Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

Amanda S. Nouwens1,{dagger}, Scott A. Beatson2,{ddagger}, Cynthia B. Whitchurch2,§, Bradley J. Walsh1, Herbert P. Schweizer3, John S. Mattick2 and Stuart J. Cordwell1

1 Australian Proteome Analysis Facility, Level 4, Building F7B, Macquarie University, Australia 2109
2 ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, The University of Queensland, Australia 4072
3 Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523-1677, USA

Correspondence
Stuart J. Cordwell
s.cordwell{at}proteome.org.au


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhlRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown QS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique ‘hypothetical’ protein (PA0572), which could not be detected in the culture supernatants of {Delta}las mutants, although they were unaffected in {Delta}rhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-I) could not be detected when any of the lasRI or rhlRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of QS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a QS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.


Abbreviations: AHL, acylated homoserine lactone; 2-DE, two-dimensional gel electrophoresis; QS, quorum sensing

{dagger}Present address: Children's Medical Research Institute, Westmead, Australia 2145.

{ddagger}Present address: Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK.

§Present address: Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, CA 94143, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomially acquired infections, particularly in the immunocompromised and in individuals with cystic fibrosis (Van Delden & Iglewski, 1998; Lyczak et al., 2000). The organism maintains a large genetic capacity (approx. 5500 genes; Stover et al., 2000), amongst which are encoded a battery of virulence factors localized on the cell surface or released into the external environment. The expression of many extracellular virulence factors is controlled by quorum sensing (QS), the use of small, diffusible signalling molecules (acylated homoserine lactones, AHLs) by a bacterial population to monitor cell density (Fuqua, W. C. et al., 1994; Fuqua, C. et al., 2001; Whitehead et al., 2001; Erickson et al., 2002). In P. aeruginosa, two QS systems exist, designated las and rhl (Passador et al., 1993; Ochsner & Reiser, 1995). Each system consists of two genes, one encoding a transcription regulatory protein (lasR and rhlR), and the other encoding an autoinducer synthase (lasI and rhlI). The autoinducer synthase catalyses the synthesis of AHL from S-adenosylmethionine and an acyl–acyl carrier protein. Specific AHLs, N-(3-oxododecanoyl)-L-homoserine lactone and n-butyryl-L-homoserine lactone, are synthesized by the las and rhl systems, respectively, and these bind the corresponding regulatory protein to form a functional complex which can subsequently activate the transcription of other genes (reviewed by Fuqua, C. & Greenberg, 1998).

The las and rhl QS systems are also connected, since the LasR–AHL complex regulates expression of the lasI gene (Seed et al., 1995), and also the entire rhl QS system via positive transcription of rhlR (Latifi et al., 1996; Pesci et al., 1997). Therefore, mutants lacking a functional las system cannot fully activate the downstream genes regulated by the rhl system. Genes regulated by QS in P. aeruginosa contain specific promoter regions to which the regulatory protein–AHL complex binds (Whiteley et al., 1999; Whiteley & Greenberg, 2001). These promoter regions are referred to as ‘lux-boxes’, based on the discovery of the promoter in the luxI gene from a comparable QS system in Vibrio fischeri (Devine et al., 1989). In P. aeruginosa, QS has been proposed as a global mechanism for controlling virulence factor expression and the development of biofilms (Davies et al., 1998). Several genes are regulated by QS, including toxA (exotoxin A; Gambello et al., 1993), lasB (elastase; Brint & Ohman, 1995; Latifi et al., 1995; Passador et al., 1993; Pearson et al., 1997) and lasA (LasA protease; Toder et al., 1991), aprA (alkaline metalloproteinase; Gambello et al., 1993; Latifi et al., 1995), rhlAB, encoding proteins involved in the synthesis of rhamnolipid (Ochsner et al., 1994; Pearson et al., 1997), katA and sodA (catalase and superoxide dismutase; Hassett et al., 1999), lecA (lectin; Winzer et al., 2000) and genes involved in pyocyanin formation (Brint & Ohman, 1995; Latifi et al., 1995) and twitching motility (Glessner et al., 1999). However, studies of QS-regulated gene expression, utilizing las and/or rhl mutants, may be inaccurate due to the accumulation of secondary mutations in global regulators such as vfr and algR that are directly responsible for some of these phenotypes (e.g. twitching motility; Beatson et al., 2002b).

Mutants lacking genes of the las and/or rhl systems are significantly less virulent towards host cells (Tang et al., 1996; Wu et al., 2001), and this appears to be independent of surface colonization (Reimmann et al., 2002). As QS is cell-density-dependent, regulation of virulence factors by this mechanism may allow the organism to mount an attack only when the number of cells present is sufficient to overwhelm the host. Therefore, QS systems and the genes they regulate represent potential targets for new antimicrobial agents (Dong et al., 2001). Furthermore, the las QS system is positively regulated by the vfr (virulence factor regulator) gene-product, a homologue of the cyclic AMP receptor protein (CRP) from Escherichia coli (Albus et al., 1997; Beatson et al., 2002a), which positively regulates the transcription of lasR. Similarly, the rhl QS system is subject to regulation by other systems, such as GacA, a two-component response regulator (Reimmann et al., 1997), MvaT (Diggle et al., 2002) and by the Pseudomonas quinolone signal (PQS) molecule (McKnight et al., 2000; Calfee et al., 2001).

