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1 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA
2 Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
Correspondence
Susan E. H. West
wests{at}vetmed.wisc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The production of these factors is controlled by different regulatory proteins, among which is the global regulator Vfr (virulence factor regulator) (Lory et al., 2004; van Delden, 2004). Vfr belongs to the family of cAMP receptor proteins (CRPs), and has 90 % similarity to the Escherichia coli CRP (West et al., 1994b). Vfr was originally described as a P. aeruginosa factor that is required for the production of exotoxin A (ETA) and protease IV (West et al., 1994b). Further studies have demonstrated that Vfr activates the transcription of several other virulence genes, such as genes encoding different components of the type III secretion system; as well as the quorum sensing (QS) genes lasR and rhlR, and rpoS, which encodes the stationary-phase sigma factor RpoS (Albus et al., 1997; Beatson, 2002; Bertani et al., 2003; Wolfgang et al., 2003). In addition, Vfr negatively regulates the production of the P. aeruginosa QS molecule PQS (Pseudomonas quinolone signal), and it downregulates the production of the major flagellar regulator FleQ (Dasgupta et al., 2002). Kanack et al. (2006) recently showed that Vfr specifically binds to the upstream regions of its target genes.
ETA is an ADP-ribosylating extracellular protein that is produced by P. aeruginosa through a complicated process that involves several regulators (Hamood et al., 2004). One of these regulators is PtxR, which enhances the transcription of the ETA gene toxA by four- to fivefold (Hamood et al., 1996). The 35 kDa PtxR, encoded by ptxR, belongs to the LysR family of transcriptional activators (Hamood et al., 1996). Available evidence suggests that PtxR regulates toxA expression through regA (Hamood et al., 1996, 2004). In addition to regulating toxA, PtxR enhances the expression of the QS gene lasI and its target lasB; however, it reduces the expression of the QS gene rhlI and its targets rhlAB and the pqsA–E operon, which is involved in PQS production. It also reduces expression of the pyocyanin genes, which are the target of the pqsA–E operon (Carty et al., 2006). Colmer-Hamood et al. (2006) showed that the ptxR upstream region contains potential binding sites for several regulators, including Vfr and the iron-starvation sigma factor PvdS. In this study, we examine the relationship of Vfr to ptxR. We show that Vfr enhances ptxR expression by specifically binding to the ptxR upstream region near the ptxR P2 promoter, and that loss of Vfr impacts the ability of PtxR to enhance synthesis of ETA and LasB. These findings suggest that Vfr fine tunes or modulates ETA and LasB production through PtxR.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. For routine growth and for the LasB assay, strains were grown in Luria–Bertani (LB) broth (Miller, 1972). For the ETA assay, strains were grown in TSB-DC (trypticase soy broth dialysate with 1 % (v/v) glycerol and 0.5 M monosodium glutamate) (Ohman et al., 1980). TSB-DC, which contains 2 µg Fe3+ ml–1, is considered to be an iron-deficient medium; for iron-sufficient medium, FeCl3 was added to TSB-DC to yield 25 µg Fe3+ ml–1. If needed, antibiotics were added at the following concentrations: carbenicillin, 300 µg ml–1; kanamycin, 300 µg ml–1; streptomycin, 300 µg ml–1; gentamicin, 40 µg ml–1.
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). The 354, 134 and 49 bp fragments used as probes in the electrophoretic mobility shift assay (EMSA) were synthesized by PCR (primers are listed in Table 2), and end-labelled with [
-32P]ATP, using T4 polynucleotide kinase (Sambrook & Russell, 2001). EMSAs were performed as described by DeVault et al. (1991), with minor modifications. Briefly, each 20 µl reaction contained approximately 15 pM radiolabelled DNA fragment, standardized to approximately 1000 c.p.m., and 10 ng purified r-Vfr in EMSA binding buffer [10 mM Tris/HCl, pH 7.4, 1 mM EDTA, 10 mM KCl, 5 % (v/v) glycerol, 1 mM DTT and 20 µM cAMP, with 50 µg BSA ml–1 and 5 µg poly(dI-dC) ml–1]. Reactions were carried out for 20 min at room temperature, and the reactions were separated by 5 % PAGE in 0.5x Tris borate EDTA buffer for 16 h at 4 °C. To promote Vfr binding, 20 µM cAMP was added to the buffer in the upper chamber. Gels were dried, and exposed to film.
