ expression or restores pyocyanin production(Déziel et al., 2005), both molecules control pyocyaninproduction through MvfR. In contrast, vesicle formationoccurs if PQS (Mashburn and Whiteley, 2005), but notHHQ (R. Hazan and L. G. Rahme, unpubl. data), is addedto mvfR– mutant cells. On the other hand, HHQ is requiredfor P. aeruginosa virulence in burn wound infections butPQS is dispensable. In accordance is the presence andlevels of HHQ in burn wound infection sites shown in Fig. 8.More importantly, the in vivo data presented here show thatthe concentration of HHQ increases over time, indicatingthat HHQ is not entirely converted into PQS, as observed invitro (Fig. 8). Nevertheless, both HHQ and PQS are foundin burn wound infection sites and cystic fibrosis airways(Machan et al., 1992; Collier et al., 2002), suggesting thatboth molecules function in P. aeruginosa pathogenesis.

Our results also show that PQS is more potent than HHQin inducing MvfR binding to the pqsA promoter; and pqsA-Etranscription in the pqsA–/pqsH– mutant. HHQ versus PQSrelative potency to induce MvfR LBD precipitation sug-gests that these molecules induce different MvfR confor-mational changes, which could subsequently result indifferent binding affinities to the same MvfR target promot-ers, and differences in MvfR-regulated gene transcription.One difference between HHQ and PQS that may explaintheir actual relative potency to induce MvfR LBD precipita-tion is the significantly higher hydrophobic character ofPQS. Indeed, PQS is less polar than HHQ (data not shown)despite the presence of an additional hydroxyl group inPQS. Here we show that with the exception of phzA1, HHQis capable of fully activating the expression of all QSMvfR-controlled genes in the pqsH– mutant, and iron-related genes (data not shown) previously shown to beactivated by PQS (Bredenbruch et al., 2006); and that PQSis fully dispensable for their expression, as they are unaf-fected by PQS absence, or by PQS addition. The ability ofHHQ to replace PQS function in the activation of these

B

A

Fig. 7. PQS is required for both full pyocyanin production andphzA1-G1 transcription.A. Pyocyanin levels in PA14 and pqsH– cells, minus and plus10 mg l-1 PQS, at OD600 = 3.0.B. b-Galactosidase activity expressed from phzABC1–lacZ in PA14and pqsH– cells at different cell densities, minus and plus 10 mg l-1

PQS.

Fig. 8. HHQ is produced to high levels in vivo. Using a thermalinjury and infection mouse model, the in vivo levels of HHQ, PQSand HQNO, the three principal members of the HAQ family wereassessed by LC/MS at 12, 15, 24 and 35 h post burn and infection.

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genes in pqsH– cells could be potentially attributed to thehigher concentrations of HHQ in pqsH– versus wild-typecells, as they cannot convert HHQ into PQS (Gallagheret al., 2002; Déziel et al., 2004), and significantly, HHQrestores the pathogenicity of these mutant cells. Nonethe-less, a qualitative difference between PQS and HHQ existsbecause even the higher concentrations (4 ¥) of HHQ inthe pqsH– cells (Fig. 2B) does not fully restore wild-typepyocyanin production (Fig. 7A), nor vesicle formation(Mashburn and Whiteley, 2005). Interestingly, Dietrichet al. (2006) have shown that pyocyanin is required for theactivation of the mexGHI-opmD genes; and here we showthat these genes are fully activated in the pqsH– cells,indicating that the amount of pyocyanin produced is suffi-cient for their full activation.