The P. aeruginosa PAO1 genome has been fully sequenced and annotated (Croft et al., 2000; Stover et al., 2000). Nearly 50 % of the open reading frames (ORFs) have no known function. Transcriptomics and proteomics (reviewed by Harrington et al., 2000; Cordwell et al., 2001) can be applied to gauge the expression of mRNA and protein gene-products in response to altered genetic or biological conditions. Both approaches have recently been used to monitor the molecular basis of biofilm development in P. aeruginosa (Whiteley et al., 2001; Sauer et al., 2002). The effects of QS on gene expression have not yet been fully determined, although it has been hypothesized that approximately 4 % of the P. aeruginosa genome (over 200 genes) may be QS-regulated (Whiteley et al., 1999). Therefore, global approaches such as transcriptomics and proteomics are the most appropriate ways to examine the influence of QS and to identify novel QS-regulated genes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
The P. aeruginosa strains used in this study are listed in Table 1. Strains were prepared as described previously (Beatson et al., 2002a, b). Bacteria were grown in Luria–Bertani (LB) broth for 16 h at 37 °C with shaking and were monitored by optical density at 420 nm using a SmartSpec 3000 spectrophotometer (Bio-Rad). To ensure that no secondary mutations in vfr or algR were present, cultures were checked for the twitching motility phenotype using the subsurface agar method (Beatson et al., 2002b).


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Table 1. P. aeruginosa strains used in this study

 
Protein isolation.
Extracellular proteins were isolated by trichloroacetic acid (TCA)/methanol precipitation (Hirose et al., 2000). Briefly, whole cells were removed from the culture by centrifugation (6000 g, 10 min at 4 °C) using a Sorvall SS-34 rotor in a Sorvall RC-5B centrifuge. The supernatant was then filtered through a 0·2 µm low protein-binding Millex filter (Millipore). TCA (20 %, w/v) was added and the acidified mixture was left on ice for 30 min to precipitate the proteins. The mixture was centrifuged (20 000 g, 20 min at 4 °C) to isolate the proteins, and the pellet was washed three times with an equal volume of ice-cold methanol to remove residual TCA.

Two-dimensional gel electrophoresis (2-DE).
Proteins were solubilized in a 2-DE compatible buffer, consisting of 5 M urea, 2 M thiourea, 2 % (w/v) CHAPS, 2 % (w/v) sulfobetaine 3–10, 2 mM tributyl phosphine, 1 % (v/v) pH 3–10 carrier ampholytes (Bio-Rad), 40 mM Tris and a trace amount of bromophenol blue. The mixture was vortexed briefly, and centrifuged (20 000 g, 10 min at 20 °C) to remove any insoluble material prior to isoelectric focusing. Proteins were loaded by in-gel rehydration on 17 cm pH 4–7 immobilized pH gradient (IPG) strips (Bio-Rad) using a maximum volume of 500 µl (500 µg protein ml-1). pH 6–11 IPGs (Amersham Biosciences) were rehydrated in sample solution for a minimum of 6 h. An aliquot of the protein sample (100 µl; 1 mg ml-1) was cup-loaded at the acidic end of the IPG prior to focusing. Isoelectric focusing was conducted as described previously (Nouwens et al., 2000). For pH 4–7 gradients, proteins were focused for a total of 25 kV h. For pH 6–11 gradients, proteins were focused for a total of 60 kV h. Following isoelectric focusing, IPG strips were reduced, alkylated and detergent-exchanged, and second dimension SDS-PAGE was carried out as described previously (Nouwens et al., 2000). Gels were fixed in 40 % (v/v) methanol/10 % (v/v) acetic acid for 1 h, and then stained with Sypro Ruby (Molecular Probes) overnight. Gels were de-stained for 2 h in 10 % (v/v) methanol/7 % (v/v) acetic acid and imaged using a Bio-Rad Molecular Imager Fx. After scanning, gels were double-stained using Coomassie G-250 (Cordwell, 2002). For comparative analyses, statistical data were acquired using PD-QUEST Version 7.0 (Bio-Rad) and Z3 (Compugen) as described previously (Cordwell et al., 2002). Briefly, proteins were acquired from separate duplicate cultures and each gel was run in duplicate. Statistical analyses were performed on triplicate gels.

Peptide mass mapping.
Protein spots were excised and placed in a 96-well microtitre plate. Gel pieces were washed three times with 120 µl of 25 mM ammonium bicarbonate (pH 7·8)/50 % (v/v) acetonitrile and dried using a SpeedVac (Savant Instruments) centrifuge for 20 min. Gel pieces were rehydrated with 8 µl of porcine modified trypsin (Promega, 10 ng µl-1) in 25 mM ammonium bicarbonate (pH 7·8) and left for 10 min at room temperature. Once gel pieces were rehydrated, proteins were digested at 37 °C for 16 h. Peptides were extracted from the gel in 8 µl of 50 % acetonitrile/1 % trifluoroacetic acid (TFA) by sonication (10 min). A small aliquot (1·0 µl) of each extract was spotted onto a target plate, and 1 µl of {alpha}-cyano-4-hydroxycinnamic acid (Sigma, 8 mg ml-1 in 50 % acetonitrile/1 % TFA) was placed on top and allowed to dry. Trypsin, wash, extraction and matrix solutions were delivered to the microtitre and target plates using a Multiprobe 104 robotic system (Packard Instruments). Peptide mass maps of tryptic peptides were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using a Micromass TOF-Spec 2E. All spectra were obtained in reflectron mode using an accelerating voltage of 20 kV. Mass calibration was performed using [Glu1] Fibrinopeptide B, 1570·68 [M+H+], as an external standard.