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), and Pfu polymerase (Stratagene). SalI and XhoI restriction enzyme sites were added to the 5' and 3' ends of the fragment, respectively, to facilitate labelling of one DNA strand. Digested fragments were 5'-end-labelled with [-32P]TTP [70 µCi (2.59 MBq) per 25 µl reaction] (PerkinElmer Life Science) using T7 Sequenase (GE Healthcare Bio-Sciences), according to the manufacturer's directions. Binding reactions were performed by incubating 2.9 µM r-Vfr with the radiolabelled DNA fragments in 25 µl EMSA binding buffer, without poly(dI-dC), for 20 min at room temperature. Reactions were incubated with 15 µg DNase I ml–1, as described by Ross et al. (1990), and separated on 10 % acrylamide sequencing gels. Maxam–Gilbert chemical sequencing reactions (Maxam & Gilbert, 1980) were also performed.
β-Galactosidase assays.
The level of β-galactosidase activity produced throughout the growth cycle of PAO1 and PAO9001 (isogenic vfr mutant) carrying different plasmids was determined as previously described (Colmer-Hamood et al., 2006). Briefly, 2 ml aliquots from overnight cultures were pelleted, washed, and resuspended in TSB-DC medium to an OD600 of 0.03–0.05. Cells were grown for 14 h at 32 °C, with shaking at 250 r.p.m., and 1 ml samples were obtained every 2 h beginning 4 or 6 h post-inoculation, depending on the fusion plasmid tested (pJAC24 and pJH2, respectively). Cells in the samples were lysed, and the level of β-galactosidase activity was determined (Miller, 1972; Stachel et al., 1985).
Assays for ETA and LasB.
For ETA analysis, overnight cultures of the P. aeruginosa strains were subcultured in TSB-DC, and grown for 14 h at 32 °C. Supernatant fractions were separated, and the level of ETA within each fraction was determined by sandwich ELISA, as previously described (Gaines et al., 2005). Values were standardized by dividing the amount of ETA (pg µl–1) in each supernatant by the OD600 of the culture from which the fraction was obtained.
To determine LasB activity, overnight cultures of the P. aeruginosa strains were subcultured into LB broth, and grown for 14 h at 37 °C. The supernatant fractions were separated, and the level of LasB activity in each fraction was determined by the elastin Congo red assay, as previously described (Schaber et al., 2004). Values were standardized by dividing the LasB activity obtained in each supernatant (A495) by the OD600 of the culture from which the fraction was obtained.
Analysis of the pvc-related product.
PtxR regulates the production of a pigment (a coumarin derivative termed pseudoverdine) produced by the pvc operon. The pigment production can be analysed by measuring the absorbance of the supernatant at 405 nm (Stintzi et al., 1996). Analysis of the pvc-related product (PVC) was accomplished by growing the P. aeruginosa strains for 14 h at 32 °C in iron-deficient and iron-sufficient TSB-DC. Supernatant fractions were separated, and the A405 of each fraction was determined. Values for PVC were standardized as described for measurement of LasB activity.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In addition, computer analysis of the ptxR-upstream region revealed the presence of a potential Vfr-binding sequence located at 186–210 bp 5' of the PtxR ATG codon, and 20–44 bp 5' of the ptxR T2 transcriptional start site (Fig. 1). The sequence contains most of the conserved nucleotides within the recently described Vfr consensus sequence (Kanack et al., 2006). To verify Vfr binding to the ptxR upstream region, we conducted EMSAs using purified r-Vfr and a 354 bp fragment that carries 340 bp 5' of ptxR plus the first coding sequence for the first four amino acids of PtxR. As shown in Fig. 2(a), r-Vfr produced a specific gel shift band with the 354 bp fragment. The intensity of the band was reduced in the presence of excess unlabelled probe (Fig. 2a). To further localize the binding, we divided the 354 bp fragment into a 49 bp fragment, which carries the potential Vfr-binding sequence, and a 134 bp fragment. r-Vfr specifically bound to the 49 bp fragment, but did not bind to the 134 bp fragment; this again confirmed the specificity of Vfr binding (Fig. 2b).
