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Journal of Bacteriology, June 2007, p. 4367-4374, Vol. 189, No. 12
0021-9193/07/$08.00+0     doi:10.1128/JB.00007-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Regulation of the Pseudomonas aeruginosa Quorum-Sensing Regulator VqsR

Luen-Luen Li, Jane E. Malone, and Barbara H. Iglewski^*

Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received 2 January 2007/ Accepted 11 April 2007

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Bacteria communicate with each other to regulate cell density-dependent gene expression via a quorum-sensing (QS) cascade. In Pseudomonas aeruginosa, two known QS systems, las and rhl, control the expression of many factors that relate to virulence, pathogenicity, and biofilm development. Microarray studies of the las and rhl regulons led to our hypothesis that a complicated hierarchy in the QS regulon is composed of multiple transcriptional regulators. Here, we examined a QS-regulated gene, vqsR, which encodes a probable transcriptional regulator with a putative 20-bp operator sequence (las box) upstream. The transcriptional start site for vqsR was determined. The vqsR promoter was identified by examining a series of vqsR promoter-lacZ fusions. In addition, an Escherichia coli system where either LasR or RhlR protein was expressed from a plasmid indicated that the las system was the dominant regulator for vqsR. Electrophoretic mobility shift assays (EMSA) demonstrate that purified LasR protein binds directly to the vqsR promoter in the presence of 3O-C₁₂-HSL. Point mutational analysis of the vqsR las box suggests that positions 3 and 18 in the las box are important for vqsR transcription, as assayed with a series of vqsRp-lacZ fusions. EMSA also shows that positions 3 and 18 are important for binding between the vqsR promoter and LasR. Our results demonstrate that the las system directly regulates vqsR, and certain nucleotides in the las box are crucial for LasR binding and activation of the vqsR promoter.

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Pseudomonas aeruginosa is an opportunistic human pathogen and a major cause of nosocomial infections. It infects immunocompromised individuals and patients with the pulmonary disorder cystic fibrosis (10). The expression of many P. aeruginosa virulence factors is controlled by a regulatory mechanism known as quorum sensing (QS) (5, 23). QS is a form of intercellular communication that allows bacteria to coordinate gene expression in response to cell density. In many gram-negative bacteria, QS is accomplished by the production of diffusible signal molecules in the form of acyl homoserine lactones, or autoinducer, and a transcriptional regulatory protein (R protein) that serves as a signal receptor and transcriptional regulator. Both autoinducer and R protein are produced at basal levels when the population density is low. As cell density increases, autoinducer molecules accumulate. As a threshold concentration of autoinducer is reached, it forms a complex with the R protein. The R protein-autoinducer complex is able to regulate its target genes (9).

In P. aeruginosa, two primary QS systems, las and rhl, have been identified. The las system includes LasI and LasR, and the rhl system includes RhlI and RhlR. LasI catalyzes the synthesis of the N-(3-oxododecanoyl)-L-homoserine lactone (3O-C₁₂-HSL) signal molecule, which binds to the transcriptional regulator protein LasR. RhlI catalyzes the synthesis of N-butanoyl-L-homoserine lactone (C₄-HSL), which binds to the RhlR protein. These two systems form a hierarchical cascade that regulates the transcription of multiple structural and regulatory genes (17, 26). A conserved dyad symmetry DNA sequence, termed the las box, has been found upstream of some genes known to be directly regulated by the las or the rhl system (7, 37). Many studies suggest that the las and rhl systems regulate their target genes by recognizing the las box (2, 6, 20, 29).

In addition to the las and rhl systems, other QS signal molecules and regulators have been identified. Yet, their roles in QS are not fully understood. For example, the P. aeruginosa quinolone signal is regulated by the las system (25) and also regulates the rhl system (19). Also, QscR, a LuxR homolog, uses 3O-C₁₂-HSL to control a specific regulon that partially overlaps the las and rhl regulon (3, 18). These add a further level of complexity to the QS circuit.