Database searching and protein identification.
Data generated from peptide mass maps were compared to the complete translated ORFs for P. aeruginosa PAO1 (http://www.pseudomonas.com) using PROTEINLYNX software (Micromass). The parameters for a successful identification included a mass tolerance of 150 p.p.m. per peptide and a minimum of three matching peptides. Generally, a set of matching peptides leading to a total sequence coverage of greater than 30 % was required to provide further confidence in the obtained match, although low and high mass proteins, and those that are the result of protein fragmentation, may not always conform to this rule. For proteins generating poor spectra, a concentration and desalting step as described in Gobom et al. (1999) was used and the concentrated peptides were again characterized by MALDI-TOF MS. Sequences from identified proteins were examined for a predicted signal sequence using the program SIGNALP (http://www.cbs.dtu.dk/services/SignalP/).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Extracellular proteins
Wild-type and P. aeruginosa QS mutants were grown in LB broth to stationary phase. Proteins were collected by TCA/methanol precipitation and aliquots were subjected to quantification by acid hydrolysis amino acid analysis and Lowry protein assay (data not shown). These values were used to ensure that near-identical quantities of protein were loaded onto the 2-D gels for comparative purposes.

Comparative 2-DE of extracellular proteins
Extracellular proteins from all strains of P. aeruginosa were solubilized and separated using 2-DE across the pH ranges 4–7 and 6–11. This provided an effective separation width of 35 cm in the first dimension with a single pH unit overlap between pH 6 and 7 (Fig. 1). The major extracellular proteins of strain PAO1, based on summed spot intensities, were elastase (lasB), LasA protease (lasA) and an aminopeptidase (PA2939). LasB appeared to be extensively processed, with 11 spot ‘variants' visible on 2-D gels. These included a major region corresponding to the mature 33 kDa enzyme (spots 1.1–1.6 in Fig. 1). Proteins from stationary phase culture supernatants were used to perform comparative 2-DE. Image and statistical analyses, using the PD-QUEST and Z3 software packages, were performed to detect differences in protein expression between wild-type and mutant strains (Fig. 2). An increase or decrease in visible spot intensity of greater than twofold, averaged over three gels and normalized using the intensities of 10 spots from each pH range with no apparent change in abundance, defined the selection of differentially expressed proteins.



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Fig. 1. Extracellular proteins from P. aeruginosa PAO1. Proteins were separated by 2-DE using IPG ranges of pH 4–7 and 6–11. Proteins discussed in the text are highlighted and were identified by peptide mass mapping. Numbers represent the spot identifications listed in Table 2.

 


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Fig. 2. Statistical analysis of changes in protein abundance in the culture supernatants of P. aeruginosa PAO1 and {Delta}las/{Delta}rhl mutants. (a) LasB; (b) LasA; (c) aminopeptidase PA2939; (d) endoproteinase PrpL; (e) PA0572; (f) AprA; (g) LipA; (h) PA0041. The remaining proteins were either absent from culture supernatants derived from all mutants or present only in the mutants.

 
Loss of a functional vfr gene resulted in identical protein profiles to strains containing las mutations (data not shown). Thirty-eight protein spots, corresponding to 13 unique ORFs, were absent from some or all of the 2-D gels representing the extracellular proteins from nine las and/or rhl mutants (Table 2A). These spots were characterized by MALDI-TOF MS, following in-gel tryptic digest. Eleven spots representing elastase prepropeptide, mature protease and one apparently non-specific fragment were present at lower abundance or were absent in the mutant strains. Mature LasB was present on 2-D gels from all mutants, at significantly lower abundance, except those that combined deletions from both the las and rhl QS systems ({Delta}lasI{Delta}rhlI, {Delta}lasR{Delta}rhlR and {Delta}lasRI{Delta}rhlRI; Fig. 2a). Similar results were obtained for LasA protease (lasA), which was also absent only from the three combined {Delta}las/{Delta}rhl mutants (Fig. 2b). Four proteins, aminopeptidase (PA2939; Figs 2c, 3a), endoproteinase (prpL/PA4175; Figs 2d, 3b), a hypothetical protein (PA0572; Fig. 2e) and alkaline metalloproteinase (aprA; Fig. 2f), were significantly downregulated or absent from all 2-D gels derived from strains containing a mutation in either lasI or lasR. However, no effect on the expression of these proteins was observed in strains containing a mutation in the rhl system alone. PA0572 is a protein of approximately 100 kDa and has no close homologues in the public sequence databases. However, it does contain an Xcp-dependant signal sequence and a 10 residue motif (693 GESHELGHNL 702) commonly found in zinc metalloproteinases (data not shown). The remaining seven proteins were detected only in the culture supernatants derived from the PAO1 strain. These included three fragments of higher mass proteins (N-acetylmuramoyl-L-alanine amidase, PA0807; a two-partner secretion transporter, PA0040; and a probable multi-drug efflux transporter, PA4233) and a further four whole proteins (azurin; chitin-binding protein, CbpD; PA4944, a hypothetical protein with sequence similarity to host factor I, HF-I; and hypothetical protein PA3611).


