The Pseudomonas aeruginosa global regulator MvaT specifically binds to the ptxS upstream region and enhances ptxS expression

Landon W. Westfall1, A. Marie Luna1,2, Michael San Francisco2, Stephen P. Diggle3, Kathryn E. Worrall3, Paul Williams3, Miguel Cámara3 and Abdul N. Hamood1

1 Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
2 Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, USA
3 Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, UK

Correspondence
Abdul N. Hamood
abdul.hamood{at}ttuhsc.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Exotoxin A production in Pseudomonas aeruginosa is regulated positively or negatively by several genes. Two such regulatory genes, ptxR and ptxS, which are divergently transcribed from each other, have been described previously. While computer analysis suggested that the ptxR-ptxS intergenic region contains potential binding sites for several regulatory proteins, the mechanism that regulates the expression of either ptxR or ptxS in P. aeruginosa is not known. The presence of a P. aeruginosa protein complex that specifically binds to a segment within this region was determined. In this study the binding region was localized to a 150 bp fragment of the intergenic region and the proteins that constitute the binding complex were characterized as P. aeruginosa HU and MvaT. Recombinant MvaT was purified as a fusion protein (MAL-MvaT) and shown to specifically bind to the ptxR-ptxS intergenic region. A PAO1 isogenic mutant defective in mvaT, PAO{Delta}mvaT, was constructed and characterized. The lysate of PAO{Delta}mvaT failed to bind to the 150 bp probe. The effect of mvaT on ptxS and ptxR expression was examined using real-time PCR experiments. The expression of ptxS was lower in PAO{Delta}mvaT than in PAO1, but no difference was detected in ptxR expression. These results suggest that MvaT positively regulates ptxS expression by binding specifically to the ptxS upstream region.


Abbreviations: EMSA, electrophoretic mobility shift assay; QS, quorum sensing


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that survives under different environmental conditions. It is also an opportunistic pathogen that causes serious infections in immunocompromised hosts, including severely burned patients, cancer patients, HIV-infected patients and individuals with cystic fibrosis (Govan & Deretic, 1996; Lyczak et al., 2000; Pollack, 1995; Van Delden & Iglewski, 1998). The ability of P. aeruginosa to cause these infections is due to its capacity to produce several cell-associated and extracellular virulence factors. Cell-associated virulence factors include lipopolysaccharides, flagellum, pili and alginate (Bergan, 1981; Doring et al., 1987; Pollack, 1995). Extracellular virulence factors include exotoxin A, elastases, alkaline protease, exoenzyme S, pyocyanin and several other factors (Hirakata et al., 1995; Nicas & Iglewski, 1985; Pollack, 1995).

The production of different virulence factors by P. aeruginosa is controlled by the cell-to-cell communication system [the quorum sensing (QS) system] (Dekievit & Iglewski, 2000; Latifi et al., 1995; Rumbaugh et al., 2000; Winson et al., 1995). The QS system, which is cell-density-dependent, relies on small diffusible molecules (autoinducers) through which individual bacteria communicate with each other and coordinate their production of different virulence factors (Dekievit & Iglewski, 2000; Rumbaugh et al., 2000; Winson et al., 1995). P. aeruginosa contains two complete QS systems; las and rhl (Dekievit & Iglewski, 2000; Rumbaugh et al., 2000). The las system consists of LasR, a transcriptional activator and LasI, an autoinducer synthase that produces the homoserine lactone 3O-C12-HSL (Dekievit & Iglewski, 2000; Pearson et al., 1994; Rumbaugh et al., 2000). The rhl system consists of the transcriptional activator RhlR and the autoinducer synthase RhlI, which synthesizes the homoserine lactone C4-HSL (Dekievit & Iglewski, 2000; Rumbaugh et al., 2000; Winson et al., 1995). In addition to these two autoinducers, P. aeruginosa produces a third autoinducer, the Pseudomonas quinolone signal (PQS) (McKnight et al., 2000). The QS systems control the production of several P. aeruginosa virulence factors, including LasB, LasA, alkaline protease, pyocyanin, exotoxin A, cytotoxic lectin, catalase and superoxide dismutase (Dekievit & Iglewski, 2000; Rumbaugh et al., 2000). In addition, the QS systems influence the ability of P. aeruginosa to form biofilms (Davies et al., 1998). The expression of the QS systems is controlled by several transcriptional activators, including gacA, vfr, rpoS and rsmA (Albus et al., 1997; Latifi et al., 1996; Pessi et al., 2001; Reimmann et al., 1997; Wagner et al., 2003; Winzer et al., 2000).