Over the past few years, global gene expression modulated by the las and rhl QS regulons in P. aeruginosa have been analyzed (11, 31, 36). Transcription profiling suggests that the las and/or rhl regulon influences the transcription of 3 to 11% of the P. aeruginosa genome. However, many of the genes in this regulon appear to lack a las box in their promoter region (36). This implies that the expression of many QS genes may be indirectly controlled by the las and/or rhl system. For instance, las and/or rhl may regulate additional transcriptional regulator(s). Further analysis of the hierarchical cascade in the QS regulon will be required to test this hypothesis and may elucidate one or more currently unknown critical regulatory factors other than the LasR:3O-C₁₂-HSL and RhlR:C₄-HSL complexes. These regulatory factors, by themselves or together with the las and rhl systems, could be potential targets for disruption for altering virulence.

In a P. aeruginosa strain isolated from a cystic fibrosis patient, inactivation of vqsR abrogated the production of acyl homoserine lactone signal molecules, decreased the production of virulence factors, and reduced the pathogenicity in a nematode infection model system (15). Preliminary transcriptome analysis also suggested that vqsR may regulate QS-controlled genes (15, 16). Expression of vqsR itself appears to be regulated by las and/or rhl in P. aeruginosa PAO1 (11, 31, 36). The goal of the present study was to determine if vqsR is directly regulated by the las and/or rhl system. The regulation of vqsR was studied in vivo with vqsRp-lacZ transcriptional fusions. A direct interaction between the vqsR promoter and purified LasR protein in vitro was also demonstrated. This work establishes the importance of the las box found in the vqsR promoter region.

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Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are shown in Table 1. P. aeruginosa strains were cultured at 37°C in peptone-tryptic soy broth (PTSB) (22) or modified FAB [0.1 mM CaCl₂, 0.1 mM FeSO₄, 0.15 mM (NH₄)₂SO₄, 0.33 mM Na₂HPO₄, 0.2 mM KH₂PO₄, 0.5 mM NaCl, 40 mM KNO₃, 10 mM glucose, 0.17% yeast extract (pH 7.0)] (4). When appropriate, carbenicillin (200 µg/ml), exogenous 3O-C₁₂-HSL, or C₄-HSL was added where indicated. Escherichia coli strains containing plasmids were grown at 37°C in Luria-Bertani (LB) broth with 100 µg/ml ampicillin (for cloning) or modified A medium (26) with 100 µg/ml ampicillin (for assaying ß-galactosidase [ß-Gal] activity).

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TABLE 1. Bacterial strains and plasmids used

DNA manipulations. Standard techniques were used for purification and manipulation of DNA (30). Restriction endonucleases, DNA ligase, and terminal deoxynucleotidyl transferase (TdT) were purchased from Invitrogen (Carlsbad, CA) or New England Biolabs (Beverly, MA). Platinum Pfx DNA polymerase and large (Klenow) fragment DNA polymerase I were purchased from Invitrogen (Carlsbad, CA). The oligonucleotides used for PCR were synthesized by Invitrogen. Calf intestine alkaline phosphatase was purchased from Roche (Indianapolis, IN). Plasmids were isolated using QIAprep spin miniprep kits (QIAGEN, Chatsworth, CA), and DNA fragments were purified using QIAquick PCR purification kits or QIAquick gel extraction kits (QIAGEN). DNA was sequenced at the DNA core facility, Functional Genomics Center, at the University of Rochester (Rochester, NY).

Construction of P. aeruginosa rhlI and lasI rhlI mutant strains. A 3-kb, HindIII-digested PCR fragment carrying the rhlI open reading frame (ORF) and flanking regions (amplified by primers 5'-AAAAAAGCTTGACCAGGCACCAGGATGGG-3' and 5'-AAAAAAGCTTCGGGCCAATTCTGCTGTGATGC-3' from PAO1 chromosomal DNA) was ligated into the vector pEX18ApEK. The resulting plasmid, pEX18Ap-rhlI, was digested with EcoRI and KpnI to make a 251-bp deletion in the rhlI ORF, treated with Klenow fragment, and then ligated with a SmaI-digested Gm^r Gfp FRT cassette to generate pJEM-rhlI. Plasmid pJEM-rhlI was transferred into P. aeruginosa PAO1 and JP1 (24) to generate an isogenic rhlI mutant (JM100) and a lasI rhlI mutant (JM2) by using allelic exchange and the Flp-FRT recombination protocol as previously described (12). The rhlI deletion in both JM100 and JM2 was confirmed by DNA sequencing.