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Table 2. Identification of proteins with altered expression levels in las and rhl QS mutants

(A) Proteins downregulated or absent in one or more QS mutants; (B) proteins upregulated or present in one or more QS mutants. Proteins were identified using MALDI-TOF MS peptide mass mapping. Spot no. refers to numbers shown in Fig. 1; numbers in parentheses indicate total number of pI variants of that spot.

 


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Fig. 3. Protein expression in QS mutants. (a) Aminopeptidase (PA2939) was detected in the wild-type and all {Delta}rhl mutants, but was absent from all {Delta}las mutants. FliC levels were unaffected. (b) Endoproteinase (PrpL) was detected in the wild-type and in {Delta}rhl mutants and was present in {Delta}lasR and {Delta}lasRI at significantly reduced levels. PrpL was not detected in any combined {Delta}las/{Delta}rhl mutant.

 
Eighteen protein spots, corresponding to 14 unique P. aeruginosa ORFs, were present at significantly greater abundance in the stationary phase culture supernatants of strains lacking QS genes (Table 2B). Several of these belong to the outer membrane or periplasm, or are part of outer membrane macromolecular structures (such as flagella). This may reflect an inhibition of protein secretion caused by the loss of functional QS systems and the concurrent stability of such proteins in the extracellular environment. Therefore, an increase in the visible spot ‘noise’ level, due to a reduction in secretion and following the release of surface-associated and cellular material via autolysis, could account for some of these differences. Nine of the 14 unique protein spots (the products of phoA, glpQ, aotJ, dsbA, PA0688, PA2635, PA4625, PA5153 and PA5369) were present at apparently increased abundance levels in all mutants and therefore may be a result of the discrepancy between the concentration of secreted proteins in QS mutants compared to strain PAO1.

Flagellar hook-associated protein (flgK) was absent from PAO1 culture supernatants but was present in all other strains. However, {Delta}rhlI, {Delta}rhlR and {Delta}rhlRI showed a different cluster of spot variants corresponding to FlgK than were detected in strains containing a non-functional las system (Fig. 4). Furthermore, the las deletion FlgK variant was also present in {Delta}las/{Delta}rhl combined mutants. A cluster of spots, identified as fragments of type B flagellin (fliC), was only present on 2-D gels from strains deficient in the las system. Lipase (lipA) was not present in wild-type or {Delta}rhl deletion strains; however, two major LipA variants were present in all strains that contained a las deletion, including those that also contained rhl deletions (Fig. 5). A 95 kDa fragment of PA0041 (two-partner secretion exoprotein) was only present in strains that did not contain a functional regulator of either QS system ({Delta}lasR, {Delta}lasRI, {Delta}rhlR, {Delta}rhlRI, {Delta}lasR{Delta}rhlR and {Delta}lasRI{Delta}rhlRI).



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Fig. 4. Expression of FlgK variants in QS mutants. FlgK was not observed in wild-type PAO1 and was present as two different spot clusters in {Delta}las mutants and {Delta}rhl mutants. Expression of FlgK reverted to the {Delta}las type when genes from both the las and rhl QS systems were absent. AprA (absent from all {Delta}las mutants), PA2939 (absent from all {Delta}las mutants) and mature FliC (landmark protein) are also shown.

 


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Fig. 5. Expression of lipase (LipA) in QS mutants. LipA was detected only in {Delta}las mutants. AotJ (present in all mutants) and LasB (absent only from combined {Delta}las/{Delta}rhl mutants) are also shown.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The influence of the las and rhl QS systems on the extracellular protein profile in P. aeruginosa was examined by 2-DE and mass spectrometry. All mutant strains showed a significant reduction in the total concentration of secreted protein, suggesting that the lack of any las or rhl gene severely disrupts protein secretion, and/or expression of previously abundant extracellular constituents. This is consistent with previous studies showing that both the las and rhl systems regulate Xcp-mediated type II secretion in P. aeruginosa (Chapon-Herve et al., 1997), and that loss of QS results in reduced expression of several secreted proteins, including elastase (lasB) (Passador et al., 1993; Pearson et al., 1997). Since LasB is the major constituent of PAO1 culture supernatants (accounting for approximately 60 % of the total visible protein abundance in this strain; Nouwens et al., 2002), a reduction in lasB expression combined with inhibited secretion, both caused by the loss of QS, may explain the dramatic reduction in extracellular protein concentration seen here.

The products of 13 genes were present at reduced extracellular abundance in our QS mutants, including known QS-regulated genes such as lasB, lasA (Toder et al., 1991) and aprA (Gambello et al., 1993), as well as genes not previously associated with QS (including PA2939 and prpL). Other well-characterized QS-regulated extracellular virulence determinants most probably require specific interactions with host factors for their expression and were not identified here. PA2939 encodes a recently characterized, secreted, zinc-dependent metalloprotease (Cahan et al., 2001). Culture supernatants derived from mutants of the las QS system ({Delta}lasR, {Delta}lasI, {Delta}lasRI) and {Delta}vfr did not contain any of the major isoforms of this protein. PA2939 contains a putative signal peptide indicating that it is secreted via the Xcp-mediated apparatus and {Delta}xcp mutants appear to lack PA2939 in their culture supernatants (Braun et al., 1998). Several xcp genes are regulated by QS (Chapon-Herve et al., 1997), suggesting the loss of PA2939 in {Delta}las mutants may be due to defective or inhibited secretion. However, PA2939 levels were unaffected in {Delta}rhl mutants, despite the role of the rhl QS system in regulating Xcp-mediated secretion. Furthermore, several other proteins secreted via the Xcp machinery could be detected in the culture supernatants from our {Delta}las mutants. We suggest, therefore, that a reduction in Xcp-mediated secretion is not solely responsible for the absence of PA2939 in {Delta}las mutants. Furthermore, preliminary analysis of membrane and cytosolic protein fractions from these mutants did not reveal an intracellular increase in PA2939 levels consistent with inhibited secretion (data not shown).