Why are there two MvfR ligands? These two mol-ecules are the main representatives of two distinctclasses of the HAQ family (Déziel et al., 2004), withHHQ the precursor of PQS. Although they derive fromthe same pathway, their production is under differenttranscriptional regulators: MvfR for HHQ, and LasR viaPqsH expression for PQS. Furthermore, PQS and HHQconcentrations peak at different growth stages; PQS butnot HHQ production depends on HHQ synthesis andconversion by PqsH; and their localization seems to bedifferent, as PQS, but not HHQ, is primarily found inmembrane vesicles (Mashburn and Whiteley, 2005). Inaddition, we have previously shown that exogenousHHQ may act as a messenger molecule in a cell-cellcommunication pathway (Déziel et al., 2004); and HHQaccumulation peaks at the end of the log phase andthen decreases while PQS accumulates and peaks atthe mid-stationary phase (Lépine et al., 2003; Dézielet al., 2004). In accord, pqsH transcription is higher atlater growth stages (Chugani et al. 2001). Thus, HHQand PQS may have different primary functions depend-ing on the growth stage, with HHQ serving as the MvfRin vivo ligand at the earlier stages. This possibility isstrongly supported by the high in vivo levels of HHQ(Fig. 8). In addition, if HHQ and PQS were perfectlyinterchangeable, then the pqsH gene would not havebeen maintained in the evolution. Interestingly, the studyby Diggle et al., which supports our previous (Dézielet al., 2004) and current findings, proposes that theability of P. aeruginosa to respond to both HHQ andPQS may provide an ecological advantage against non-PQS producing bacterial pathogens (Diggle et al.,2006a,b). These authors show that exogenous HHQrestores Burkholderia pseudomallei hhqA mutant pheno-types (Diggle et al., 2006b), suggesting that HHQ mayact as a signal in this bacterium, as indeed has beenshown for P. aeruginosa (Déziel et al., 2004).

Here our findings demonstrate a new role for HHQ inP. aeruginosa QS-regulation and virulence. The discovery

that another HAQ besides PQS functions in MvfR activa-tion opens the way for a better understanding of MvfRpathway regulation; and its relationship to other QS regu-latory systems, as the MvfR pathway is the first LysR-typeQS system whose activation is mediated by quinolinederivatives. That MvfR function is regulated in vivo bycompounds belonging to two different HAQ families mightenhance QS regulation and this finding could aid touncover the biological role of precursors of other QSsignalling molecules involved in bacterial cell-cell commu-nication and controlled group behaviours. Also further useof E. coli to study MvfR regulation and HAQ productionmay aid in the dissection of the P. aeruginosa MvfRpathway, and provide a system to screen and identifyinhibitors of MvfR function and its pathway, which couldprove useful anti-infective compounds to limitP. aeruginosa infections in humans.

Experimental procedures

Materials

Oligonucleotides were from MWG Biotech (High Point, NC).Restriction endonucleases and T4 DNA ligase were fromNew England Biolabs (Cambridge, MA). Qiaquick PCR puri-fication kit, Qiaquick gel extraction kit, Qiaprep spin miniprepkit, RNeasy Mini kit and Rnase-free DNase I were fromQiagen (Chatsworth, CA). Expand High Fidelity PCR systemwas from Roche (Indianapolis, IN). HisBind® Kit was fromNovagen (Madison, WI). Precasted Tris-HCl PAGE gels werefrom Bio-Rad (Hercules, CA). 32P-g-ATP was from Perki-nElmer (Boston, MA). HHQ and PQS were synthesized asdescribed (Lépine et al., 2003); and anthranilic acid andHQNO were from Sigma-Aldrich (Oakville, Can).

Strains and growth conditions

Table 1 lists bacterial strains. P. aeruginosa and E. coli cellswere grown at 37°C in Luria–Bertani (LB) broth or on LB agar.E. coli JM109 was routinely used for subcloning and plasmidpropagation. For plasmid selection and maintenance, ampi-cillin (100 mg ml-1) and tetracycline (10 mg ml-1) were used forE. coli, and carbenicillin (300 mg ml-1) and tetracycline(50 mg ml-1) for P. aeruginosa.

A PA14 pqsH– mutant strain was generated by insertingthe aacC1 cassette (Gmr) into the PA14 PA2587 gene(pqsH). A pqsH 368 bp chromosomal DNA fragment wasPCR-amplified with a 5′ primer, pqsHGmF (5′-CGGGATCCCGGCTAGGATGGACATTTTT-3′), containing a BamHIsite, and a 3′ primer, pqsHGmR (5′-ACGCGTCGACAGACTGGAAGGCATCGACAT-3′), containing a SalI site;and cloned into BamHI and SalI sites of pNTPS138 to gen-erate pNTPS138pqsH. A 1.8 kb EcoRI fragment containingthe aacC1 gene in pUC7Gm was ligated into the EcoRI site ofpNTPS138pqsH to generate pNTPS138pqsHGm. This con-struct contains the aacC1 gene inserted 54 bp downstream ofthe pqsH translational start site. The aacC1 insertion muta-tion was confirmed by restriction digestion and PCR.