Mapping the transcriptional start site. For RNA isolation, P. aeruginosa PAO1 cultures were collected at the mid-logarithmic growth phase (optical density at 660 nm [OD₆₆₀], 0.5) and early stationary-growth phase (OD₆₆₀, 1.0). RNA was isolated using RNAwiz and a DNA-Free kit (Ambion, Austin, TX) as previously described (36). Rapid amplification of cDNA ends (5'RACE) was performed to identify the transcriptional start site of vqsR by using the 5'RACE system (Invitrogen) according to the manufacturer's instructions. Briefly, gene specific primer 5'-TCCGTCACCACATAATGC-3', RNA from P. aeruginosa PAO1, and reverse transcriptase were used for first-strand cDNA synthesis. A homopolymeric tail was then added to the 3' end of the cDNA by using TdT and dCTP/dATP. PCR amplification was accomplished using a nested gene-specific primer (5'-CGACAGTTCCTGGATACC-3'), a novel deoxyinosine-containing anchor primer, and the poly(C) tail cDNA as a template. PCR products were sequenced, and transcriptional start sites were identified.

Plasmid construction and mutagenesis. (i) Construction of deletion mutations in the vqsR promoter region. To construct pLL3, a HindIII-XbaI vqsR promoter fragment from nucleotide (nt) –248 to +170 (relative to the translational start codon) was generated from a P. aeruginosa genomic DNA template by using PCR and inserted into HindIII- and XbaI-digested vector pLP170 (27). To construct pLL4, pLL3 was digested with ApaLI and SmaI, filled in with the Klenow fragment, and religated to create a promoter-lacZ fusion from nt –166 to +170. For the construction of pLL5, the same PCR fragment containing the vqsR promoter, which was used to construct pLL3, was digested with ClaI and gel purified. A 235-bp fragment recovered from the gel was treated with Klenow fragment, followed by XbaI digestion, and then inserted into a SmaI- and XbaI-digested pLP170 plasmid to create a promoter-lacZ fusion from nt –65 to +170.

(ii) Site-directed mutagenesis of the vqsR las box. Plasmids pLL3A through pLL3F, containing point mutations in the vqsR las box, were constructed by using pLL3 as a template and a QuikChangeXL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The oligonucleotides used in the construction of vqsRp-lacZ fusion plasmids are listed in Table 2. The promoter sequences of vqsR in pLL3 and pLL3A-pLL3F were verified by DNA sequencing.

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TABLE 2. Oligonucleotides used for construction of vqsRp-lacZ fusions

(iii) Construction of vqsRp-lacZ plasmids for expression in E. coli. XmnI DNA restriction fragments containing vqsRp-lacZ fusions from pLP170, pLL3, and pLL3A through pLL3F were inserted into pECP8 (treated with BamHI, Klenow fragment, and alkaline phosphatase according to the manufacturers' protocols) and pJPP8 (treated with NdeI, Klenow fragment, and alkaline phosphatase according to the manufacturers' protocols) separately to construct plasmids pLL6, pLL7, pLL8, pLL8A through pLL8F, and pLL9.

Monitoring promoter activity in P. aeruginosa. Promoter activity in P. aeruginosa was analyzed according to previously described methods (26). Briefly, cultures were grown in PTSB (supplemented with 200 µg/ml carbenicillin) at 37°C with shaking for 16 h and subcultured into the same medium to a starting OD₆₆₀ of 0.05. When indicated, 3O-C₁₂-HSL or C₄-HSL was added at the start of subculturing. When cultures were grown to an OD₆₆₀ of 1, cells were collected, resuspended in phosphate-buffered saline, and assayed for ß-Gal activity as described previously (21).