Aminopeptidases, such as PA2939, act by generating free amino acids from short peptides under nutrient limitation. Any linkage of this protein to QS-regulated virulence may be via the complementary role of endoproteinases, which cleave host-surface proteins into smaller peptides. PA4175 contains sequence similarity to bacterial endoproteinases and was recently renamed PrpL (PvdS-regulated endoprotease; Wilderman et al., 2001). PrpL is able to cleave lactoferrin, transferrin, elastin and casein, suggesting that it may have a role in virulence. prpL is under the transcriptional control of PvdS, an alternate sigma factor that regulates genes involved in the production of pyoverdine (Cunliffe et al., 1995; Leoni et al., 2000; Wilson et al., 2001). PrpL was present in reduced abundance or absent from the culture supernatants of strains containing a {Delta}las mutation, but was unaffected in {Delta}rhlI, {Delta}rhlR and {Delta}rhlRI. Several pyoverdine synthetases appear to be QS-regulated (Whiteley et al., 1999); however, it is unknown whether QS directly regulates the expression of these genes, or whether it does so via an intermediary factor. The reduction of PrpL levels seen here could reflect direct QS regulation of prpL or a secondary effect of QS on regulators, such as pvdS.

Two apparently QS-regulated proteins (PA0572 and PA3611), which were previously designated as hypothetical, were detected. PA0572 is absent from strains containing a {Delta}las mutation, yet is present in strains containing only a {Delta}rhl mutation, while PA3611 is only present in the wild-type. PA0572 contains some similarity to zinc metalloproteinases (S. A. Beatson, personal communication). Three other proteins (azurin, CbpD and PA4944) were detected only in wild-type supernatants. Chitin-binding protein (CbpD) has adhesin-like properties and is found primarily in clinical isolates of P. aeruginosa (Folders et al., 2000). The 43 kDa mature form of CbpD is protected from the proteolytic activity of elastase when bound to chitin. Elastase cleaves CbpD into smaller products (including 30 and 23 kDa forms) that are incapable of binding chitin (Folders et al., 2000). The 30 and 23 kDa products were both identified here, which is not surprising since no specific efforts to inhibit elastase activity were undertaken. Regulation of cbpD by QS has been characterized by Whiteley et al. (1999), who showed that the loss of either the las or rhl system reduced cbpD transcription to 15 % ({Delta}las) and 1 % ({Delta}rhl) of that measured in the wild-type. We were unable to observe CbpD in any mutants suggesting that (i) the reduction in cbpD transcription results in levels of CbpD that are too low to be detected by fluorescence staining of 2-DE separated proteins and/or (ii) the secretion system required to export CbpD also requires both QS systems. All mutants were deficient in the expression of PA4944, a protein with significant sequence similarity to host factor I (HF-I), an RNA-binding protein that regulates enterotoxin production in Yersinia enterocolitica (Nakao et al., 1995) and several virulence factors in Brucella abortus (Robertson & Loop, 1999). Previous reports have suggested that genes under the control of QS may also be regulated by other factors, including the alternative sigma factors RpoS (Van Delden & Iglewski, 1998) and PvdS (Ochsner et al., 1996). RpoS may regulate QS itself via repression of rhlI transcription post-stress (Whiteley et al., 2000). PA4944 may be another protein in this family of regulators.

PA0807 (N-acetylmuramoyl-L-alanine amidase), PA0040 and PA4233 (a hypothetical protein with similarity to multi-drug efflux transporters) low-mass fragments were present in culture supernatants derived from PAO1, but were not present in culture supernatants derived from the mutant strains. Since they are membrane-associated, rather than truly secreted proteins, and since we could not identify the whole product of any of these fragments, we cannot distinguish between the possibility that the genes encoding these products are truly regulated by QS, or that the proteins themselves are the substrates of QS-regulated extracellular proteases. PA0040 and PA0041 are linked together both genetically and functionally (Bordetella pertussis filamentous haemagglutinin-like two-partner secretion transporter and exoprotein, respectively; Croft et al., 2000; Jacob-Dubuisson et al., 2001), yet they display different regulation here. A possible hypothesis for these apparently conflicting data is that the transporter (PA0040) is enzymically processed in strain PAO1 (hence the presence of a PA0040 fragment) by QS-regulated proteases (e.g. LasB). In QS mutants, this processing might not occur, allowing the complete transporter to export substantially greater amounts of the exoprotein (PA0041). Increased levels of a second two-partner secretion exoprotein, PA4625, were also detected in several QS mutants.

