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Mutation analysis of the Pseudomonas aeruginosa mvfR and pqsABCDE gene promoters demonstrates complex quorum-sensing circuitry

Department of Surgery, Microbiology and Molecular Genetics, Harvard Medical School, Department of Surgery, Massachusetts General Hospital, and Shriners Burns Institute, Boston, MA 02114, USA

Correspondence
Laurence G. Rahme
rahme{at}molbio.mgh.harvard.edu

	ABSTRACT

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The LysR-type transcriptional regulator MvfR (PqsR) (multiple virulence factor regulator) plays a critical role in Pseudomonas aeruginosa pathogenicity via the transcriptional regulation of multiple quorum-sensing (QS)-regulated virulence factors. LasR activates full mvfR transcription, and MvfR subsequently activates pqsA–E expression. This study identifies and characterizes the key cis-regulatory elements through which mvfR and pqsA–E transcription is regulated in the highly virulent P. aeruginosa strain PA14. Deletion and site-directed mutagenesis indicate that: (1) LasR activates mvfR transcription by binding to a las/rhl box, CTAACAAAAGACATAG, centred at –513 bp upstream of the MvfR translational start site; and (2) RhlR represses pqsA transcription by binding to a las/rhl box, CTGTGAGATTTGGGAG, centred at –311 bp upstream of the pqsA transcriptional initiation site. Furthermore, it is shown that MvfR activates pqsA–E transcription by binding to a LysR box, TTCGGACTCCGAA, centred at –45 bp relative to the pqsA transcriptional initiation site, demonstrating that this LysR box has a critical role in the physical interaction between the MvfR protein and the pqsA promoter. These results provide new insights into the regulatory relationships between LasR and mvfR, and between MvfR/RhlR and the pqs operon, and elucidate further the complex regulation of the P. aeruginosa QS circuitry.

	INTRODUCTION

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Pseudomonas aeruginosa is an opportunistic pathogen that causes deleterious infections in patients with reduced immunity and with cystic fibrosis (Lyczak et al., 2000). This Gram-negative bacterium produces and responds to diffusible intercellular signals and autoinducers that mediate pathogenicity via the coordinated expression of a large array of virulence genes (Rumbaugh et al., 2000; Smith & Iglewski, 2003). This cell-density-dependent regulation is termed quorum sensing (QS) (Fuqua et al., 2001), as autoinducer concentrations increase, up to a threshold, with increasing cell density. QS regulation is a potential target for limiting P. aeruginosa pathogenicity.

The best studied QS regulatory cascade components are the LasR and RhlR transcriptional regulators that are recognized and activated by their respective autoinducers (Pesci & Iglewski, 1999). The activated LasR and RhlR proteins specifically and/or cooperatively regulate multiple genes by binding to las/rhl boxes located in target promoter regions. These elements have the consensus sequence CT-[N]₁₂-AG and occur in one or more copies in QS target gene promoters (McKnight et al., 2000; Whiteley & Greenberg, 2001).

The transcriptional regulator MvfR plays a critical role in P. aeruginosa pathogenicity. mvfR mutants exhibit decreased virulence in different host models, including plants, insects and mammals (Cao et al., 2001; Gallagher & Manoil, 2001; Lau et al., 2003; Mahajan-Miklos et al., 1999; Rahme et al., 1997). MvfR is required to produce virulence factors, such as pyocyanin and hydrogen cyanide (Gallagher & Manoil, 2001; Rahme et al., 1997), and a large family of 4-hydroxy-2-alkylquinolines (HAQs), including the intercellular signals 3,4-dihydroxy-2-heptylquinoline (PQS) and 4-hydroxy-2-heptylquinoline (HHQ) (Déziel et al., 2004; Gallagher et al., 2002; Lépine et al., 2004), which function in the transcriptional regulation of multiple virulence genes (Déziel et al., 2004; Gallagher et al., 2002; McKnight et al., 2000; Pesci et al., 1999).