Autoinducer sensitivity assays. (i) P. aeruginosa. The autoinducer sensitivity of the vqsR promoter in P. aeruginosa was assayed as described previously (24). Briefly, cells from overnight PTSB-carbenicillin (200 µg/ml)-grown cultures were subcultured into the same medium to an OD₆₆₀ of 0.05 and then grown at 37°C with shaking to an OD₆₀₀ of 0.3. At this time, 1-ml aliquots of culture were transferred to tubes containing various concentrations of autoinducer. Growth was continued for 1 h, and the cultures were assayed for ß-Gal activity (21).

(ii) E. coli. The autoinducer sensitivity of the vqsR promoter in E. coli was assayed as described previously (24). Briefly, cultures were grown in medium A supplemented with 100 µg/ml ampicillin at 37°C with shaking overnight and subcultured into the same medium to a starting OD₆₀₀ of 0.08. After growth at 37°C with shaking to an OD₆₀₀ of 0.3, IPTG (isopropyl-ß-D-thiogalactoside) was added to a final concentration of 0.2 mM, and then 1-ml aliquots of culture were transferred to tubes containing various concentrations of autoinducer. Growth was continued for 90 min, and the cultures were assayed for ß-Gal activity (21).

Overproduction and purification of the LasR protein. For LasR expression, the culture was grown as previously described (32), with some modifications. Briefly, overnight colonies of E. coli BL21 carrying pECP8 on LB-ampicillin plates were used to inoculate 500 ml LB containing 100 µg/ml ampicillin and 10 µM 3O-C₁₂-HSL. The culture was incubated at 37°C until its OD₆₀₀ reached 0.5. Then, the culture was rapidly chilled to 20°C, induced with 0.2 mM IPTG (final concentration), and incubated at 17°C overnight. Cells were harvested from the overnight culture and frozen at –80°C. All subsequent steps for purification of LasR were performed as previously described (32). The resulting LasR preparation was verified by Western blot analysis with anti-LasR antibody and was 96% pure as judged by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a densitometric scan. The Bradford assay (Bio-Rad) was used to determined protein concentrations.

EMSA. A 309-bp DNA probe of the rsaL promoter region (nt –278 to +31) and a 292-bp DNA probe of the vqsR promoter region (nt –247 to +45) were generated by PCR from PAO1 genomic DNA. A 201-bp nonspecific DNA probe was also generated by PCR amplification of DNA encompassing the rsaL coding region and the intergenic region between rsaL and lasR (nt +601 of lasR to +144 of rsaL). All probes were labeled at the 3' end by using [-³²P]CTP and TdT according to the manufacturers' protocols. Electrophoretic mobility shift assay (EMSA) experiments were based on a protocol by Urbanowski et al. (34), and the reaction condition for LasR-DNA interaction was according to that previously described (32).

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Determination of the vqsR transcriptional start site. The transcriptional start site of vqsR was determined by 5'RACE as described in Materials and Methods. With the poly(C) tail cDNA used as a template, PCR amplification with the nested gene-specific primer and the novel deoxyinosine-containing anchor primer resulted in a single-band PCR product. This PCR product was analyzed with DNA sequencing, and the result revealed that the vqsR transcriptional start site was at 51 bp upstream of the translation start site (Fig. 1A). A putative las box is located from 115 bp to 96 bp upstream of the translation start site (as indicated in Fig. 1A) and exhibits substantial homology with other reported las box sequences (7, 37).

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FIG. 1. Identification of the probable promoter region of vqsR. (A) Nucleotide sequence of the vqsR promoter region. Numbering is relative to the vqsR GTG start codon, which is indicated with a gray box. The vqsR mRNA start site, mapped by 5'RACE, is at position –51 (G), indicated by a black box with white type. The potential las box is as indicated. (B) Three vqsRp-lacZ fusions, pLL3, pLL4, and pLL5, were constructed as indicated. Numbering is relative to the translational start site. (C) ß-Gal expression of pLL3, pLL4, pLL5, and the control vector in the wild-type strain PAO1 (shaded bar), JM2 (black bar), and JM2 with exogenous 3O-C12-HSL and C4-HSL (white bar) is shown. Data represent the means for three independent experiments, each done in triplicate. PAI, Pseudomonas aeruginosa autoinducer.

