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Journal of Bacteriology, May 2006, p. 3365-3370, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3365-3370.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Distinct QscR Regulon in the Pseudomonas aeruginosa Quorum-Sensing Circuit{dagger}

Yannick Lequette,1 Joon-Hee Lee,1 Fouzia Ledgham,2 Andrée Lazdunski,2 and E. Peter Greenberg1*

Department of Microbiology, University of Washington, Seattle, Washington 98195,1 Laboratoire d'Ingénierie des Systèmes Macromoléculaires, IBSM/CNRS, Marseille 13402, Cedex 20, France2

Received 12 October 2005/ Accepted 1 February 2006


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ABSTRACT
 
The opportunistic pathogen Pseudomonas aeruginosa possesses two complete acyl-homoserine lactone (acyl-HSL) signaling systems. One system consists of LasI and LasR, which generate a 3-oxododecanoyl-homoserine lactone signal and respond to that signal, respectively. The other system is RhlI and RhlR, which generate butanoyl-homoserine lactone and respond to butanoyl-homoserine lactone, respectively. These quorum-sensing systems control hundreds of genes. There is also an orphan LasR-RhlR homolog, QscR, for which there is no cognate acyl-HSL synthetic enzyme. We previously reported that a qscR mutant is hypervirulent and showed that QscR transiently represses a few quorum-sensing-controlled genes. To better understand the role of QscR in P. aeruginosa gene regulation and to better understand the relationship between QscR, LasR, and RhlR control of gene expression, we used transcription profiling to identify a QscR-dependent regulon. Our analysis revealed that QscR activates some genes and represses others. Some of the repressed genes are not regulated by the LasR-I or RhlR-I systems, while others are. The LasI-generated 3-oxododecanoyl-homoserine lactone serves as a signal molecule for QscR. Thus, QscR appears to be an integral component of the P. aeruginosa quorum-sensing circuitry. QscR uses the LasI-generated acyl-homoserine lactone signal and controls a specific regulon that overlaps with the already overlapping LasR- and RhlR-dependent regulons.


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INTRODUCTION
 
The opportunistic pathogen Pseudomonas aeruginosa can be found free living in water and soil. This bacterium also causes infections in a variety of animals and plants (7). Quorum-sensing systems control hundreds of P. aeruginosa genes, including genes that code for exoenzymes and extracellular virulence factor synthesis. Quorum sensing also plays a role in biofilm development (4, 5, 13, 17, 22, 28). There are two well-studied P. aeruginosa acyl-homoserine lactone (acyl-HSL) quorum-sensing systems. One system is comprised of the LasR signal receptor, which responds to the LasI-generated signal N-3-oxododecanoyl-homoserine lactone (3OC12-HSL). The other system is comprised of the RhlR signal receptor, which responds to the RhlI-generated signal N-butanoyl-homoserine lactone (C4-HSL) (5, 13, 18, 19, 26, 29). The rhlI and rhlR genes are among the functions activated by LasR and LasI. Both systems are also integrated in regulatory networks that respond to environmental cues other than cell density (20, 21, 30).

In addition to LasR and RhlR, there is a third, orphan LasR-RhlR homolog, QscR, for which there is no cognate acyl-HSL synthase gene (3). A qscR mutant is hypervirulent. The influence of QscR on the expression of a few genes controlled by the LasR-I and RhlR-I systems has been examined. These genes are prematurely activated in a qscR mutant and include genes in the phz1 and phz2 phenazine synthesis operons; hcnAB, the hydrogen cyanide synthesis operon; lasB, which codes for elastase; rhlI; and lasI (3, 14). The mechanism for transient repression of these genes by QscR is not clear. At low acyl-HSL concentrations, QscR can form heterodimers with LasR and RhlR. This might inactivate LasR and RhlR (3, 14). It is also possible that QscR sequesters acyl-HSL signals and thereby delays the expression of LasR- and RhlR-dependent genes (3, 14). To develop a better view of the role of QscR in P. aeruginosa gene regulation, we employed microarray technology to assess the influence of QscR on the transcriptome. We show that QscR affects transcript levels of over 400 genes, most of which are not affected by the LasR-I or RhlR-I systems. Our microarray studies and subsequent reporter gene experiments indicate that there is a specific QscR regulon. We believe that QscR can directly influence specific genes in response to the LasI-generated signal 3OC12-HSL.