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). As shown in Fig. 3(a), in the presence of 2.9 µM r-Vfr, the site 5'-ACCGTCTGAAGCAGTTCTCATTTAT-3' on the ptxR coding strand was protected from DNase I cleavage. In addition, r-Vfr protected the corresponding sequence on the opposite strand of the DNA fragment (data not shown). Out of the 21 conserved nucleotides within the Vfr consensus sequence, 17 were matched within the protected sequence (Fig. 3b). In addition, the location of the protected sequence matched that of the potential Vfr-binding sequence that was identified by computer analysis (Fig. 1). Within this sequence, we located sites with increased hypersensitivity to DNase I treatment; the sites were located at positions 9, 10, 18 and 19 of the 25 nt protected sequence (Fig. 3a). These sites indicate a perturbance in the DNA helical structure that could include a kink or a bend (Kanack et al., 2006). Similar perturbances have been detected in the Vfr-binding analyses with the upstream regions of toxA, regA and lasR (Albus et al., 1997; Kanack et al., 2006). The DNase I footprinting experiments, together with the EMSA, indicate that Vfr binds at a specific site within the ptxR–ptxS intergenic region. The proximity of this sequence to the ptxR T2 transcription initiation site suggests that Vfr regulates ptxR expression rather than ptxS expression.
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; Kanack et al., 2006), the Vfr-BS within the ptxR upstream region is located 5' of the putative ptxR T2 initiation site (Colmer-Hamood et al., 2006; Vasil et al., 1998). However, the spacing between the Vfr-BS and the T2 initiation site appears to be different from the typical E. coli CRP-activated promoters. The Vfr-BS is centred at –32.5 bp upstream of the T2 initiation site. In E. coli promoters that are activated by CRP, the CRP-binding sites are usually centred at –42, –62 and –72 bp upstream of the transcription initiation sites (Kolb et al., 1993). The Vfr-BS within the lasR promoter is centred at –47.5 and –77.5 bp upstream of the T2 and T1 transcription initiation sites (Albus et al., 1997). However, Albus et al. (1997) have shown that increasing the spacing by 4 or 8 bp is important, but not critical, for lasR expression. The identified Vfr-BS within the ptxR upstream region overlaps with the putative binding site for the iron-regulated P. aeruginosa iron-starvation sigma factor PvdS (Colmer-Hamood et al., 2006; Gaines et al., 2007). PvdS, which is required for the expression of toxA and the pyoverdine genes under aerobic conditions, is synthesized in P. aeruginosa at a high level in iron-deficient medium (Ochsner et al., 1996). Our recent EMSA has demonstrated that the PvdS–RNA-polymerase complex specifically binds to the 354 bp sequence within the ptxR upstream region that contains the iron-starvation box (Fig. 1) (Colmer-Hamood et al., 2006; Gaines et al., 2007). Moreover, in an iron-deficient medium under aerobic conditions, ptxR expression from P2 is significantly lower in the pvdS deletion strain PAO
pvdS than in its parent strain (Gaines et al., 2007).