MvfR, LasR and RhlR are components of a complex regulatory network. While MvfR controls the pqsABCDE and phnAB operons, which encode proteins mediating HAQ biosynthesis (Déziel et al., 2004; Gallagher et al., 2002), the final step of PQS production, HHQ hydroxylation, appears to require the LasR-regulated pqsH gene (Déziel et al., 2004; Gallagher et al., 2002). Furthermore, although MvfR regulates multiple P. aeruginosa QS-controlled genes, it is not involved in lasRI/rhlRI expression, or in homoserine lactone autoinducer signal production (Déziel et al., 2005). Finally, while the las system activates mvfR and pqsA–E transcription, the rhl system appears to repress their expression (McGrath et al., 2004; Schuster et al., 2003). Recent studies indicate that the mvfR promoter contains a putative las/rhl box that is required for mvfR transcriptional activation (Wade et al., 2005).

The MvfR protein has an N-terminal DNA-binding domain and a C-terminal ligand-binding domain, belonging to the LysR-type transcriptional regulator (LTTR) protein family. The prototypical LTTR, upon binding its corresponding small activator molecule, both represses its own gene expression, and activates the transcription of target genes whose regulatory regions carry a LysR box having the consensus palindromic sequence T-[N]₁₁-A (Schell, 1993). Recent studies show that PQS enhances the in vitro binding of MvfR to a pqsA–E promoter DNA fragment, suggesting that it could be the in vivo MvfR co-inducer (Wade et al., 2005).

Here, we further interrogate and characterize the key cis-regulatory elements via which mvfR and pqsA–E transcriptional regulation is mediated in the highly virulent P. aeruginosa strain PA14. Our results provide new insights into the molecular mechanism of MvfR regulation by characterizing the regulatory relationships between LasR and mvfR, and between MvfR/RhlR and the pqs operon.

	METHODS

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Bacterial strains, growth conditions and plasmids.
Table 1 lists the bacterial strains and plasmids. P. aeruginosa and Escherichia coli were grown at 37 °C in Luria–Bertani (LB) broth and on LB agar. E. coli JM109 was used for subcloning and plasmid propagation. Ampicillin (100 µg ml^–1) for E. coli, and carbenicillin (300 µg ml^–1) and tetracycline (50 µg ml^–1) for P. aeruginosa were used for plasmid selection and maintenance.

DNA manipulations.
pQF50 is the parent plasmid for all of the lacZ transcription fusion constructs (Farinha & Kropinski, 1990). mvfR and pqsA promoter fragments were PCR-amplified from cosmid clone pH44 (He et al., 2004), digested with KpnI and HindIII, and subcloned into pQF50, to generate pGX2 from primers GX30 and GX31, pGX5 from primers GX32 and GX36, pGX6 from primers GX33 and GX36, and pGX7 from primers GX34 and GX36 (Table 2). Sequential, PCR-based, site-directed mutagenesis was used to introduce mutations into the mvfR and pqsA promoter fragments, which were then digested with KpnI and HindIII, and subcloned into pQF50, to generate pGX3 from primers GX29, GX52, GX53 and GX31. Briefly, pGX3 was generated using pGX1 as a template, and GX29 and GX52, and GX53 and GX31 were used, respectively, to amplify two overlapping PCR products, which were purified and mixed with the template to amplify the final mutated PCR product, using GX29 and GX31. The mutated nucleotide was then introduced using GX52 and GX53. The final PCR products were digested with KpnI and HindIII, and subcloned into pQF50. Following the same procedure, pGX4 was generated using GX29, GX54, GX55 and GX31; pGX9 using GX34, GX56, GX57 and GX36; pGX10 using GX34, GX58, GX59 and GX36; pGX11 using GX32, GX60, GX61 and GX36; and pGX12 using GX32, GX210, GX209 and GX36 (Table 2). Constructs were confirmed by DNA sequencing, and electroporated into E. coli and P. aeruginosa PA14 cells.

-Galactosidase activity assay.