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MATERIALS AND METHODS
 
Bacterial strains and plasmids. We used the isogenic P. aeruginosa strains PAO1 and PAO-R3 (3), and we used Escherichia coli DH5{alpha}. The P. aeruginosa strains overexpressing qscR (YL113) and a qscR 3' deletion (YL117) were constructed in PAO-R3 as follows. A qscR overexpression plasmid (pJN105-QscR) was constructed as described elsewhere previously (15). An in-frame deletion of the qscR 3' end was created as follows. We amplified an XbaI-SacI fragment extending from 45 bp upstream of the qscR start codon through codon 182 (PCR-1). We amplified a second SacI-XbaI fragment extending from codon 219 to 67 bases past the stop codon (PCR-2). The PCR-1 and PCR-2 products were cloned together into XbaI-digested pJN105 to yield a plasmid coding for a QscR DNA-binding mutant polypeptide missing amino acid residues 183 to 218 (pYL135). The SalI-SacI (with the SacI site blunt ended) fragment from pJN105-QscR and the SalI-NotI fragment (with the NotI site blunt ended) from pYL135 were cloned into SalI-SspI-digested mini-CTX-lacZ to yield pYL129 and pYL137. These plasmids were used to insert qscR alleles into the P. aeruginosa chromosomal attB site by standard techniques (10, 11). The resulting P. aeruginosa strains had unmarked chromosomal copies of a qscR allele. All primers used in this study are described in Table S1 in the supplemental material.

Plasmids with point mutations in qscR were constructed with PCR products as follows. Mutations were constructed in two PCR steps. The first PCR used one flanking primer and an internal primer containing a point mutation. The second PCR used the other flanking primer and a complementary internal primer with the desired point mutation. Gel-purified PCR fragments were annealed and amplified by PCR using flanking primers, which included XbaI overhangs. The resulting PCR product was digested with XbaI and cloned into pJN105. The PA1897-lacZ reporter plasmid pJL101 has been described elsewhere previously (15).

Microarray analysis. All strains were grown under identical conditions in Luria-Bertani (LB) broth containing 50 mM MOPS (morpholinepropanesulfonic acid) (pH 7.0) as described elsewhere previously (22), except that we included 0.2% L-arabinose in every case. We isolated RNA from 2 x 109 cells from cultures at optical densities at 600 nm of 0.5, 0.8, 1.4, 2.0, and 3.5. The RNA purification, cDNA synthesis, fragmentation, labeling, and processing of P. aeruginosa microarrays (Affymetrix) were performed as described previously (22). Experiments with strains PAO1 and PAO-R3 were done in duplicate. Experiments with strains YL113 and YL117 were done once. Data were processed with Affymetrix software suite 1.1, CYBER-T (http://www.visitor.ics.uci.edu/genex/cybert/) (1, 8), and GeneSpring 6.1. To identify those genes with expression significantly different between PAO1 and PAO-R3 at different culture densities, we used CYBER-T. The Bayesian prior estimate was 10, and the sliding-window size was 101. The P value threshold was 0.001, the posterior probability of differential expression was >0.95, and the severalfold change was >2.5.

Real-time PCR. Primers (see Table S1 in the supplemental material) were designed using Primer Express software (Taqman). Total RNA was extracted from P. aeruginosa cultures at optical densities (600 nm) of 0.4, 1, 2.0, 3.0, and 4.0. PCRs included 1 ng of cDNA, and primers at a concentration of 300 nM in 25 µl of SYBR green PCR amplification master mix (Applied Biosystems). Real-time PCR conditions were as follows: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C (denaturation step), and 1 min at 60°C (annealing and extension steps). Genomic DNA was used as a standard, and nadB (PA0761) was used as an internal control.