QS has not previously been reported to negatively regulate gene expression in P. aeruginosa; however, such regulation has been shown in Yersinia paratuberculosis (Atkinson et al., 1999). In our study, several proteins appeared only in QS mutant strains. The majority of these proteins belonged to the Gram-negative outer membrane or periplasm (FlgK, FliC, PhoA, AotJ, DsbA and GlpQ). The possibility that some of these proteins are negatively regulated by QS must take into account the following arguments: (i) the reduction in abundance of truly secreted proteins (e.g. LasB) in QS mutants enhances their detection by 2-DE; (ii) that QS mutations lead to structural damage to the outer membrane causing hyper-release of these proteins; and (iii) that QS-regulated proteases are absent in QS mutants allowing these proteins a longer half-life in the culture supernatant following cell autolysis. However, QS-regulated genes that encode for products in the V. fischeri periplasm have been determined previously (Callahan & Dunlap, 2000).

The differences in electrophoretic mobility for FlgK and the absence of FliC fragments in the wild-type and {Delta}rhl mutants suggest an involvement of QS in P. aeruginosa swimming and/or swarming (Köhler et al., 2000). Both {Delta}las and {Delta}rhl mutants display defective swarming (Köhler et al., 2000) and this may partly be due to effects on flagella and pili; however, {Delta}rhl mutants remain capable of swimming and thus the negative swarming phenotype may be independent of effects on flagella. Furthermore, exogenous AHL destruction in the presence of P. aeruginosa does not affect flagella or twitching motility (Reimmann et al., 2002). Lipase (lipA) is regulated by the rhl QS system (Reimmann et al., 1997) and was not detected in wild-type extracellular fractions, or in those from any {Delta}rhl mutant, but was clearly identified as two major spots in supernatants derived from all {Delta}las mutants, including those that also contained a {Delta}rhl mutation. This result suggests that lipA is negatively regulated by the las system or that the las system represses a downstream inducer of lipA. The net effect of this lipA–las interaction is stronger than the positive control of the rhl system on lipA. Our results appear to conflict with those seen by Reimmann et al. (1997), who have shown that increased levels of RhlR and LasR result in enhanced levels of LipA; however, this may be a result of increased RhlR and independent of the presence of LasR.

This study has been the first to examine the role of QS in P. aeruginosa extracellular protein production using a proteomics approach. Similar studies conducted on whole cells of V. fischeri (Callahan & Dunlap, 2000) discovered five novel QS-regulated proteins. We examined only the extracellular fraction with the aim of discovering potential QS-regulated secreted virulence factors. The products of 13 genes, including seven not previously associated with QS, were identified. We conclude that the role of QS in P. aeruginosa gene expression is yet to be fully understood and that further global analyses, such as proteome analysis of intracellular and membrane-associated proteins, and transcriptomics will be needed to shed further light on this complex regulatory mechanism.


   ACKNOWLEDGEMENTS
 
This work has been facilitated by access to the Australian Proteome Analysis Facility, established under the Australian Government Major National Research Facility programme, and was supported in part by the National Health and Medical Research Council (C. B. W. and J. S. M.). H. P. S. was supported by Cystic Fibrosis Foundation grant SCHW99I0. A. S. N. is the recipient of an Australian Postgraduate Award and an Australian Proteome Industry Research and Development Award. This work has been funded in part by a Macquarie University Postgraduate Award (A. S. N.).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Albus, A. M., Pesci, E. C., Runyen-Janecky, L. J., West, S. E. H. & Iglewski, B. H. (1997). Vfr controls quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179, 3928–3935.[Abstract]

Atkinson, S., Throup, J. P., Stewart, G. S. & Williams, P. (1999). A hierarchical quorum-sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping. Mol Microbiol 33, 1267–1277.[CrossRef][Medline]

Beatson, S. A., Whitchurch, C. B., Sargent, J. L., Levesque, R. C. & Mattick, J. S. (2002a). Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 184, 3605–3613.[Abstract/Free Full Text]

Beatson, S. A., Whitchurch, C. B., Semmler, A. B. T., Young, M. D. & Mattick, J. S. (2002b). Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa. J Bacteriol 184, 3598–3604.[Abstract/Free Full Text]

Braun, P., de Groot, A., Bitter, W. & Tommassen, J. (1998). Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa. J Bacteriol 180, 3467–3469.[Abstract/Free Full Text]

Brint, J. & Ohman, D. (1995). Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR–RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR–LuxI family. J Bacteriol 177, 7155–7163.[Abstract]

Cahan, R., Axelrad, I., Safrin, M., Ohman, D. E. & Kessler, E. (2001). A secreted aminopeptidase of Pseudomonas aeruginosa – identification, primary structure and relationship to other aminopeptidases. J Biol Chem 276, 43645–43652.[Abstract/Free Full Text]

Calfee, M. W., Coleman, J. P. & Pesci, E. C. (2001). Interference with Pseudomonas quinolone signal synthesis inhibits virulence factor expression by Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 98, 11633–11637.[Abstract/Free Full Text]

Callahan, S. M. & Dunlap, P. V. (2000). LuxR- and acyl-homoserine-lactone-controlled non-lux genes define a quorum-sensing regulon in Vibrio fischeri. J Bacteriol 182, 2811–2822.[Abstract/Free Full Text]

Chapon-Herve, V., Akrim, M., Latifi, A., Williams, P., Lazdunski, A. & Bally, M. (1997). Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa. Mol Microbiol 24, 1169–1178.[Medline]