DNA mobility gel-shift assay.
Lysate from E. coli cells carrying pDN18–mvfR, which overexpresses MvfR, was mixed with 2 nM ³²P-labelled wild-type or mutated pqsA promoter fragments in a total volume of 15 µl, incubated at room temperature for 20 min in the presence of 40 pM PQS, and analysed by electrophoresis on 5 % nondenaturated polyacrylamide gels at 200 V at 4 °C.

	RESULTS AND DISCUSSION

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A las/rhl box functions in mvfR transcriptional activation
Microarray and lacZ-fusion data suggest that LasR, and not RhlR, upregulates mvfR transcription (Hentzer et al., 2003; Schuster et al., 2003; Wade et al., 2005). A putative las/rhl box, centred at –513 bp from the MvfR translational start site (Wade et al., 2005; Fig. 1A), shares 81.3 % identity with the las/rhl box of the promoter region of the QS-regulated rsaL gene (Whiteley & Greenberg, 2001; Fig. 1B). The las/rhl-box sequence has nearly all the conserved sequence elements of the prototypical las-specific promoter (Schuster et al., 2004). To determine if this putative element functions in mvfR regulation, mvfR'–lacZ transcriptional fusions were generated. The DNA fragment from the 746 bp upstream to the first 160 nucleotides of the mvfR gene was fused to a promoterless lacZ gene in pQF50, to generate pGX1 (Déziel et al., 2005). As a control, we fused the mvfR –447 to +160 region, which lacks the putative las/rhl box, to lacZ (pGX2). These two constructs were separately introduced into strain PA14, with pGX1 also introduced into lasR^– mutant cells. Fig. 1(C) shows that the -galactosidase activity from both fusions increased with increasing PA14 cell density. Furthermore, pGX1 gave significantly higher levels in strain PA14 versus pGX2, suggesting that its putative las/rhl box functions in mvfR regulation.

View larger version (13K): [in this window] [in a new window]   Fig. 1. A las/rhl box is critical for mvfR transcriptional activation. (A) Sequence and localization of the putative las/rhl box in the nadA–mvfR intergenic region. Bold nucleotides are the most highly conserved in known and putative las/rhl boxes. Only the substituted nucleotides are shown in the C1T and A11T las/rhl box mutants. – and + indicate position relative to the MvfR translational start site. Lines are not drawn to scale. (B) Comparison between the las/rhl boxes of mvfR and rsaL and a proposed las-specific recognition sequence (Schuster etal., 2004). The highly conserved consensus sequence in CT-[N]12-AG is shown in bold. (C) MvfR–lacZ expression in strain PA14 is altered by deletion of the –746 to –448 region, which contains the putative las/rhl box, and by the C1T and A11T las/rhl box substitutions. -Galactosidase activity was measured at different cell densities for strain PA14 wild-type (WT) : : pGX1 (mvfR'–lacZ, –746, ), pGX2 (mvfR'–lacZ, –447, ), pGX3 (mvfR'–lacZ, –746, C1T, ), pGX4 (mvfR'–lacZ, –746, A11T, ), and PA14 lasR– : : pGX1 (mvfR'–lacZ, –746, ).

If LasR regulates mvfR expression only via elements between –746 and –448 bp upstream of the translational start site, deletion of this region should be equivalent to a lasR mutation, which completely abrogates mvfR expression (Wade et al., 2005; Fig. 1C). In contrast, complete deletion of this region, or mutagenesis of two conserved las-box nucleotides, results in limited mvfR expression in strain PA14 (Fig. 1C, pGX2–4 constructs). These results indicate that an unknown LasR-dependent transcriptional regulator(s) activate(s) mvfR expression via a regulatory site(s) within the –447 to +160 bp region that lacks las/rhl box homology, or contains a LasR cryptic site. VqsR could be a candidate for this unknown regulator, as LasR controls its expression, and VqsR affects mvfR transcription (Juhas et al., 2004; Schuster et al., 2003). In addition, Fig. 1(C) (and data not shown) demonstrate that the pGX1–4 constructs only display activity in lasR^– mutant cells during late growth, suggesting LasR-independent transcription factors also function in mvfR regulation.