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pNTPS138pqsHGm was introduced into PA14 by conjugationwith E. coli S17-1. Single crossover exconjugants wereselected on LB plus 100 mg ml-1 gentamicin. Double-crossover exconjugants resistant to gentamicin and sucrose,and sensitive to 200 mg ml-1 kanamycin, were counterselected on LB agar plus 5% sucrose. The second recombi-nation event and chromosomal insertion was confirmed byPCR.

A pqsA–/pqsH– double mutant was obtained by transfer ofthe chromosomal pqsH::Gm mutation into the pqsA::TnphoAmutant, essentially as described (Choi et al., 2006). Briefly,approx. 1010 electrocompetent pqsA– cells resuspended in100 ml of 300 mM sucrose were electroporated in the pres-ence of total genomic DNA from a 2 ml overnight culture ofpqsH– cells, and the double mutant was selected on LB pluskanamycin (200 mg ml-1) and gentamicin (100 mg ml-1). Thedouble mutant is unable to produce HHQ, nor convert HHQinto PQS.

Plasmid construction

Plasmids are listed in Table 3. Standard DNA techniqueswere used. All constructs were confirmed by DNA sequenc-ing, and introduced into E. coli and PA14 by electroporation.

For pET16B-mvfRC87, the mvfR gene from nucleotide 259to the stop codon was PCR-amplified from pLGR34B12 usingprimers F3 (5′-CTTCAATCTCCATATGGGTCCGCGCAATCTCC-3′) and R3 (5′-GGTCTCGAGTGCCTGGCCGATGCCGATGG-3′); and subsequently cloned into the NdeI/XhoI sites ofpET16B.

b-Galactosidase, DNA mobility gel-shift and HAQassays

PA14 or isogenic mutant cultures carrying pGX5 (pqsA–lacZ) were grown overnight at 37°C, diluted to OD600 = 0.01,and grown at 37°C; and b-galactosidase activity was mea-sured (Miller, 1972) at different time points. Results,expressed in Miller Units (MU), correspond to the mean oftriplicate experiments � standard deviation.

For the DNA mobility gel-shift assay, the pqsApromoter fragment from -89 to +85 bp relative to the pqsAtranscriptional initiation site was PCR-amplified from pGX5using primers GX34 (5′-GGATCGGTACCCGTGGTTCTTCTCCCCG-3′) and GX129 (5′-TCAGGTTGGCCAATGTGGACATGA-3′). T4 polynucleotide kinase (PNK)and 32P-g-ATP were used to label the PCR products,which were then purified using the Qiagen PCR purificationkit. Lysate from E. coli cells carrying pDN18-mvfR wasmixed with 2 nM 32P-labelled pqsA promoter fragment in atotal volume of 15 ml; incubated at room temperature for20 min in the presence of 10 mg l-1 HHQ or PQS; and sepa-rated on 5% non-denaturated polyacrylamide gels at 200 V,4°C.

HAQs were quantified from cultures using LC/MS, as pre-viously described (Lépine et al., 2003).

MvfR protein overexpression and purification

(His)10-MvfRC87 protein was purified from E. coli using theHis·Bind® Kit following the manufacturer’s instruction.

Table 3. Bacterial strains and plasmids.