Based on the results presented in this study, the Vfr-BS within the ptxR upstream region may belong to the class II CRP-dependent promoters in which the site from which CRP activates transcription overlaps the DNA-binding site of the RNA polymerase (Busby and Ebright, 1999; Kanack et al., 2006). However, the putative Vfr-BS within the ptxR upstream region differs from the typical class II CRP-dependent promoters in its location with respect to the –35 region sequences. In class II CRP-dependent promoters, the CRP-binding site partially overlaps the –35 region sequence (Busby & Ebright, 1999). However, the Vfr-BS within the ptxR upstream region overlaps the entire –35 region sequence (Fig. 1). Whether this represents a possible variation in class II CRP-dependent promoters is not known. At this time, we consider the T1 and T2 transcriptional initiation sites within the ptxR upstream region to be putative. Additional experiments (including primer extension analysis) may refine the exact position of one or both sites. This refinement may also adjust the above-discussed distance between the Vfr-BS and the T2 initiation site.
Based on 10 Vfr-BSs, Kanack et al. (2006) have developed a Vfr consensus binding sequence (5'-ANWWTGNGAWNY:AGWTCACAT-3') that contains two, more conserved, half-sites: TGNGA (left halfsite) and TCACA (right half-site) (Fig. 3b). Experimental evidence has indicated that Vfr activates the expression from the lasR, regA and toxA promoters, but represses the expression from the fleQ promoter (Albus et al., 1997; Dasgupta et al., 2002; West et al., 1994b). Efficient regulation of lasR and fleQ by Vfr depends on specific nucleotides that exist in the conserved half-sites of the Vfr-BSs for each gene. While lasR contains all the conserved nucleotides in each half-site, fleQ contains only three of five conserved nucleotides in the left half-site (Albus et al., 1997; Dasgupta et al., 2002; Kanack et al., 2006). Albus et al. (1997) have shown that a single base pair mutation of the left half-site (TGNGA to TCNCA) and the right half-site (TCACA to TGAGA) (2 bp at a time) in the lasR Vfr-BS results in fivefold reduction in lasR expression, while changing the outermost nucleotides of each half-site has little effect. Similarly, Dasgupta et al. (2002) have shown that mutation of the right half-site from TCACA to TCCGC obviates Vfr binding, and that expression of fleQ from this mutated promoter in PAO1 is increased compared with the wild-type, and is not repressed by overexpression of vfr from a plasmid.
Varying numbers of nucleotides that match the ones within the conserved half-sites are found in the Vfr-BS of ptxR (4/5 in the left half-site, and 4/5 in the right half-site), regA (3/5 and 4/5), and toxA (4/5 and 4/5) (Fig. 3b) (Kanack et al., 2006). Despite the variations in the conserved nucleotides, each half-site for these genes contains one nucleotide conserved in all the Vfr-BSs; the right halves contain a C toward the 5' end (xCxxx), while the left halves contain A at the 3' end (xxxxA). These two conserved nucleotides may be critical for Vfr binding and/or regulation of these genes by Vfr. Future in vitro mutagenesis analysis will be necessary to determine if changing the conserved A or C, or both, within the Vfr-BS of ptxR, regA and toxA, interferes with Vfr binding to the upstream regions of these genes.
Vfr enhances ptxR expression in PAO1
To determine if Vfr regulates ptxR or ptxS expression, we compared the levels of expression of each gene in PAO1 and its vfr isogenic mutant PAO9001 using ptxR and ptxS translational fusion plasmids (pJAC24 and pBS8-4, respectively; Table 1). The level of β-galactosidase activity was determined throughout the growth cycle of PAO1 and PAO9001 carrying these plasmids, as previously described (Colmer & Hamood, 1998; Swanson et al., 1999). Throughout their growth cycles, PAO1/pBS8-4 and PAO9001/pBS8-4 produced comparable levels of β-galactosidase activity (data not shown), thus eliminating the possibility that Vfr regulates ptxS expression in PAO1. However, the level of ptxR expression was significantly (P<0.001) higher in PAO1/pJAC24 than in PAO9001/pJAC24 throughout the growth cycle (Fig. 4a), indicating that Vfr enhances ptxR expression in P. aeruginosa. Strains containing the cloning vector pSW205 produced no detectable levels of β-galactosidase activity (data not shown). In addition, there was no variation in the growth pattern of PAO1 and PAO9001 carrying the plasmids throughout the growth cycle (data not shown).