Identification of the probable promoter region of vqsR. To understand how vqsR is regulated, we constructed three vqsRp-lacZ fusions: pLL3 (which contains vqsR upstream, from nt –248 to +170), pLL4 (which contains vqsR upstream, from nt –166 to +170), and pLL5 (which contains vqsR upstream, from nt –65 to +170) (the numbering is relative to the translational start site) (Fig. 1B). These three fusions were introduced into the wild-type strain PAO1 and JM2 (lasI rhlI), and ß-Gal activity was measured to assay promoter activity. Compared to that in the control vector pLP170, ß-Gal activity increased 14-fold in PAO1/pLL3, 10-fold in PAO1/pLL4, and minimally (control levels) in PAO1/pLL5 (Fig. 1C). A similar trend of promoter activity was observed in JM2 with both exogenous 3O-C₁₂-HSL and C₄-HSL. However, in the JM2 strain without added autoinducers, only background levels of ß-Gal activity were detected with all three plasmid constructs. These findings suggested that vqsR is regulated by the las and/or rhl QS system, and the vqsR upstream region from nt –248 to +170 contains the promoter and elements for maximum transcription of vqsR. Thus, the fusion construct pLL3 was used for further transcriptional studies of vqsR.

QS control of vqsR. To identify which QS system of P. aeruginosa regulates vqsR, plasmid pLL3 (vqsRp-lacZ) was introduced into JP1 (lasI) and JM100 (rhlI), and ß-Gal activity was assayed. Compared with the ß-Gal activity in PAO1/pLL3, only 15% ß-Gal activity can be detected in JP1/pLL3, but 69% ß-Gal activity can be detected in JM100/pLL3 (Fig. 2A). To investigate the response of the vqsR promoter to different doses of autoinducers in these two strains, we added exogenous 3O-C₁₂-HSL into JP1/pLL3 culture or C₄-HSL into JM100/pLL3 cultures to a final concentration from 1 nM to 10 µM. As shown in Fig. 2B, in both JP1/pLL3 and JM100/pLL3 cultures, with increasing amounts of exogenous autoinducer in the cultures, higher ß-Gal activities were detected. For strain JP1/pLL3, with exogenous 3O-C₁₂-HSL from 0 to 10 µM, the ß-Gal activity increased 5-fold; for strain JM100/pLL3, with exogenous C₄-HSL from 0 to 10 µM, the ß-Gal activity increased only 1.5-fold.

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FIG. 2. Regulation of vqsR by the las and/or rhl QS system in either P. aeruginosa or E. coli. (A) ß-Gal activities demonstrated the response of the vqsR promoter in P. aeruginosa PAO1 (white bar), JP1 (dotted bar), and JM100 (black bar) without exogenous autoinducer. (B) The response of the vqsR promoter in JP1 with increasing doses of 3O-C12-HSL (dashed line) or in JM100 with increasing doses of C4-HSL (solid line). (C) Response of vqsR promoter in an E. coli background. Symbols: , LasR plus 3O-C12-HSL; , LasR plus C4-HSL; , RhlR plus C4-HSL; , RhlR plus 3O-C12-HSL. The ß-Gal activity values shown are normalized by subtracting the background levels (ß-Gal values of pLL6 and pLL7 in E. coli with increasing doses of 3O-C12-HSL or C4-HSL). Data represent the means for three independent experiments, each done in triplicate. (D) EMSA for the vqsR promoter and purified LasR. All lanes contained 10 fmol of an equimolar mixture of the two probes (negative control and positive control or the vqsR promoter) and the indicated concentration of LasR in the presence of 10 µM 3O-C12-HSL. (Left) Positive control (the rsaL promoter); (right) the vqsR promoter. The film was exposed for 24 h. PAI, Pseudomonas aeruginosa autoinducer.