Exotoxin A, which is considered the most virulent of the proteins secreted by P. aeruginosa, is an ADP-ribosyl transferase enzyme that catalyses the transfer of the ADP-ribosyl moiety of NAD+ onto elongation factor 2 in eukaryotic cells leading to a cessation of protein synthesis and eventual cell death (Iglewski & Kabat, 1975; Liu, 1966). The regulation of Exotoxin A production by P. aeruginosa is a complicated process that involves both positive and negative regulators, including the ptxR and ptxS genes. ptxR encodes a 34 kDa protein that belongs to the LysR family of transcriptional activators (Hamood et al., 1996). In the presence of a ptxR plasmid, toxA transcription in P. aeruginosa is enhanced four- to fivefold (Hamood et al., 1996). The exact mechanism of toxA regulation by ptxR is not known at this time. ptxS encodes a 38 kDa protein that belongs to the GalR family of transcriptional repressors (Colmer & Hamood, 1998). Available results suggest that ptxS interferes with the effect of ptxR on toxA transcription (Colmer & Hamood, 1998). ptxR and ptxS, which are divergently transcribed from each other, share an intergenic region of about 500 bp (Colmer & Hamood, 1998). Computer analysis, as well as experimental evidence, indicated the presence of binding sites for several potential ptxR and/or ptxS regulatory proteins within this shared region (Hamood & Colmer, 2000). The present study describes the isolation of one of these proteins, P. aeruginosa MvaT.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and media.
The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli and P. aeruginosa strains were grown in LB medium (Miller, 1972). Cultures were grown at 37 °C with vigorous aeration. To examine the level of ptxR expression, P. aeruginosa was grown at 32 °C in Chelex-treated trypticase soy broth to which 1 % glycerol (v/v) and 0·05 M monosodium glutamate were added (Ohman et al., 1980). Antibiotics (Sigma or Research Products International) were used at the following concentrations: for E. coli, 100 µg carbenicillin ml–1, 50 µg ampicillin ml–1 and 50 µg kanamycin ml–1; for P. aeruginosa, 300 µg carbenicillin ml–1 and 300 µg chloramphenicol ml–1.


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Table 1. Bacterial strains and plasmids used in this study

 
DNA manipulations.
Routine genetic manipulations were conducted following standard protocols as described by Sambrook & Russell (2001). Enzymes were purchased from Promega. Chromosomal DNA was extracted from P. aeruginosa as described by Goldberg & Ohman (1984). Plasmid DNA was introduced into PAO1 by electroporation (Smith & Iglewski, 1989). Gel purification of PCR products was done using the Qiaex II Gel Extraction Kit (Qiagen). DNA sequence analysis of the PCR products was conducted at the Biotechnology Core Facility at Texas Tech University.

Preparation of cell extracts.
Whole-cell extracts were prepared as follows. Cells were grown overnight in LB broth at 37 °C and a 1 ml aliquot of the culture was pelleted and then resuspended in 100 µl distilled water. The cells were disrupted by sonication (Virsonic), pelleted and the supernatant fraction (whole-cell lysate) was separated and utilized in electrophoretic mobility shift assays (EMSAs).

EMSAs.
The assays were done using one of the following protocols.

(1) DNA fragments containing different segments of the ptxR-ptxS intergenic region were obtained by either restriction digestion or PCR (White, 1993). The fragments were labelled with [{gamma}-32P]ATP (New England Nuclear) by the end-labelling technique using T4 polynucleotide kinase (Promega) (Ausubel et al., 1988). EMSAs were performed as follows: binding reactions were set up in DNA-binding buffer (10 mM Tris/HCl, pH 7·4/1 mM EDTA/10 mM KCl/0·1 mM DTT/5 % glycerol) plus 50 mg BSA and 1 mg poly(d[I-C]) (ml binding buffer)–1 (Sambrook & Russell, 2001). Each reaction contained 20–50 µg of PAO1 lysate proteins and 105–107 c.p.m. of radiolabelled probe. Reactions were incubated for 1 h at 32 °C and separated by 5 % SDS-PAGE. Gels were dried and exposed to X-ray film or developed by phosphorimaging (Typhoon 9410; Molecular Dynamics).

(2) The ptxR-ptxS intergenic region was amplified using DIG-labelled primers (MWG Biotech). The DIG-labelled DNA was incubated with the indicated amount of the purified MAL-MvaT fusion protein and DNA-binding buffer (100 mM HEPES, pH 7·6/50 mM (NH4)2SO4/5 mM DTT/15 %, v/v, Tween 20/150 mM NaCl) at room temperature for 10 min. The binding mixture was then electrophoresed on 5 % polyacrylamide gels and the separated DNA and DNA-bound proteins were transferred onto nylon membrane. The membrane was treated with blocking reagent and incubated with anti-DIG antibody. Prior to development, the blot was treated with CDP-Star Chemiluminescence. The blot was developed by exposure to X-ray film. The blocking reagent, anti-DIG antibody and CDP-Star were supplied by Roche Applied Science.

Enrichment of putative ptxR-ptxS intergenic region binding proteins.
P. aeruginosa strain PAO1 was grown in 1 litre LB medium at 37 °C for 16 h. Cells were pelleted and resuspended in 10 ml distilled water (10x concn). Cells were lysed by passing them twice through a French pressure cell at 1000 p.s.i. (American Instrument Company). Lysed cells were centrifuged at 100 000 g and the supernatant fraction (cell lysate) was isolated. Using this approach, the cell lysates from 10 separate 1 litre cultures of PAO1 were collected. The proteins within these individual whole-cell lysates were concentrated further by ammonium sulfate precipitation (55 %, w/v). Precipitated proteins were resuspended in 5 ml distilled water and dialysed exhaustively against several changes of DNA-binding buffer at 4 °C. The dialysed mixture was then loaded onto a heparin Sepharose column (Amersham Biosciences) with a 5 ml bed volume at a flow rate of 0·5 ml min–1. The column was extensively washed with DNA-binding buffer and proteins were eluted from the column using a potassium chloride step gradient of 0·2–2·0 M (Baynham et al., 1999). Eluted fractions were concentrated further using Microcon-100 spin columns (Amicon) and the amount of protein in each fraction was determined using the Lowry method.