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). Similar to the expression from pJAC24, the level of ptxR expression from pJH2 was significantly (P<0.001) higher in PAO1 than in PAO9001 (Fig. 4b) throughout the growth cycle. We then examined ptxR expression from plasmid pJAC90, which carries only the ptxR P1 promoter (Colmer-Hamood et al., 2006), to eliminate the possibility that Vfr affects the expression from ptxR P1. We detected no significant difference in the level of β-galactosidase activity produced by PAO1/pJAC90 and PAO9001/pJAC90. The level of β-galactosidase activity produced by PAO1/pJAC90 was 7.6±0.37 units (mean±SEM), while that produced by PAO9001/pJAC90 was 6.3±0.92 units. Plasmid pJAC90 carries a ptxR P1 transcriptional fusion while pJAC24 carries a ptxR P1/P2 translational fusion (Table 1). Thus, to eliminate any possible post-translational influence on our results shown in Fig. 4, we utilized pJAC47, which carries a ptxR P1/P2 transcriptional fusion (Table 1). We obtained results similar to those shown in Fig. 4(a) (data not shown). These results confirm that Vfr influences ptxR expression through the ptxR P2 promoter.
PtxR and RegA regulate toxA expression, and their expression is positively regulated by Vfr (Fig. 4) (Hamood et al., 1996; Hindahl et al., 1987; Kanack et al., 2006). Similar to ptxR, regA is expressed from two separate promoters (P1 and P2) that are differentially regulated by iron (Frank et al., 1989; Storey et al., 1990). While iron does not affect the expression from the P1 promoter of either gene, it negatively regulates the expression from both P2 promoters (Frank et al., 1989; Storey et al., 1990; Vasil et al., 1998). However, the two genes differ in the location of the Vfr-BS with respect to the two promoters, and in the influence of Vfr on their P1 and P2 promoters. While the Vfr-BS is centred 32.5 bp 5' of the ptxR P2 promoter (Fig. 1), it is centred 63 bp 5' of the regA P1 promoter (Kanack et al., 2006). Our expression analysis suggests that Vfr is required for ptxR P2 expression throughout the growth cycle in iron-deficient medium (Fig. 4b). In contrast, Vfr regulates the expression of regA P1 rather than P2 (West et al., 1994a). The expression of regA P1 is iron insensitive, and occurs at early stages of growth, whereas that of regA P2 is iron repressible (detected in iron-deficient medium only) and occurs during later stages of growth (Frank et al., 1989). It is known that RegA regulates toxA expression in iron-deficient medium throughout the growth cycle (Frank et al., 1989; Storey et al., 1990). Vfr is required for efficient ETA production at late stages of growth, and in iron-deficient medium (Table 3) (West et al., 1994a). In addition, Vfr specifically binds to the toxA upstream region (Kanack et al., 2006). Whether Vfr regulates toxA expression directly through this binding at later stages of growth and in iron-deficient medium is yet to be determined.
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; West et al., 1994a). In comparison with its parent strain, the vfr mutant PAO9001 produced significantly lower levels of ETA and LasB (Table 3) (West et al., 1994a). PtxR also positively affects toxA and lasB expression and production (Table 3) (Carty et al., 2006; Hamood et al., 1996). Therefore, PtxR may be one of the factors through which Vfr regulates the expression of these genes. Thus, we explored the possibility that pJAC7-1, a high-copy ptxR plasmid, would partially or completely complement the defect of PAO9001 in ETA and LasB production. Plasmid pJAC7-1 increased ETA production in PAO1 by tenfold, but did not restore ETA production in PAO9001 (Table 3). Similarly, pJAC7-1 did not restore LasB activity in PAO9001 (Table 3).