In strain JM100, only the las system is functional because of the mutation in rhlI. In strain JP1, however, the lack of a functional las system causes a failure to activate the rhl system. Thus, both the las and the rhl systems are absent (6, 25). Therefore, in order to test the direct effect of the las or the rhl system on vqsR regulation, it is necessary to examine the vqsR promoter activity in an environment that can separately provide either the las or the rhl system. P. aeruginosa homoserine lactone autoinducers as well as lasR and/or rhlR are absent in E. coli DH5 (33). Using E. coli DH5 as a host, we analyzed the direct effect of the las and rhl QS systems on vqsR expression. To supply the las system in trans, a fragment containing the vqsRp-lacZ fusion (from pLL3) was inserted into pECP8, which contains an IPTG-inducible tacp-lasR fusion, to construct plasmid pLL8. Similarly, plasmid pLL9 was constructed with insertion of the vqsRp-lacZ fusion into pJPP8, which contains the tacp-rhlR fusion. A promoterless lacZ fragment was also inserted into plasmids pECP8 and pJPP8 to construct pLL6 and pLL7 for use as controls. The autoinducer sensitivity of the vqsR promoter was assayed by pairing pLL8 or pLL9 with various concentrations of either 3O-C₁₂-HSL or C₄-HSL. As shown in Fig. 2C, without their cognate autoinducer, neither LasR nor RhlR could activate the vqsR promoter in E. coli DH5. In the presence of a relatively low concentration of 3O-C₁₂-HSL (1 nM), the vqsR promoter was activated by the las system more than twofold compared to the control. However, the rhl system did not activate the vqsR promoter in the presence of 1 nM C₄-HSL. With a higher concentration of C₄-HSL (100 nM), the vqsR promoter showed modest activation by the rhl system. This modest activation by the rhl system was threefold less than the activation by the las system at the same concentration of autoinducer. These results suggest that the vqsR promoter is more sensitive to activation by the las system than the rhl system.

Interaction between the vqsR promoter and the las system. To investigate whether the LasR:3O-C₁₂-HSL complex binds to the vqsR promoter directly, EMSAs were performed. Based on protocols by Schuster et al. (32), the LasR protein was purified to 96% homogeneity as judged by a silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a densitometric scan (data not shown), and the rsaL promoter region was used as a positive-control DNA for the EMSA. A DNA fragment encompassing the rsaL coding region and the intergenic region between rsaL and lasR, which does not bind to LasR (as shown by Schuster et al. [32]), was also included in each reaction as a negative control (see Materials and Methods for details of sequences). As shown in Fig. 2D, in the presence of 3O-C₁₂-HSL, LasR caused a shift in the migration of the vqsR promoter DNA fragment similar to that for the positive-control fragment (a DNA that contains a known las box and binds to LasR, as shown by Schuster et al. [32]).

The importance of the las box for vqsR transcription. (i) In a P. aeruginosa background. Conserved nucleotides of the las box are critical for QS control of lasB (29). To examine whether the apparent las box sequence/motif in the vqsR promoter is critical for QS regulation, a series of vqsRp-lacZ fusions containing point mutations in the apparent las box were constructed. These plasmids were introduced into P. aeruginosa PAO1, and ß-Gal activity was measured. According to the study of Rust et al. (29), the expression of lasBp-lacZ decreased dramatically when point mutations at positions 3, 18, and both 3 and 18 were introduced in the OP1 las box of lasB. Here, we report that in P. aeruginosa PAO1, a similar effect was also observed in the same point mutations in the vqsR las box. The levels of expression were reduced to 7% of that for the wild-type vqsR las box when the mutation was at position 3 (nt –113; C to T; pLL3A), 18% when the mutation was at position 18 (nt –98; G to A; pLL3B), and 12% when mutations were introduced at both position 3 and position 18 (pLL3C) (Table 3). These data suggest that both position 3 and position 18 of the vqsR las box are critical for vqsR transcription. We also examined the expression levels of vqsR with point mutations in the las box at position 1 (nt –115; A to G; pLL3D) and position 20 (nt –96; T to C; pLL3E) and double-point mutations at both position 1 and position 20 (pLL3F). The levels of expression were reduced to 40% of that for the wild-type vqsR las box when the mutation was at position 1 and 46% when the mutations were introduced at both position 1 and position 20. The level of expression showed no significant change only when the mutation was at position 20. (The Student t test was used to identify mutations that showed significant changes of promoter activity [P < 0.05].)