Amino acid sequencing of MvaT.
Proteins within the 0·6 M KCl fraction were further concentrated using Centricon YM-100 spin columns (Amicon). About 40 µg protein was separated on denaturing 15 % polyacrylamide gels, transferred to PVDF membrane (Bio-Rad) and stained with Coomassie brilliant blue. Bands corresponding to each protein were excised from the membrane and submitted to the Biotechnology Core Facility (Texas Tech University) for amino terminus sequence analysis. The 15 aa sequence identified for each protein was utilized to search the P. aeruginosa genome (http://www.ncbi.nlm.nih.gov) for the ORF encoding each protein

Cloning and expression of mvaT in the T7 system.
For expression in the T7 system, mvaT was cloned from the chromosome of PAO1 by PCR using primers mvaTF (5'-AGAAACATCGCGAAAGGCCGCCATTCTA-3') and mvaTR (5'-TACGCCGCGGAACTGCGTCAACGCTATT-3'). The synthesized 750 bp product contains 250 bp upstream of the mvaT initiation codon, the intact mvaT ORF and 120 bp downstream of the translational stop codon. Nucleotide sequence analysis confirmed that the correct sequence was synthesized. The 750 bp fragment was then cloned into the T7-expression vector pT7-5 with the mvaT start codon in the correct orientation with respect to the T7 gene 10 promoter (Tabor & Richardson, 1985). Recombinant plasmid pLW1 was transformed into the E. coli expression strain K38(pGP1-2) and expression analysis was conducted as described by Tabor & Richardson (1985). Additional expression experiments were conducted omitting radiolabelling. The cell lysates from these experiments were utilized in EMSAs. For complementation analysis, recombinant plasmid pLW3 was constructed by cloning the 750 bp fragment that carries intact mvaT into the P. aeruginosa shuttle vector pUCP19 (Table 1).

Purification of MvaT as a MalE fusion protein.
A 400 bp fragment containing the entire mvaT gene was amplified by PCR using two mvaT-specific primers (5'-TACCTCAGCTGTCCCTGATCAACGAA-3' and 5'-TTCGTCGACCTGACTGGTTTAGCCGA-3'). The PCR product was cloned into the protein fusion vector pMAL-c2x (New England Biolabs), producing the recombinant plasmid pMAL : : mvaT. Expression experiments showed that upon the induction of E. coli DH5{alpha}(pMAL : : mvaT) cultures by the addition of IPTG, a 59 kDa fusion protein was detected. The MAL-MvaT fusion protein was purified according to the manufacturer's recommendations (New England Biolabs) with slight modifications. Briefly, an overnight culture of DH5{alpha}(pMAL : : mvaT) was inoculated (1 : 100) in 10 ml LB broth. The cells were grown for 2 h, induced by the addition of IPTG at 1 mM concentration and the growth continued for an additional 2 h. The cells were lysed by gentle sonication and the lysate fraction was passed through a column containing 10 ml amylose resin (New England Biolabs) equilibrated with PBS. MAL-MvaT was eluted with PBS containing 10 mM maltose (1 ml fractions). The amount of protein in each fraction was determined by measuring A280 (DU-70 Spectrophotometer; Beckman) and the purity of MAL-MvaT was examined by SDS-PAGE.

Construction of the mvaT isogenic mutant.
PAO{Delta}mvaT was constructed using the suicide vector pDM4, which carries the chloramphenicol resistance gene and the sacB gene that confers sucrose sensitivity, as described by Milton et al. (1996). A Gram-negative bacterium carrying the sacB gene is usually killed by the product of this gene when grown in sucrose medium. Plasmid pDM4{Delta}mvaT was conjugated from E. coli strain S17-1 into PAO1 (Diggle et al., 2002). To enrich for the mvaT isogenic mutants that arise through double crossover events, PAO1(pDM4{Delta}mvaT) was grown in nutrient broth containing 5 % (w/v) sucrose. Cells that grew in the 5 % sucrose broth were serially diluted and plated on nutrient agar containing 5 % sucrose. Individual colonies were then screened for chloramphenicol susceptibility, indicative of potential mvaT isogenic mutants. The construction of PAO{Delta}mvaT was confirmed by PCR analysis.