Measurement of lacZ transcription in recombinant E. coli. To monitor lacZ transcription, we measured ß-galactosidase activity by using a Galacto-Light Plus kit (Tropix) as described elsewhere previously (28). Results are given in units of ß-galactosidase per optical density at 600 nm.

Microarray accession number. Microarray analysis data have been deposited in the GEO database (http://www.ncbi.nlm.nih.goc/geo) under accession number GSE4026.


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RESULTS
 
The P. aeruginosa QscR regulon. We first compared transcriptomes of the P. aeruginosa PAO-R3 qscR mutant and the parental strain, PAO1, at several points during growth (optical density at 600 nm of 0.5, 0.8, 1.4, 2.0, and 3.5). We identified 424 genes and eight intergenic regions showing statistically significant differences of at least 2.5 (see Tables S2, S3, and S4 in the supplemental material). Most of the 424 differentially regulated genes appeared to be repressed by QscR (329 genes), but 76 genes appeared to be induced by QscR. Nineteen genes were discontinuously regulated during growth. We assume that the discontinuously regulated genes are controlled via multiple inputs, with QscR representing one input.

Our transcriptome analysis and real-time PCR analyses confirmed previous reports (3, 14) that QscR repressed hcnABC and the two phenazine operons (phzI and phzII) in logarithmic phase. Moreover, we found that QscR induced hcnA in stationary phase and repressed lasB until early stationary phase. We grouped the QscR-controlled genes into five classes based on the timing of QscR-dependent regulation (Fig. 1; see Tables S2 to S4 in the supplemental material): there are 98 class I genes regulated by QscR in exponential phase, 195 class II genes showing differential transcript levels during the transition from logarithmic to stationary phase, 94 class III genes regulated by QscR in stationary phase, 18 class IV genes regulated at all times during growth, and 19 class V genes showing discontinuous regulation during growth.


Figure 1
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  		FIG. 1. GeneSpring cluster analysis of P. aeruginosa QscR-regulated genes and examples of transcript levels for members of each class of expression pattern at different culture densities. (A) Cluster analysis. Red indicates negative changes in transcript levels of the parent compared to the qscR null mutant, and green indicates positive changes in transcript levels of the parent compared to the qscR null mutant. Class I, genes regulated in logarithmic phase; class II, genes regulated during the transition from logarithmic to stationary phase; class III, genes regulated in stationary phase; class IV, genes regulated throughout growth; class V, genes discontinuously regulated. (B) Transcript levels (x1,000; units as determined by array software) for representative members of each class. Transcript levels of the qscR null mutant ({circ}) and wild type ({triangledown}) are shown. The gene numbers or gene names are indicated according to the Pseudomonas Genome Project website (http://www.pseudomonas.com).

Role of the QscR DNA-binding domain in regulation of gene expression. The formation of inactive QscR-LasR and QscR-RhlR heterodimers cannot explain the QscR activation we observed in our transcriptome analysis, nor can it explain the QscR repression of genes that are not regulated by the LasR-I or RhlR-I systems. Rather, our observations suggest that QscR might be capable of binding to certain promoters directly. The QscR protein has a DNA-binding domain and an acyl-HSL recognition domain (3). We predicted that a mutant QscR with a defect in the DNA-binding domain might retain an ability to repress genes by forming heterodimers with LasR or RhlR or by sequestering acyl-HSLs, but it should not be able to control gene expression by a direct interaction with promoters. Thus, we constructed P. aeruginosa qscR mutants containing a chromosomal copy of either an L-arabinose promoter-driven qscR allele (pbad-qscR) or an L-arabinose promoter-driven qscR deletion that codes for a polypeptide with a truncation of the C-terminal DNA-binding domain (pbad-qscR-{Delta}dbd) (see Materials and Methods). Under the conditions of our experiments, the L-arabinose promoter-controlled qscR alleles were both expressed at a level about 50-fold higher than that of qscR in the parental strain, PAO1 (data not shown). We identified 38 genes (Table 1) that were at least threefold differentially expressed in the strain containing the L-arabinose promoter-driven qscR allele compared to the strain containing the L-arabinose promoter-driven qscR-{Delta}dbd allele and were also at least threefold differentially expressed in the parent strain, PAO1, compared to the strain containing the qscR null mutation. We suspect that QscR controls at least some of these 38 genes directly. There are many other ways to sort these genes. We chose this analysis as a way to derive a minimum number of genes with some likelihood of being controlled by QscR directly.