Cordwell, S. J. (2002). Acquisition and archiving of information for bacterial proteomics: from sample preparation to database. Methods Enzymol 358, 207–227.[Medline]

Cordwell, S. J., Nouwens, A. S. & Walsh, B. J. (2001). Comparative proteomics of bacterial pathogens. Proteomics 1, 461–472.[CrossRef][Medline]

Cordwell, S. J., Larsen, M. R., Cole, R. T. & Walsh, B. J. (2002). Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100. Microbiology 148, 2765–2781.[Abstract/Free Full Text]

Croft, L., Beatson, S. A., Whitchurch, C. B., Huang, B., Blakeley, R. L. & Mattick, J. S. (2000). An interactive web-based Pseudomonas aeruginosa genome database: discovery of new genes, pathways and structures. Microbiology 146, 2351–2364.[Abstract/Free Full Text]

Cunliffe, H. E., Merriman, T. R. & Lamont, I. L. (1995). Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor. J Bacteriol 177, 2744–2750.[Abstract]

Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295–298.[Abstract/Free Full Text]

Devine, J. H., Shadel, G. S. & Baldwin, T. O. (1989). Identification of the operator of the lux regulon from the Vibrio fischeri strain ATCC 7744. Proc Natl Acad Sci U S A 86, 5688–5692.[Abstract]

Diggle, S. P., Winzer, K., Lazdunski, A., Williams, P. & Camara, M. (2002). Advancing the quorum in Pseudomonas aeruginosa: MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression. J Bacteriol 184, 2576–2586.[Abstract/Free Full Text]

Dong, Y.-H., Wan, L.-H., Xu, J.-L., Zhang, H.-B., Zhang, X.-F. & Zhang, L.-H. (2001). Quenching quorum sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411, 813–817.[CrossRef][Medline]

Erickson, D. L., Endersby, R., Kirkham, A., Stuber, K., Vollman, D. D., Rabin, H. R., Mitchell, I. & Storey, D. G. (2002). Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis. Infect Immun 70, 1783–1790.[Abstract/Free Full Text]

Folders, J., Tommassen, J., Van Loon, L. C. & Bitter, W. (2000). Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa. J Bacteriol 182, 1257–1263.[Abstract/Free Full Text]

Fuqua, C. & Greenberg, E. P. (1998). Self perception in bacteria: quorum sensing with acylated homoserine lactones. Curr Opin Microbiol 1, 183–189.[CrossRef][Medline]

Fuqua, C., Parsek, M. R. & Greenberg, E. P. (2001). Regulation of gene expression by cell–cell communication: acyl-homoserine lactone quorum sensing. Annu Rev Genet 35, 439–468.[CrossRef][Medline]

Fuqua, W. C., Winans, S. C. & Greenberg, E. P. (1994). Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 176, 269–275.[Medline]

Gambello, M. J., Kaye, S. & Iglewski, B. H. (1993). LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. Infect Immun 61, 1180–1184.[Abstract]

Glessner, A., Smith, R. S., Iglewski, B. H. & Robinson, J. B. (1999). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility. J Bacteriol 181, 1623–1629.[Abstract/Free Full Text]

Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R. & Roepstorff, P. (1999). Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom 34, 105–116.[CrossRef][Medline]

Harrington, C. A., Rosenow, C. & Retief, J. (2000). Monitoring gene expression using DNA microarrays. Curr Opin Microbiol 3, 285–291.[CrossRef][Medline]

Hassett, D. J., Ma, J.-F., Elkins, J. G. & 10 other authors (1999). Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol Microbiol 34, 1082–1093.[CrossRef][Medline]

Hirose, I., Sano, K., Shioda, I., Kumano, M., Nakamura, K. & Yamane, K. (2000). Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional electrophoretic study. Microbiology 146, 65–75.[Abstract/Free Full Text]

Jacob-Dubuisson, F., Locht, C. & Antoine, R. (2001). Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins. Mol Microbiol 40, 306–313.[CrossRef][Medline]

Köhler, T., Curty, L. K., Barja, F., Van Delden, C. & Pechère, J.-C. (2000). Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signalling and requires flagella and pili. J Bacteriol 182, 5990–5996.[Abstract/Free Full Text]

Latifi, A., Winson, M. K., Foglino, M., Bycroft, B. W., Stewart, G. S., Lazdunski, A. & Williams, P. (1995). Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol Microbiol 17, 333–343.[Medline]

Latifi, A., Foglino, M., Tanaka, K., Williams, P. & Lazdunski, A. A. (1996). A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhlR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol 21, 1137–1146.[Medline]

Leoni, L., Orsi, N., de Lorenzo, V. & Visca, P. (2000). Functional analysis of PvdS, an iron starvation sigma factor of Pseudomonas aeruginosa. J Bacteriol 182, 1481–1491.[Abstract/Free Full Text]

Lyczak, J. B., Cannon, C. L. & Pier, G. B. (2000). Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2, 1051–1060.[CrossRef][Medline]

McKnight, S. L., Iglewski, B. H. & Pesci, E. C. (2000). The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 182, 2702–2708.[Abstract/Free Full Text]

Nakao, H., Watanabe, H., Nakayama, S. & Takeda, T. (1995). yst gene expression in Yersinia enterocolitica is positively regulated by a chromosomal region that is highly homologous to Escherichia coli host factor 1 gene (hfq). Mol Microbiol 18, 859–865.[Medline]