Real-time PCR analysis.
Cells were grown in LB broth and total RNA was extracted by the hot phenol method (Frank et al., 1989). Real-time PCR analysis was conducted as described by Carty et al. (2003). To compensate for differences in the number of cells harvested and the efficiency of extracting RNA, the measurement of the 16S rRNA was used as an internal standard. Results of the quantitative analysis of ptxS were expressed as numbers of copies per 1010 copies of 16S rRNA.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Localizing the binding activity within the PAO1 lysate to a 150 bp fragment within the ptxR-ptxS intergenic region
We previously identified within the lysate of the P. aeruginosa strain PAO1 a protein or a complex of proteins that specifically bound to the ptxR–ptxS intergenic region. The binding was initially localized to a 304 bp fragment and then to a 201 bp fragment within the ptxR-ptxS intergenic region (Fig. 1a) (Swanson et al., 1999). To analyse this protein complex and the region to which it binds, we tried first to localize the binding activity to a smaller DNA fragment using EMSAs (Sambrook & Russell, 2001; Swanson et al., 1999). The 201 bp fragment was divided into two fragments of 102 and 98 bp each, which were synthesized from the 201 bp fragment by PCR. Specific gel shift bands were detected with both fragments (Fig. 1a). The intensity of these bands was considerably less than that produced by the binding of the 201 bp fragment, suggesting that regions from each of the smaller fragments (Fig. 1a) are necessary for efficient binding of the protein complex. To examine such a possibility, a 150 bp fragment that overlaps the 102 and 98 bp fragments was synthesized and used as a probe. As shown in Figs 1(a) and (b), a strong gel shift band was detected with the 150 bp fragment. This band is as intense as the one that we usually detected with the 201 bp fragment (Fig. 1a), suggesting that the essential sequence for efficient binding of the protein complex is localized within 150 bp fragment of the ptxR-ptxS intergenic region. To localize the binding to a smaller region within the 150 bp fragment, two non-overlapping 72 and 71 bp fragments were synthesized. However, neither fragment produced a strong gel shift band (data not shown). Therefore, throughout the rest of the study, we utilized the 150 bp fragment in the EMSAs. The specificity of binding to the 150 bp fragment was confirmed by competition experiments; in the presence of excess unlabelled 150 bp fragment the gel shift band was eliminated (data not shown).



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Fig. 1. (a) Schematic diagram of the ptxR-ptxS intergenic region showing the different fragments used as probes in EMSAs to localize binding of the putative PAO1 regulatory proteins. Restriction sites: B, BamHI; D, DpnI; Hc, HincII; K, KpnI. + indicates the presence of a strong gel shift band; ± indicates a weak gel shift band. (b) EMSA showing binding of PAO1 proteins to the 150 bp fragment. The radiolabelled probe was incubated with 50 µg PAO1 lysate proteins. Lanes: 1, probe alone; 2, probe+PAO1 lysate. F, free probe; C, DNA–protein complex.

 
Enrichment and identification of the DNA-binding proteins
To identify the proteins within the binding complex, we tried to chemically enrich for DNA-binding proteins. Briefly, the pellets from 10 separate 1 litre cultures of PAO1 were collected and the cells were lysed using a French pressure cell. Proteins within the lysate fractions were concentrated as described in Methods. The concentrated proteins were applied to a heparin-Sepharose column and the proteins were eluted from the column using a 0·2–2·0 M KCl step gradient (Baynham et al., 1999). Proteins from the individually eluted fractions were concentrated and their binding to the 150 bp was examined using EMSAs. In addition, the proteins from each fraction were separated by SDS-PAGE and the gels were stained with silver stain (Bio-Rad). As shown in Fig. 2(a), a specific gel shift band was detected with proteins eluted with 0·6 M KCl. Migration of these bands paralleled that of the gel shift band that was initially detected when the 150 bp probe was incubated with PAO1 lysate (data not shown). Analysis of the protein profile of the 0·6 M KCl fraction revealed the presence of only two proteins of 9 and 16 kDa (Fig. 2a). A search of the P. aeruginosa genome with the amino acid sequences obtained from amino terminus sequencing showed that the 9 kDa protein is the P. aeruginosa HU protein, which is encoded by the hupB gene (Delic-Attree et al., 1995), while the 16 kDa protein is the P. aeruginosa transcriptional regulator MvaT (Diggle et al., 2002), a homologue of the MvaT beta subunit in Pseudomonas mevalonii (Rosenthal & Rodwell, 1998). Three other column fractions produced gel shift bands that varied in their migration distance when compared to the 0·6 M KCl fraction (data not shown). In addition, the protein profiles of these fractions revealed the presence of several proteins that varied in their molecular masses from HU and MvaT (data not shown). Further experiments will be conducted to identify the proteins within these fractions that bind to the 150 bp fragment.



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Fig. 2. Separation and identification of DNA-binding proteins within the PAO1 lysate by heparin Sepharose chromatography. Proteins were eluted from the column using a 0·2–2·0 M KCl step gradient. Each eluate was used in EMSA with the 150 bp probe to determine the presence of specific DNA-binding proteins. (a) SDS-PAGE analysis of the proteins within different eluate fractions. Proteins were separated by 15 % SDS-PAGE and stained with silver stain. Lanes: 1, molecular mass standards; 2, 0·5 M KCl fraction; 3, 0·3 M KCl fraction; 4, 0·6 M KCl fraction. (b) EMSA from the 0·6 M KCl fraction. Lanes: 1, probe alone; 2, probe+0·6 M KCl fraction. F, free probe; C, DNA–protein complex.

 
Using DNase I protection experiments, we tried to identify the specific nucleotide sequence within the 150 bp fragment to which MvaT binds. Initial analysis indicated a 25 bp protected sequence (5'-CGATTATTCGGTTTTCATGAACAAC-3') (data not shown). This 25 bp sequence does not carry a recognizable unique feature such as a direct repeat, indirect repeat or a palindromic sequence.