Strains/plasmids Relevant characteristics References/source

StrainsP. aeruginosa

PA14 Clinical isolate UCBPP-PA14 Rahme et al. (1995)PA14 mvfR– PA14 containing an mvfR nonsense point mutation Cao et al. (2001)PA14 pqsA– TnphoA insertion mutation in pqsA of PA14; Kmr Déziel et al. (2004)PA14 pqsH– PA14 containing an aacC1 cassette inserted into pqsH; Gmr This studyPA14 pqsA–/pqsH– PA14 pqsA– containing an aacC1 cassette inserted into pqsH; Gmr, Kmr This study

E. coliJM109 F–(traD36 proAB+lacIqlacZ DM15) endA1 reacA1 hsdR17

(rK– mK

+) supE44 thi-1 gyrA96 relA1 D(lac-proAB)Laboratory collection

BL21(DE3) F–ompT hsdSB(rB– mB

–) gal dcm (DE3) NovagenS17-1 Smr Tprmod+res thi pro recA hsdR17, integrated plasmid

RP4-TC::Mu-Km::Tn7 into genomeSimon et al. (1983)

PlasmidspNTPS138 pLitmus38-derived suicide vector, oriT, sacB; Kmr M. R. K. AlleypUC7Gm Source of aacC1 cassette; Gmr S. LorypLG12 pUCP18 carrying pqsABCD operon and its native promoter; Apr Gallagher et al. (2002)pGX5 pQF50 carrying pqsA–lacZ transcriptional fusion; Apr Xiao et al. (2006)pDN18 Expression vector; Tetr Nunn et al. (1990)pDN18-mvfR pDN18 with the lac promoter fused to the mvfR gene; Tetr Déziel et al. (2004)pH44 Cosmid carrying PA14 genomic DNA from PA0963 to PA1004; Apr He et al. (2004)pET16B Expression vector; Apr NovagenpLGR34B12 Cosmid containing the mvfR gene; Apr Rahme et al. (1997)pET16B-mvfRC87 pET16B with mvfR nucleotide 259 through the stop codon

under the control of the T7 promoter; AprThis study

pMW303 pEX1.8 carrying phzABC1–lacZ transcriptional fusion; Apr Whiteley et al. (2000)

Kmr, kanamycin-resistant; Apr, ampicillin-resistant; Tetr, tetracycline-resistant; Gmr, gentamicin-resistant.

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HAQ-mediated precipitation of (His)10-MvfRC87

HQNO, HHQ and PQS were each dissolved in ethyl acetateand dried under nitrogen. A solution containing (His)10-MvfRC87 was added to each HAQ to achieve maximal con-centration, and the mixture was incubated at roomtemperature for 30 min before centrifugation, or as indicated.Supernatants were loaded onto 12% SDS-PAGE gels forprotein analysis.

Animal studies

Animal pathogenicity was assessed using the thermal injurymouse model (Stevens et al., 1994) as described (Rahmeet al., 1995). Animal mortality protocols were reviewed andapproved by the Institutional Animal Care and Use Committeeof the Massachusetts General Hospital. For HAQs quantifica-tion in vivo, animals were burned and infected with 5 ¥ 105

PA14 cells, and biopsies of the underlying muscle(Rahme et al., 1995) were collected at 12, 15, 24 and35 h post infection. Five hundred microlitres of a methanolicsolution of 0.2 mg ml-1 5,6,7,8-tetradeutero-3,4-dihydroxy-2-heptylquinoline (PQS-D4) and 5,6,7,8-tetradeutero-4-hydroxy-2-heptylquinoline (HHQ-D4) as internal standards(Lépine et al., 2003) was added to the collected tissue in a 2 mlEppendorf tube. The tissues were homogenized, centrifugedand the supernatant collected. Eighty ml of the supernatantwere injected to a C18 reverse-phase column connected to atriple quadrupole mass spectrometer with a water/acetonitrilegradient (Lépine et al., 2003). HAQ analysis was performed inpositive electrospray in MRM mode using the transitions: HHQ244 > 159; HHQ-D4 248 > 163; HQNO 260 > 159; PQS260 > 175; and PQS-D4 264 > 179. The collision gas wasargon at 2 ¥ 10–3 mTorr, and the collision energy was 30 V.

Acknowledgements

We thank E. P. Greenberg for the plasmid pMW303; ColinManoil for pLG12; and former and current laboratorymembers for discussion and critical reading of themanuscript. This work was supported by The Claflin Distin-guished Scholar Award, Shriners Research Grant # 8850 and1 R01 AI063433-01A2 to L.G.R.

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