A las/rhl and a LysR box are critical for pqsABCDE transcriptional regulation
MvfR binds to the promoter region of the pqsA–E operon (Wade et al., 2005). Fig. 2(A) shows that the pqsA promoter region carries a putative LysR box, with a perfect dyad symmetry centred at –45 bp relative to the pqsA transcription initiation site, and two putative las/rhl boxes centred respectively at –151 and –311. While the –311 las/rhl box, pqsA-1, has nearly all the conserved sequence elements of the prototypical rhl-responsive promoter (Schuster et al., 2004), the –151 las/rhl box, pqsA-2, is less conserved (Fig. 2B). These elements suggest that, in addition to MvfR, LasR and/or RhlR may also regulate pqsA–E transcription. To this end, we generated and assayed pqsA'–lacZ deletion constructs, starting from pGX5 (Fig. 2A), which carries the pqsA promoter region from –486 to +231 relative to the pqsA transcriptional initiation start site, fused to lacZ. This 717 bp fragment includes the 5' 160 nt of the pqsA ORF and, presumably, the entire pqsA–E regulatory region. pGX6 and pGX7 carry, respectively, the fragments –246 to +231 and –89 to +231 fused to lacZ.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Analysis of pqsA regulatory region mutants shows that a LysR box and a las/rhl box are critical for pqsA–E transcriptional regulation. (A) Localization in the ogt and pqsA intergenic region of the putative las/rhl (–311 pqsA-1 and –151 pqsA-2) and LysR boxes (–45), and the putative LysR box sequence. The pqsA transcriptional start occurs 71 bp upstream of the translational start codon (McGrath et al., 2004). Bold nucleotides are the most highly conserved in all known and putative LysR boxes. Only the substituted nucleotide is shown for the T1C LysR box mutant. The remaining sequence is for the half LysR box mutant deletion. The position –1 C of the 5' pqsA-1 las/rhl box was substituted with T to generate the C1T las/rhl box mutant. The whole pqsA-1 las/rhl box was deleted to create the del mutant. – and + indicate position relative to the pqsA transcriptional initiation site. Lines are not drawn to scale. (B) Comparison between the las/rhl boxes of pqsA and rhlA and a proposed rhl-responsive motif (Schuster et al., 2004). The highly conserved consensus sequence in CT-[N]12-AG is shown in bold. Only the substituted nucleotide is shown for the C1T las/rhl box mutant (pGX11). (C) Strain PA14 expression of wild-type, LysR box and las/rhl box mutants. The effect was determined by deleting the sequences –486 to –247, –246 to –90, the pqsA-1 las/rhl box and half of the LysR box, and substitutions of the pqsA-1 las/rhl box C1T, and the LysR box T1C, on pqsA–lacZ expression in PA14. -Galactosidase activity was measured at different cell densities for strain PA14 : : pGX5 (pqsA'–lacZ, –486, ), pGX6 (pqsA'–lacZ, –246, ), pGX7 (pqsA'–lacZ, –89, ), pGX9 (pqsA'–lacZ, –89, T1C, ), pGX10 (pqsA'–lacZ, –89, half, -x-), pGX11 (pqsA'–lacZ, –486, C1T, ), and pGX12 (pqsA'–lacZ, –486, del, ). (D) Expression of wild-type and a las/rhl box mutant instrain PA14 WT, rhlR– and lasR– mutants. The effect of deleting the pqsA-1 las/rhl box on pqsA–lacZ expression in PA14 WT, rhlR– and lasR– mutants. -Galactosidase activity was measured at different cell densities for pGX5 (pqsA'–lacZ, –486) and pGX12 (pqsA'–lacZ, –486, del) in PA14 wild-type, rhlR– and lasR– mutants, respectively. , pGX5, WT; , pGX12, WT; , pGX5, rhlR–; , pGX12, rhlR–; , pGX5, lasR–; , pGX12, lasR–.