Expression of the P. aeruginosa MvaT homologue in E. coli
Based on the above results, the direct binding to the 150 bp probe may represent binding of the MvaT homologue, the HU protein or both. Our initial aim was to determine if P. aeruginosa MvaT alone specifically binds to the 150 bp probe. Using primers that correspond to sequences flanking the MvaT ORF, a 750 bp fragment that carried intact mvaT was synthesized from the PAO1 chromosome by PCR. Nucleotide sequence analysis confirmed that the isolated fragment carried mvaT. The 750 bp fragment was then cloned in the correct orientation in the E. coli expression plasmid pT7-5, producing the recombinant plasmid pLW1. Expression experiments were conducted as described in Methods. As shown in Fig. 3(a), a single 16 kDa protein was detected in the lysate from E. coli K38(pGP1-2)(pLW1). No product was detected in the K38(pGP1-2) strain carrying the pT7-5 expression vector (control) (Fig. 3a). We also examined whether the overexpressed MvaT in the lysate of K38(pGP1-2)(pLW1) binds to the 150 bp probe. As shown in Fig. 3(b), a gel shift band was detected when this lysate was incubated with the 150 bp probe. No gel shift band was detected when the control lysate of K38(pGP1-2)(pT7-5) was incubated with the 150 bp probe (Fig. 3b). Competition experiments using an excess of unlabelled 150 bp fragment confirmed the specificity of binding (data not shown). These results indicate that the P. aeruginosa MvaT homologue binds to a specific sequence within the ptxS-ptxR intergenic region.



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Fig. 3. (a) SDS-PAGE analysis of the translated product of mvaT using the E. coli T7 expression system. The mvaT ORF was expressed from the T7 gene 10 promoter in pLW1. Expression experiments were performed as described by Tabor & Richardson (1985). Lanes: 1, K38 (pGP1-2)(pT7-5) (negative control); 2, K38(pGP1-2)(pLW1). Molecular mass standards are shown to the left of the autoradiogram. (b) Binding of overexpressed P. aeruginosa MvaT to the 150 bp fragment. Approximately 20 µg of lysate proteins was mixed with labelled 150 bp probe for the EMSA. Lanes: 1, probe alone; 2, probe+lysate of K38(pGP1-2)(pT7-5); 3, probe+lysate of K38(pGP1-2)(pLW1). F, free probe; C, DNA–protein complex.

 
Purification and DNA binding of MvaT
Purified MvaT was required for additional EMSAs. To accomplish this, the mvaT ORF was fused in-frame with the malE gene in the expression vector pMAL-c2x as described in Methods. The fusion protein encoded by the recombinant plasmid contains the intact MvaT fused with the carboxy terminus arginine of MalE. When E. coli DH5{alpha} containing pMAL : : mvaT was grown under inducing conditions, a 59 kDa fusion protein was detected (data not shown) that corresponds closely to the predicted molecular mass of MAL-MvaT (43 plus 16 kDa). When MAL-MvaT was purified using amylose affinity chromatography, as described in Methods, pure fusion protein was detected (data not shown). MAL-MvaT contains a single cleavage site for the factor X protease that would allow for the separation of MvaT from MalE. However, despite several attempts, we were not able to obtain a sufficient amount of purified MvaT. It is possible that the treatments of the fusion protein with factor X protease degraded MvaT. A similar phenomenon was previously reported by Hovey & Frank (1995) during their attempts to purify the P. aeruginosa type III secretion system regulator ExsA from the ExsA-MalE fusion protein.

We then examined the binding of purified MAL-MvaT to the ptxR-ptxS intergenic region. A clear gel shift band was detected when the fragment was incubated with either 500 or 250 ng of the protein, although binding was reduced when the binding reaction contained only 125 or 62 ng of the fusion protein (data not shown). No binding was observed when MAL-MvaT was replaced by MalE alone (data not shown). The specificity of the binding band was again confirmed by competitive binding experiments using excess unlabelled intergenic region. The binding was eliminated upon the addition of excess unlabelled fragment (data not shown). In contrast, the binding band was not eliminated by the addition of excess unlabelled non-specific DNA ({lambda} DNA) (data not shown). These results confirm that MvAT specifically binds to the ptxR-ptxS intergenic region and that HU protein is not essential for this binding.

Characterization of the PAO{Delta}mvaT mutant
Diggle et al. (2002) identified the P. aeruginosa MvaT protein as a global regulator of virulence factors. An mvaT mutation in their PAO1 strain caused an enhancement of lecA expression and pyocyanin production (Diggle et al., 2002). In addition, the production of LasB, LasA and the autoinducers 3O-C12-HSL and C4-HSL was altered (Diggle et al., 2002). Therefore, we aimed to determine whether a mutation in mvaT also affects the expression of ptxR, ptxS or both. To prevent any intrinsic problems associated with the use of different P. aeruginosa PAO1 strains, we constructed our own mvaT mutant, using the allelic exchange plasmid pDM4{Delta}mvaT, as described by Diggle et al. (2002). Successful construction of PAO{Delta}mvaT was confirmed by PCR analysis using the same primers that we used to clone the mvaT ORF from the PAO1 chromosome. A 750 bp fragment that carries the intact mvaT ORF was isolated from the PAO1 chromosome (data not shown). However, only a 425 bp fragment was isolated from the chromosome of PAO{Delta}mvaT carrying the 327 bp internal deletion (data not shown). Deletion of the 327 bp fragment was confirmed by nucleotide sequence analysis of the PCR product (data not shown).