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TABLE 3. The importance of the las box in the vqsR promoter region

(ii) In an E. coli background. To further examine the role of the apparent las box in regulation of the vqsR promoter by the las system, the expression levels of the vqsR promoters containing point mutations in this motif were examined using a reporter system in E. coli DH5 as described above. Promoter-lacZ fusions containing point mutations in the vqsR las box from plasmids pLL3A-F were inserted into pECP8 (containing an IPTG-induced lasR) to create pLL8A-F. Plasmids were introduced into E. coli DH5, and ß-Gal activities were measured. As shown in Table 3, when position 3 of the vqsR las box was mutated (pLL8A), the level of expression was reduced to 10% of that for the wild-type vqsR las box. When position 18 was mutated (pLL8B), the level of expression was reduced to 24%. When both position 3 and position 18 were mutated (pLL8C), the level of expression was reduced to 7%. However, the effect of mutating position 1 (pLL8D) or 20 (pLL8E) was less severe; the levels of expression were reduced to 88% and 46% separately. These data indicate that within the conserved las box sequence, these positions are crucial for las regulation of vqsR transcription.

The importance of the vqsR las box for LasR binding. The involvement of the vqsR las box for LasR binding was further tested by performing EMSA. DNA fragments containing mutations in the conserved las box of the vqsR promoter were generated by PCR amplification of plasmids pLL3A through pLL3F and were labeled with ³²P as described in Materials and Methods. As shown in Fig. 3, a mutation at vqsR las box position 3 appears to slightly decrease the binding of LasR to the vqsR promoter compared to the wild-type vqsR las box. A similar result was also observed when a point mutation was introduced at las box position 18. Single mutations at position 1 and position 20 and a double mutation at positions 1 and 20 slightly enhance the LasR binding. Interestingly, a las box containing mutations at both position 3 and position 18 completely abolished binding by LasR. These results further support the idea that the las box of vqsR is important for binding of LasR to the vqsR promoter and show that conserved positions 3 and 18 in the las box are the most crucial for binding of LasR to the vqsR promoter.

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FIG. 3. Importance of the las box for the binding of LasR to the vqsR promoter. Shown are EMSA results for the binding of LasR to the vqsR promoter with point mutations in the las box at the positions indicated. All lanes contained 10 fmol of an equimolar mixture of the two probes and purified LasR (the concentration is indicated on the top of each panel) in the presence of 10 µM 3O-C12-HSL. The films were exposed for 48 h.

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Previous transcriptome analyses have suggested that the fourth LuxR homologue in P. aeruginosa, VqsR, is controlled by the las and/or rhl QS system (11, 31, 36). VqsR also appears to be involved in the regulation of virulence and QS-controlled genes (15, 16). Therefore, VqsR is part of the QS circuit in P. aeruginosa. We hypothesized previously that a complicated hierarchy in the QS regulon is composed of multiple transcriptional regulators. The las and/or rhl system may control additional transcriptional regulator(s), and VqsR might be among these regulators.

In this study, we determined the vqsR transcriptional start site. We also examined the vqsR upstream region to identify the promoter. A 20-bp lux box-like element that matches the minimal consensus sequence and position for the P. aeruginosa las box (37) was found upstream of the transcriptional start site (Fig. 1A). This region was confirmed to contain the probable promoter region of vqsR by ß-Gal activity analysis of serially truncated upstream region-lacZ fusions (Fig. 1B).

Our next goal was to specifically address the involvement of the las and the rhl systems in regulation of the vqsR promoter. As shown in the result, the vqsR promoter appeared more sensitive to activation by the las system. When lasR was expressed, the vqsR promoter displayed relatively robust activation by 1 nM 3O-C₁₂-HSL. In comparison, when rhlR was expressed, only a modest induction was detectable, even when 100 nM C₄-HSL was added (Fig. 2B). These data suggest that the las system directly regulates vqsR and is the dominant regulator of vqsR. We further tested the idea that LasR directly regulates vqsR by performing EMSA with purified LasR and vqsR promoter DNA. Our results indicate that in the presence of 3O-C₁₂-HSL, purified LasR binds to the vqsR promoter. The shift was comparable to that for the positive control (the rsaL promoter DNA) (Fig. 2C).