RhlR represses pqsA expression (McGrath et al., 2004), therefore, pqsA transcriptional repression can be mediated via RhlR binding to the pqsA-1 element. To this end, we compared the LacZ activities of pGX5 and pGX12 in strain PA14 versus PA14 rhlR^– mutant cells (Fig. 2D). Fig. 2(D) shows that pqsA expression in rhlR^– mutant cells is unaffected with or without the presence of the pqsA-1 element. These results suggest that RhlR binds to this las/rhl box to repress pqsA transcription, and that the highly conserved C-1 nucleotide is unimportant for this DNA–protein interaction.

Fig. 2(A) presents two pGX7 derivatives used to assess the importance of the putative LysR box. pGX9 (T1C) was generated by replacing the highly conserved 1-T nucleotide (Schell, 1993) with a C, and pGX10 (half) was generated by deleting the 5' 6 nt of pGX7, to destroy its dyad symmetry. Fig. 2(C) shows that the T1C substitution severely reduced pqsA activation, and that this activation was completely abrogated by the LysR box half deletion. Since all the altered nucleotides are 5' of position –45, these mutations likely do not perturb RNA polymerase binding. These data further demonstrate that the putative LysR box centred at –45 is a critical pqsA–E regulatory element, likely via MvfR recognition and binding (Wade et al., 2005). Nonetheless, Fig. 2(D) also shows that pqsA expression in lasR^– mutant cells (lasR/pGX5) increases later in growth, in agreement with the mvfR expression kinetics in lasR^– mutant cells (Fig. 1C, lasR/pGX1). These data concur with previous results for late PQS and pyocyanin production in these cells (Diggle et al., 2003), further suggesting that, in the absence of LasR, an unknown LasR-independent transcription factor(s) activate(s) mvfR, and, consequentially, pqsA–E during late growth.

MvfR binds to a LysR box to activate pqsABCDE transcription
PQS potentiates MvfR binding to a pqsA–E promoter DNA fragment (Wade et al., 2005). To determine if the two putative LysR box mutations affect the DNA binding of MvfR, we separately mixed ³²P-labelled wild-type and mutated pqsA promoter fragments with E. coli cell lysate expressing MvfR in the presence of PQS (Fig. 3A, B). Consistent with the Fig. 2(C) expression results, the T1C substitution was seen to reduce MvfR binding. Furthermore, binding to the half LysR box deletion was significantly reduced, resulting in a protein–DNA complex with higher mobility and weaker affinity. These results suggest that an MvfR monomer weakly binds the right-half LysR box, and that MvfR dimerization is important for the biologically productive protein–DNA interaction.

View larger version (35K): [in this window] [in a new window]   Fig. 3. The LysR box functions in MvfR–pqsA promoter DNA–protein interaction. (A) A 32P-labelled 174 bp DNA fragment of the pqsA promoter region was mixed with E. coli cell lysate containing MvfR plus 40 pM PQS (lanes 2–6, 8–10 and 12–14). Protein added per reaction: lanes 1, 7 and 11, 0 ng µl–1; lane 2, 0.6 ng µl–1; lane 3, 3 ng µl–1; lanes 4, 8 and 12, 6 ng µl–1; lanes 5, 9 and 13, 30 ng µl–1; and lanes 6, 10 and 14, 60 ng µl–1. Mixtures were electrophoresed on 5 % nondenaturated polyacrylamide gels. (B) Relative signal levels were determined by densitometry.

	ACKNOWLEDGEMENTS

 
We thank Dr E. P. Greenberg (University of Iowa) for providing pQF50 and Dr S. E. Stachel (BioDiversity LLC) for comments and editing. This work was supported by the Claflin Distinguished Scholar Award, and Shriners Research grant no. 8850, to L. G. R.

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Received 18 October 2005; revised 31 January 2006; accepted 7 February 2006.

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