Diggle et al. (2002) demonstrated that the level of pyocyanin produced by their mvaT mutant, PAO-P10, is about ninefold higher than that produced by PAO1. Therefore, we tried to determine if a similar phenotype existed in our mutant. The level of pyocyanin produced by PAO{Delta}mvaT was compared to that produced by our PAO1 parent strain. Cells were grown in LB broth and the amount of the pyocyanin within the supernatant fraction was determined by the acid/chloroform extraction procedure (Essar et al., 1990). As expected, PAO{Delta}mvaT produced significantly higher levels of pyocyanin than its parent strain (data not shown). We then tried to determine if the mvaT mutation affected the formation of the binding complex with the 150 bp probe. The lysate of either PAO1 or PAO{Delta}mvaT was mixed with the 150 bp probe and the production of a specific gel shift band was determined by EMSA. As shown in Fig. 4, the binding complex was detected when the 150 bp probe was incubated with the lysate of PAO1, but not with lysate from PAO{Delta}mvaT, further confirming that the previously observed binding complex is produced by the MvaT protein binding directly to the 150 bp probe. The failure of the lysate from PAO{Delta}mvaT to produce any binding band with the 150 bp fragment indicates that HU protein does not bind to this fragment independent of MvaT. This suggests that the association of HU protein with MvaT is essential for HU binding to the 150 bp fragment.



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Fig. 4. Characterization of the PAO{Delta}mvaT mutant. The mutant, which was confirmed by PCR, carries a 327 bp internal deletion within mvaT. Binding of the PAO1 and PAO{Delta}mvaT lysates to the 150 bp fragment was examined by EMSA. Approximately 50 µg protein from each lysate was mixed with radiolabelled 150 bp probe. Lanes: 1, probe alone; 2, probe+PAO{Delta}mvaT lysate; 3 probe+PAO1 lysate. F, free probe; C, DNA–protein complex.

 
We also examined the effect of the mvaT mutation on the expression of ptxS and ptxR using real-time PCR analysis. We have previously utilized this method to examine toxA and ptxR transcription in P. aeruginosa (Carty et al., 2003). The amount of ptxS and ptxR transcripts in PAO1 were compared with those in PAO{Delta}mvaT. The number of copies of ptxS mRNA in PAO{Delta}mvaT was twofold lower than that in PAO1 (data not shown). No difference was detected between the two strains in the number of copies of ptxR mRNA (data not shown). Complementation experiments using plasmid pLW3, which carries intact mvaT in the P. aeruginosa shuttle vector pUCP19, were conducted to further confirm the effect of mvaT on ptxS expression. PAO1 carrying pLW3 produced a significantly lower level of pyocyanin than PAO1 carrying the cloning vector (data not shown). This indicates that pLW3 encodes a functional MvaT. The presence of pLW3 in PAO{Delta}mvaT increased the number of copies of ptxS mRNA to the level detected in the PAO1 parent strain (data not shown). In addition, pLW3 increased the number of copies of ptxS mRNA in the PAO1 parent strain (data not shown). Microarray analysis of PAO1 and PAO{Delta}mvaT recently confirmed the effect on the level of ptxS mRNA (data not shown). While no difference in the level of ptxR mRNA was detected, the level of ptxS mRNA was twofold lower in PAO{Delta}mvaT than in PAO1 (L. W. Westfall & A. N. Hamood, unpublished results). These microarray experiments were conducted on PAO1 and PAO{Delta}mvaT that were grown to late stationary phase (OD600=5·4). However, in a separate microarray analysis that was conducted on PAO1 and PAO{Delta}mvaT grown to an earlier stage of growth (OD600=3·0), no difference in the expression of either gene was detected (Vallet et al., 2004). This suggests that the effect of mvaT on ptxS expression is influenced by the stage of growth of PAO1. However, in both microarray experiments, most of the affected genes were either upregulated or downregulated by two- to fivefold (L. W. Westfall & A. N. Hamood, unpublished results; Vallet et al., 2004).