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  		TABLE 1. Genes potentially dependent on the native DNA-binding domain of QscR

Evidence for direct control of PA1897 by QscR and 3OC12-HSL. To provide further evidence that QscR can regulate P. aeruginosa genes directly, we studied PA1897 gene expression in recombinant E. coli (Table 2). We chose PA1897 because we found that in P. aeruginosa PA1897, transcript levels were low in the qscR null mutant, higher in the wild type, and even higher in the qscR overexpression strain (Fig. 2). Furthermore, PA1897 is adjacent to qscR on the P. aeruginosa genome (24), it requires 3OC12-HSL for full induction (22), and LasR does not bind to the PA1897 promoter (23). Expression of a plasmid-encoded PA1897-lacZ fusion was relatively low in the absence of both a plasmid expressing qscR and added 3OC12-HSL (Table 2). In the presence of both QscR and 3OC12-HSL, lacZ expression was nearly 200-fold higher than that in the absence of QscR. LasR did not substitute for QscR, and C4-HSL did not substitute for 3OC12-HSL.


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  		TABLE 2. Acyl-HSL-dependent expression of a PA1897-lacZ fusion in E. coli containing LasR, QscR, or QscR mutant expression vectorsa


Figure 2
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  		FIG. 2. Transcript levels of PA1897-1894 in the P. aeruginosa parent strain and in P. aeruginosa qscR mutants. The qscR mutants are the qscR null mutant, the strain expressing L-arabinose promoter-driven qscR (pbad-qscR), and the strain expressing the L-arabinose promoter-driven qscR deletion that codes for a polypeptide with a truncation of the C-terminal DNA-binding domain (pbad-qscR-{Delta}dbd). Transcript levels from cultures at an optical density (600 nm) of 1.4 are the averages of transcript levels for PA1897, PA1895, and PA1894 open reading frames from our microarray data. Errors bars show the standard deviations.

We also used five distinct qscR alleles containing single point mutations in codons for amino acid residues conserved in LuxR homologs or conserved in the 3OC12-HSL recognition region of LasR (12). None of the QscR point mutant polypeptides activated the PA1897-lacZ reporter (Table 2). The QscR-dependent activation of the PA1897 promoter is consistent with a model of direct QscR function. Our experiments further indicate that QscR, which does not have a cognate acyl-HSL synthase, recognizes the 3OC12-HSL signal from the LasR-LasI system.

Relationships between the QscR, LasR, and RhlR regulons. Several groups have previously used microarrays to identify genes controlled by the LasR-LasI and RhlR-RhlI systems (9, 22, 25). We used experimental conditions comparable to those described previously by Schuster et al. (22) for our array experiments. Thus, we have compared our data to those described previously by Schuster et al. (22). We found that only 37% of the QscR regulon is activated (159 genes) by the LasR-I or RhlR-I systems. Among the 159 genes, only 12% (19 genes) were induced by QscR. All 19 of these genes depend primarily on 3OC12-HSL for expression and show little or no additional activation by C4-HSL (Fig. 3A) (22). Eight of the nine genes discontinuously regulated by QscR during growth require C4-HSL for full activation (data not shown).