According to a previous study from this laboratory (36), palindromic las box-like sequences can be found in the upstream regions of about 7% of the QS-regulated genes. Evidence from transcriptional fusion studies showed that the las box is important for the transcription of a number of QS-regulated genes. For example, the transcription of rhlI was dramatically decreased when half of the las box was deleted (6). In addition, point mutations at certain positions in the OP1 las box nearly abolished the transcription of lasB (29). In this study, we investigated the effects of point mutations in the vqsR las box. Individual mutations at positions 3 and 18 and a double mutation at both position 3 and position 18 caused the promoter activity of vqsR to be reduced by 80% to 90% compared with that of the wild-type vqsR las box, while a double mutation at both position 1 and position 20 reduced promoter activity by 50% in both a P. aeruginosa PAO1 background and an E. coli DH5 background (Table 3). These data agree with previous studies which showed that positions 3 and 18 in the las box upstream of lasB are crucial for promoter activity (29). We further studied the effects of las box mutations on the binding of LasR by using EMSA. Compared with the binding between LasR and the native vqsR promoter region, LasR binding was abolished by a double mutation at both position 3 and position 18 (Fig. 3). This is the first demonstration that mutations of certain nucleotides in the las box directly affect the binding of LasR to the vqsR promoter by using purified components.

Our results from transcriptional analysis and protein binding studies suggest that positions 3 and 18 in the vqsR las box are crucial for both LasR binding and activation of the vqsR promoter. Interestingly, our data show that mutations at positions 1 and 20 in the vqsR las box do not greatly interfere with LasR binding but still influence vqsR transcription. There are several possible reasons for this observation. First, the in vivo ß-Gal reporter system may be more sensitive than the in vitro protein-DNA binding assay. Second, positions 3 and 18 in the vqsR las box may affect the on/off rate of LasR protein more severely in EMSA than positions 1 and 20. Third, the vqsR las box is located at nt –43 to –62 relative to the transcriptional start site and potentially overlaps the nt –35 promoter determinant. The point mutation at position 20 in the las box (nt –43) may affect the promoter interaction with the RNA polymerase. Fourth, the upstream sequence of the vqsR las box (nt –62 to –113) is very low in G+C content in contrast to the overall P. aeruginosa mol% G+C of 67 (2, 14). This AT-rich region may function as the UP element for RNA polymerase CTD binding (1, 8, 28). Thus, the point mutation at position 1 (nt –62) in the las box may affect the UP element.

Previous studies have shown that E. coli DH5 is a good host for in vivo reconstitution of the P. aeruginosa transcription regulation network (6, 26), because P. aeruginosa homoserine lactone autoinducers as well as lasR and/or rhlR are absent in E. coli DH5 (33). By separately providing either the las or the rhl system in the E. coli host, we were able to examine the direct effect of the las and rhl QS systems on vqsR expression. Our results indicate that the minimal components of LasR together with 3-O-C₁₂-HSL are sufficient to reconstitute activation of the vqsR promoter in the E. coli "host" system.

In conclusion, we have demonstrated that the QS regulator VqsR is directly regulated by the las system. Our results also show that certain nucleotides in the las box are important for activation of the vqsR promoter. Therefore, in the QS cascade, we can place VqsR at the same level as RhlR and QscR, the other two LasR directly activated regulators. The results of this study provide information for a more complete understanding of the hierarchy in the QS regulon and hopefully identify a potential target for future treatment of P. aeruginosa infections.

 
We thank M. Schuster for helpful information for LasR purification and H. Ngai, K. Picardo, G. Tombline, and V. Wagner for insightful discussions and comments on the manuscript.

This work was supported by grant R37 AI033713 (to B. H. Iglewski) from the National Institutes of Health.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Box 672, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. Phone: (585) 275-3402. Fax: (585) 473-9573. E-mail: bigl{at}mail.rochester.edu

Published ahead of print on 20 April 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, June 2007, p. 4367-4374, Vol. 189, No. 12
0021-9193/07/$08.00+0     doi:10.1128/JB.00007-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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