It is possible that MvaT binding to the ptxS upstream region is sufficient to enhance ptxS expression. Alternatively, MvaT may require other P. aeruginosa factors, including HU protein, to enhance ptxS expression. To examine these possibilities, we tried to determine the effect of MvaT on ptxS expression in E. coli. We introduced plasmid pJAC69, which carries a ptxS-lacZ transcriptional fusion in the lacZ fusion vector pMP190 (Colmer & Hamood, 2001), into E. coli strain MC4100. The detection of {beta}-galactosidase activity in MC4100(pJAC69) confirmed that ptxS is transcribed in E. coli (data not shown). We then introduced either pLW3 or the cloning vector pUCP19 into MC4100(pJAC69). There was no significant difference in the level of {beta}-galactosidase produced by either MC4100(pJAC69)(pUCP19) or MC4100(pJAC69)(pLW3) (data not shown). These results suggest that besides binding to the ptxS upstream region, MvaT requires additional P. aeruginosa factors to enhance ptxS expression.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The present study has shown that MvaT enhances the expression of ptxS. This enhancement may be accomplished directly by MvaT binding to the ptxR-ptxS intergenic region (Figs 1 & 3). This is the first gene shown to be directly regulated by MvaT and is also the first known regulatory gene shown to be regulated by MvaT. In the soil bacterium Pseudomonas mevalonii, the heteromeric transcriptional activator MvaT positively regulates the transcription of the mvaAB operon which is involved in the utilization of mevalonate (Rosenthal & Rodwell, 1998). In P. aeruginosa, which does not carry a complete mvaAB operon, MvaT appears to function as a positive regulator for ptxS expression and a negative regulator of the QS systems (data not shown) (Diggle et al., 2002). PAO1 mutants that carry either a transposon insertion or a specific deletion within the mvaT gene showed a pleiotropic defect in the production of several QS-regulated factors (Diggle et al., 2002). For example, in comparison with their parent strain, the mvaT mutants produced increased levels of pyocyanin, PA-IL lectin, C4-HSL and 3O-C12-HSL (Diggle et al., 2002). Based on these results, Diggle et al. (2002) suggested that in P. aeruginosa MvaT is an important component in the control of growth-phase-dependent gene regulation, which is required to prevent the early or premature expression of certain QS-controlled genes. The exact mechanism through which it regulates the QS-related genes is still unknown. However, due to its pleiotropic effect, MvaT is likely to regulate these genes indirectly through other regulators. MvaT may regulate the expression of lasI and rhlI, which encode the autoinducer synthases. Such regulation may vary according to the growth phase of P. aeruginosa. Our recent microarray analysis of PAO1 and PAO{Delta}mvaT supports this possibility. Results of this analysis suggest that at a late stationary phase of growth, and in comparison with PAO1, the level of rhlR expression in PAO{Delta}mvaT is decreased by three- to fourfold (L. W. Westfall & A. N. Hamood, unpublished results). Therefore, MvaT may enhance ptxS expression by binding directly to the ptxS upstream region. Alternatively, due to its pleotropic effect, MvaT may enhance ptxS expression indirectly. Final confirmation of direct regulation of ptxS by MvaT requires further studies, including ptxS in vitro transcriptional analysis in the presence or absence of MvaT.

The regulation of ptxS expression by MvaT suggests the presence of a possible relationship between ptxS and the P. aeruginosa QS system. ptxS is the first gene in the P. aeruginosa 2-ketogluconate utilization (kgu) operon (Swanson et al., 2000). This operon consists of the regulator ptxS and four structural genes, kguE, kguK, kguT and kguD, which encode an epimerase, kinase, permease and dehydrogenase, respectively (Swanson et al., 2000). Microarray analysis of the QS-regulated genes in P. aeruginosa suggested that the QS system represses the expression of ptxS and the other genes of the kgu operon at different levels (Wagner et al., 2003). Wagner et al. (2003) compared gene expression in the lasI/rhlI mutant PAO-JP2 grown in the presence of the two autoinducers (3O-C12-HSL and C4-HSL) with gene expression in the absence of the autoinducers. In response to the presence of exogenous autoinducers, ptxS expression was reduced 4-fold, while kguE expression was reduced 13-fold and kguK expression was reduced 18-fold (Wagner et al., 2003). While the specifics of ptxS interaction with the QS systems are not known, we have previously identified several aspects of ptxS and kgu regulation (Swanson et al., 2000). PtxS regulates the kgu operon by binding to a 14 bp palindromic sequence located within two regions of the operon; the ptxS upstream region and between ptxS and kguE (Swanson et al., 2000). We have also shown that this regulation occurs in response to the effector molecule, 2-ketogluconate (Swanson et al., 2000). Binding of PtxS to its target sequence is abolished when P. aeruginosa is grown in a medium containing 2-ketogluconate (Swanson et al., 2000). The degree of repression of kguE and kguK by the QS systems (13- and 18-fold, respectively) highlights the importance of the kgu operon in the function of the QS systems and further analysis of this phenomenon is warranted. For example, beside their direct effect on ptxS expression, 2-ketogluconate molecules may affect the QS systems by modifying the expression of the QS genes. Currently, we are examining the level of expression of rhlI, lasI, rhlR and lasR in PAO1 and its ptxS isogenic mutant.

While the QS systems negatively regulate ptxS expression, they themselves appear to be negatively regulated by MvaT (possibly through its effect on lasI and rhlI). Diggle et al. (2002) revealed that in PAO{Delta}mvaT the levels of 3O-C12-HSL and C4-HSL were about 30 % higher than those in PAO1. The relative increase in the level of the autoinducers is inverse but comparable to the relative decrease in ptxS expression in PAO{Delta}mvaT (data not shown). One possible scenario that would accommodate the results of these different studies may lie within the nature of MvaT as a growth-phase-dependent regulator. The reported repression of ptxS, kguE and kguK by the QS systems was observed in a PAO1 strain that was grown to mid-exponential phase (Wagner et al., 2003). Similarly, the observed effect of MvaT on the production of 3O-C12-HSL and C4-HSL was determined in PAO1 and PAO{Delta}mvaT grown to late exponential/early stationary phase (Diggle et al., 2002). However, in this study, the effect of MvaT on ptxS expression was examined in PAO1 and PAO{Delta}mvaT that were grown to a late stationary phase (data not shown). Therefore, at the early stages of growth, MvaT may affect ptxS expression indirectly through the QS systems; i.e. MvaT represses the QS system thereby alleviating the QS-induced repression of ptxS. At late stages of growth, however, MvaT may enhance ptxS expression directly by binding specifically to the ptxS upstream region (Fig. 3; data not shown). One important approach to examine the above possibilities is to determine the level of expression of ptxS, rhlI and lasI at different stages of growth of PAO1. It is also important to determine if the binding of MvaT to the ptxS upstream region varies according to the stage of growth of PAO1 (EMSAs using lysates of PAO1 grown to different stages of growth as the source of MvaT).