Figure 3
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  		FIG. 3. Venn diagrams showing overlaps between QscR-repressed and QscR-activated genes and LasR-I and RhlR-I regulons. Blue, genes that require LasR-I for activation; yellow, genes that require RhlR-I for activation; gray, genes that require both LasR-I and RhlR-I for activation. (A) Green, QscR-activated genes. (B) Pink, QscR-repressed genes. There are an additional 274 QscR-dependent genes that are not induced by the LasR-I or RhlR-I systems and are not represented in these Venn diagrams.

QscR repressed 131 LasR-I- or RhlR-I-activated genes identified previously (22). Of these genes, 48% require C4-HSL for activation, 36% require both 3OC12-HSL and C4-HSL for maximal activation, and 16% require 3OC12-HSL only for activation (Fig. 3B). The six previously identified quorum-sensing genes repressed by QscR (3, 14) belong to a subset of 38 genes affected by QscR in the logarithmic phase. An example of transcript levels during growth for a gene in this subset is shown in Fig. 1B (class I). QscR represses other genes later in growth (classes II to V) (Fig. 1B). Genes activated by the RhlR-I or LasR-I systems and repressed by QscR, particularly those genes repressed by QscR in logarithmic phase, may be affected by the ability of QscR to form heterodimers with RhlR or LasR or by an ability to sequester acyl-HSL signals, but we cannot rule out the possibility of direct regulation of some of these genes at this time.

Functional classification of QscR-regulated genes. Functional classification was based on the Pseudomonas Genome Project website (http://www.pseudomonas.com). The main classes represented in the QscR regulon were energy source metabolism (13%), transport of small molecules (11%), and virulence factor biosynthesis (9%) (see Fig. S1A in the supplemental material). QscR did not regulate any genes involved in cell division, cell wall/lipopolysaccharide/capsule, or nucleotide biosynthesis and metabolism (see Fig. S1A in the supplemental material). QscR repressed some genes and activated some genes in almost every class (see Fig. S1B in the supplemental material). The exceptions were genes coding for production of secreted factors and quinolone biosynthesis genes. Genes in these two groups were repressed but not activated by QscR and are only activated by LasR and RhlR (22). QscR repressed 31 iron starvation response cistrons.


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DISCUSSION
 
Previous studies have shown that the orphan LasR-RhlR homolog QscR can influence the virulence of P. aeruginosa. A qscR mutant was hypervirulent. Furthermore, QscR was shown to delay the expression of several genes that were activated by LasR or RhlR (3, 14). There are several possible explanations for these QscR phenotypes: QscR might interact with specific LasR- or RhlR-controlled promoters directly; QscR might bind to the LasR signal, 3OC12-HSL, or the RhlR signal, C4-HSL; or QscR might form inactive heterodimers with LasR or RhlR. In fact, evidence indicates that QscR can form heterodimers with LasR and RhlR (14). Heterodimer formation could explain the QscR delays in expression of several LasR- and RhlR-activated genes. There is precedent for this type of regulatory effect. In Agrobacterium tumefaciens, there is a homolog of the quorum-sensing signal receptor TraR. This homolog, TrlR, is an orphan TraR homolog that, unlike QscR, has a mutation that eliminates its DNA-binding region. Thus, it can function only in the capacity of forming inactive heterodimers or sequestering signal (2, 16, 31).

The proposed functions of QscR are not exclusive of each other, and because, unlike the A. tumefaciens TrlR, QscR has what appears to be a functional DNA-binding domain, it could function via direct binding to specific promoter elements. To investigate the mechanisms by which QscR might influence P. aeruginosa gene expression, we compared the transcriptomes of a qscR mutant, a mutant that overexpresses QscR, a mutant that overexpresses a defective QscR with a deletion in the C-terminal DNA-binding domain, and the parent. Our results show that QscR can activate some genes and repress some genes that are not regulated by the LasR-I or RhlR-I systems. The regulation of these genes must not result from the formation of inactive LasR-QscR or RhlR-QscR dimers or from QscR competition for acyl-HSLs with LasR or RhlR. Many QscR-activated genes show higher levels of transcription in the QscR overexpression strain than in the parent (and higher levels in the parent than in the qscR null mutant). This indicates that QscR levels are limiting in the wild type under the conditions of our experiments.