Nouwens, A. S., Cordwell, S. J., Larsen, M. R., Molloy, M. P., Gillings, M., Willcox, M. D. P. & Walsh, B. J. (2000). Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa. Electrophoresis 21, 3797–3809.[CrossRef][Medline]

Nouwens, A. S., Willcox, M. D. P., Walsh, B. J. & Cordwell, S. J. (2002). Proteomic comparison of membrane and extracellular proteins from invasive (PAO1) and cytotoxic (6206) strains of Pseudomonas aeruginosa. Proteomics 2, 1325–1346.[CrossRef][Medline]

Ochsner, U. A. & Reiser, J. (1995). Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 92, 6424–6428.[Abstract]

Ochsner, U. A., Koch, A. K., Fietcher, A. & Reiser, J. (1994). Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. J Bacteriol 176, 2044–2054.[Abstract]

Ochsner, U. A., Johnson, Z., Lamont, I. L., Cunliffe, H. E. & Vasil, M. L. (1996). Exotoxin A production in Pseudomonas aeruginosa requires the iron-regulated pvdS gene encoding an alternative sigma factor. Mol Microbiol 21, 1019–1028.[Medline]

Passador, L., Cook, J. M., Gambello, M. J., Rust, L. & Iglewski, B. H. (1993). Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260, 1127–1130.[Medline]

Pearson, J. P., Pesci, E. C. & Iglewski, B. H. (1997). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179, 5756–5767.[Abstract]

Pesci, E. C., Pearson, J. P., Seed, P. C. & Iglewski, B. H. (1997). Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179, 3127–3132.[Abstract]

Reimmann, C., Beyeler, M., Latifi, A., Winteler, H., Foglino, M., Lazdunski, A. & Haas, D. (1997). The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase. Mol Microbiol 24, 309–319.[CrossRef][Medline]

Reimmann, C., Ginet, N., Michel, L. & 9 other authors (2002). Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1. Microbiology 148, 923–932.[Abstract/Free Full Text]

Robertson, G. T. & Loop, R. M., Jr (1999). The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol Microbiol 34, 690–700.[CrossRef][Medline]

Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. & Davies, D. G. (2002). Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184, 1140–1154.[Abstract/Free Full Text]

Seed, P., Passador, L. & Iglewski, B. (1995). Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. J Bacteriol 177, 654–659.[Abstract]

Stover, C. K., Pham, X. Q., Erwin, A. L. & 28 other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964.[CrossRef][Medline]

Tang, H. B., DiMango, E., Bryan, R., Gambello, M., Iglewski, B. H., Goldberg, J. B. & Prince, A. (1996). Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection. Infect Immun 64, 37–43.[Abstract]

Toder, D. S., Gambello, M. J. & Iglewski, B. H. (1991). Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR. Mol Microbiol 5, 2003–2010.[Medline]

Van Delden, C. & Iglewski, B. H. (1998). Cell-to-cell signalling and Pseudomonas aeruginosa infections. Emerg Infect Dis 4, 551–560.[Medline]

Whitehead, N. A., Barnard, A. M. L., Slater, H., Simpson, N. J. L. & Salmond, G. P. C. (2001). Quorum-sensing in Gram-negative bacteria. FEMS Microbiol Rev 23, 365–404.[CrossRef]

Whiteley, M. & Greenberg, E. P. (2001). Promoter specificity elements in Pseudomonas aeruginosa quorum-sensing-controlled genes. J Bacteriol 183, 5529–5534.[Abstract/Free Full Text]

Whiteley, M., Lee, K. M. & Greenberg, E. P. (1999). Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 96, 13904–13909.[Abstract/Free Full Text]

Whiteley, M., Parsek, M. R. & Greenberg, E. P. (2000). Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa. J Bacteriol 182, 4356–4360.[Abstract/Free Full Text]

Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R., Teitzel, G. M., Lory, S. & Greenberg, E. P. (2001). Gene expression in Pseudomonas aeruginosa biofilms. Nature 413, 860–864.[CrossRef][Medline]

Wilderman, P. J., Vasil, A. I., Johnson, Z., Wilson, M. J., Cunliffe, H. E., Lamont, I. L. & Vasil, M. L. (2001). Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa. Infect Immun 69, 5385–5394.[Abstract/Free Full Text]

Wilson, M. J., McMorran, B. J. & Lamont, I. L. (2001). Analysis of promoters recognized by PvdS, an extracytoplasmic-function sigma factor protein from Pseudomonas aeruginosa. J Bacteriol 183, 2151–2155.[Abstract/Free Full Text]

Winzer, K., Falconer, C., Garber, N. C., Diggle, S. P., Camara, M. & Williams, P. (2000). The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are controlled by quorum sensing and by RpoS. J Bacteriol 182, 6401–6411.[Abstract/Free Full Text]

Wu, H., Song, Z., Givskov, M., Doring, G., Worlitzsch, D., Mathee, K., Rygaard, J. & Hoiby, N. (2001). Pseudomonas aeruginosa mutations in lasI and rhlI quorum sensing systems result in milder chronic lung infection. Microbiology 147, 1105–1113.[Abstract/Free Full Text]

Received 29 August 2002; revised 10 January 2003; accepted 14 January 2003.