MvaT belongs to the MvaT family of proteins which exists only in Pseudomonas species (Tendeng et al., 2003) and includes MvaT of P. aeruginosa, and TurA of Pseudomonas putida (Diggle et al., 2002; Rescalli et al., 2004). Despite the lack of significant homology to other prokaryotic proteins, MvaT proteins have some structural and functional similarities to the H-NS protein of enterobacteria (Rescalli et al., 2004; Tendeng et al., 2003). The small chromatin-associated protein, H-NS, is considered a modulator that negatively or positively regulates the expression of different genes (Atlung & Ingmer, 1997). Most of these genes are regulated by environmental conditions, including temperature, osmolarity, anaerobiosis and growth phase (Atlung & Ingmer, 1997). H-NS proteins are DNA-binding proteins that bind either specifically or non-specifically to their target DNA (Atlung & Ingmer, 1997; Lucht et al., 1994). Specific binding targets usually involve stretches of AT-rich regions. Similar to H-NS, microarray analysis revealed that the MvaT protein of P. aeruginosa regulates (positively or negatively) the expression of at least 150 genes. Negatively regulated genes include fimU, mexE, algG, plcH, exsA, lecA, cupA1 and several QS system genes, while positively regulated genes include phnA, pqsH, pchF, hmgA, cyoC and ptxS (Vallet et al., 2004; L. W. Westfall & A. N. Hamood, unpublished results). In addition, MvaT specifically binds to the 150 bp fragment within the ptxS upstream region (Figs 1, 2 and 3). Initial DNase I protection analysis indicates that MvaT binds to an AT-rich 25 bp sequence within the 150 bp fragment (16 out of the 25 bp are either A or T) (data not shown). Therefore, as an H-NS homologue, MvaT may be a global modulator of different P. aeruginosa genes and not a specific QS regulator. Accordingly, and as an alternative scenario to explain our results, MvaT may regulate ptxS expression independently of the QS system; i.e. the regulation does not follow a hierarchical system. In support of this hypothesis, Diggle and co-workers have shown that besides the ptxS upstream region, MvaT specifically binds to the upstream region of the P. aeruginosa gene lecA (K. Worrall, personal communication).

The other protein that binds to the 150 bp fragment is the HU protein (Fig. 3). HU is a small basic histone-like protein that is conserved in different micro-organisms, including P. aeruginosa (Delic-Attree et al., 1995; Ferrell et al., 2001). In general, HU binding to DNA is characterized as less efficient and does not involve specific sequences (Drlica & Rouviere-Yaniv, 1987). However, the affinity of the binding is high for certain DNA structures such as DNA junctions, nicks and gaps (Castaing et al., 1995; Drlica & Rouviere-Yaniv, 1987; Pontiaggia et al., 1993). HU facilitates several cellular functions that include DNA binding, such as DNA replication, DNA recombination and transposition (Drlica & Rouviere-Yaniv, 1987). Recent analysis showed that HU functions as a cofactor that facilitates the binding of the E. coli gal repressor GalR to the bipartite gal operators OE and OI (Aki & Adhya, 1997; Aki et al., 1996). In this case, the binding is orientation-specific and depends on the binding of GalR to both OE and OI (Aki & Adhya, 1997). PtxS belongs to the GalR family of transcriptional repressors (Swanson et al., 1999). In addition, the 14 bp palindromic ptxS operator site contains most of the conserved nucleotide sequence within the GalR OE and OI sites (Swanson et al., 1999, 2000). Our analysis clearly showed that the HU protein is not essential for MvaT binding to the ptxS upstream region. Purified MvaT specifically bound to the ptxS upstream region (Fig. 3, data not shown). However, the mere binding of MvaT to the ptxS upstream region is not sufficient to enhance ptxS expression. As we have shown in this study, MvaT expressed in an E. coli background bound to the ptxS upstream region (Fig. 3), but failed to enhance ptxS expression in E. coli MC4100 (data not shown). Therefore, it is possible that P. aeruginosa HU protein or other factors may potentiate MvaT binding to the ptxS upstream region. Alternatively, HU or the other factors may modify the bound MvaT to enhance its function.


   ACKNOWLEDGEMENTS
 
The authors thank Dr Jane Colmer-Hamood for her critical review of the manuscript. This work was supported by a grant from the National Institutes of Health and Infectious Disease AI33386 to A. N. H. K. E. W. was funded by a Medical Research Council UK PhD studentship and S. P. D. was supported by grant QLK3-2000-01759 from the European Union (Vth Framework Programme).


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Received 22 April 2004; revised 22 July 2004; accepted 3 August 2004.



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