As a confirmation that QscR can activate P. aeruginosa genes directly, we studied the influence of QscR on the expression of one gene (PA1897) selected from among those activated by QscR in recombinant E. coli (Table 2). Although PA1897 has a 20-bp inverted repeat promoter element similar to those involved in the activation of some LasR-dependent genes, LasR does not bind to this element (23). In fact, PA1897 shows a strong 3OC12-HSL-dependent induction by QscR in E. coli. The substitution of C4-HSL for 3OC12-HSL is ineffective, as was the substitution of LasR for QscR. Our parallel work on purified QscR shows that in vitro, this transcription factor binds to the PA1897 inverted repeat element in a 3OC12-HSL-dependent manner (15). All of this information is consistent with the conclusion that QscR is a 3OC12-HSL-responsive transcription factor that is capable of binding to and affecting specific promoters in P. aeruginosa. It now seems evident that our previous demonstration of a small activation of the PA1897 promoter in E. coli overexpressing LasR (27) was not a reflection of promoter specificity in P. aeruginosa. Rather, it is analogous to the activation of the lasB promoter in recombinant E. coli overexpressing the Vibrio fischeri LuxR protein (6).

Although we have learned some things about the signal-binding specificity and DNA-binding specificity of QscR from our transcriptome analysis and our in vitro analysis of QscR (15), some important issues remain to be resolved. We cannot yet conclude what genes other than those described previously (15) possess QscR-binding sites. We have not succeeded to learn more about binding targets by using computational approaches (Y. Lequette and E. P. Greenberg, unpublished data). In view of recent discoveries that the promoter regions of LasR-controlled genes do not always have sequence similarity to each other (23), this comes as no surprise. We previously suggested (15) that QscR might function to recognize signals produced by other bacteria. This was based on the fact the QscR was slightly more sensitive to 3OC10-HSL than it was to 3OC12-HSL. We have little to add in this regard. The alternative is that QscR was acquired relatively recently by horizontal gene transfer and that it has evolved to a point where it now functions to respond to 3OC12-HSL and control specific genes in response to P. aeruginosa population density. We have not performed any experiments involving coculture of P. aeruginosa with a 3OC10-HSL- or a C10-HSL-producing bacterium.

Many transcripts that are influenced by overexpressed QscR are also influenced by the overexpressed QscR DNA-binding domain mutant polypeptide. We believe that QscR regulates the genes encoding these transcripts indirectly. This indirect effect could result from heterodimer formation, by competition of QscR with LasR for 3OC12-HSL or with RhlR for C4-HSL, by both mechanisms, or in some unexplained nonspecific mechanism.

We are left with the view that QscR affects the transcription of well over 7% of the more than 5,500 genes in the P. aeruginosa genome. Many of these genes are regulated by the LasR-LasI and RhlR-RhlI quorum-sensing systems and may be influenced by QscR indirectly. Others appear to be regulated by QscR together with the LasR-LasI signal 3OC12-HSL directly. Furthermore, like rhlR, qscR activity is regulated by the LasR-LasI quorum-sensing system. In the case of rhlR, LasR functions at the level of transcription. In the case of QscR, the LasR-LasI system dominates, because QscR requires the LasI-generated quorum-sensing signal 3OC12-HSL for direct control of gene expression. We conclude that QscR, LasR, and RhlR control overlapping but distinct regulons in P. aeruginosa and that QscR is capable of direct activation of a group of P. aeruginosa genes.


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ACKNOWLEDGMENTS
 
This study was supported by USPHS grant GM-59026 and by a grant from the W. M. Keck Foundation.

We thank Jessica Linton from the University of Iowa DNA core facility for microarray processing.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, HSB Room G-328, 1959 NE Pacific Street, Seattle, WA 98195-7242. Phone: (206) 616-2881. Fax: (206) 616-2938. E-mail: epgreen{at}u.washington.edu. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, May 2006, p. 3365-3370, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3365-3370.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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