Deletion of vfr may impact another of the functions of PtxR. One of the targets of PtxR is the pvc operon, whose product is not completely defined. In PAO1, pJAC7-1 enhanced the synthesis of PVC by about 2.5-fold, regardless of the level of iron in the growth medium (Table 3). The loss of Vfr led to enhancement in synthesis of PVC; however, unlike with ETA and LasB, pJAC7-1 enhanced the levels of PVC in PAO9001 in the presence or absence of iron (Table 3).
Results of this and previous studies suggest that Vfr enhances toxA expression either directly through Vfr binding to the toxA upstream region, or indirectly through Vfr binding to the upstream regions of regA and ptxR, and enhancing their expression (Figs 2, 4 and 5) (West et al., 1994a; Kanack et al., 2006). Therefore, instead of being the primary factor through which Vfr regulates toxA expression, Vfr may fine tune or modulate toxA expression through ptxR. This suggestion is based on the following observations. (1) Our results indicate that Vfr enhances the expression from the ptxR P2 promoter, but not the P1 promoter (Figs 1 and 4). Thus, an increase in the expression of the Vfr-independent ptxR P1 promoter (due to its presence on the multicopy plasmid pJAC7-1) should increase ETA production. However, as shown in Table 3, the level of ETA produced by PAO9001/pJAC7-1 was similar to that produced by PAO9001 carrying a vector control. (2) Recently, we have shown that ptxR expression from an exogenous lac promoter (plasmid pJAC5-1) does not bypass the defect of PAO9001 in ETA production (data not shown). (3) ETA production in PAOptxR, which carries an intact copy of vfr, is reduced but not eliminated (Hamood et al., 1996).
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) (Albus et al., 1997; Kanack et al., 2006). Yet, similar to toxA, Vfr may utilize PtxR to modulate lasB expression. Results of our recent analysis suggest that PtxR enhances the expression of lasI rather than lasR (Carty et al., 2006). LasI is involved in the synthesis of the homoserine lactone molecule 3OC12-HSL, which activates LasR (de Kievit & Iglewski, 2000) (Fig. 5). Thus, a modest increase in PtxR synthesis by Vfr would increase the amount of activated LasR, and enhance lasB expression in PAO1, but not PAO9001 or PAOlasR (Fig. 5, Table 3; data not shown).
With respect to PVC production, Vfr appears to have a unique effect. As shown in Table 3, vfr deletion enhanced PVC production by PAO1 in iron-deficient medium only (A405 0.08 for PAO1/18.230 versus A405 0.14 for PAO9001/p18.230). This effect is not related to PtxR, since pJAC7-1 enhanced PVC production in PAO1 and PAO9001 under both iron-deficient and iron-sufficient conditions (Table 3). Whether a possible relationship exists between Vfr, iron and PVC production is not known at this time. Several aspects of the pvc operon, including the functions of the pvc-encoded proteins and the mechanism of pvc regulation by PtxR, are yet to be determined.
In conclusion, our results show that Vfr specifically binds to the ptxR upstream region, and enhances the expression from the ptxR P2 promoter. In addition, our results suggest that Vfr modulates toxA and lasB expression in PAO1 through PtxR.
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ACKNOWLEDGEMENTS |
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Edited by: W. Bitter
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Beatson, S. A. (2002). Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 184, 3605–3613.
Bertani, I., Sevo, M., Kojic, M. & Venturi, V. (2003). Role of GacA, LasI, RhlI, Ppk, PsrA, Vfr and ClpXP in the regulation of the stationary-phase sigma factor rpoS/RpoS in Pseudomonas. Arch Microbiol 180, 264–271.[CrossRef][Medline]
Busby, S. & Ebright, R. H. (1999). Transcription activation by catabolite activator protein (CAP). J Mol Biol 293, 199–213.[CrossRef][Medline]
Carty, N. L., Layland, N., Colmer-Hamood, J. A., Calfee, M. W., Pesci, E. C. & Hamood, A. N. (2006). PtxR modulates the expression of QS-controlled virulence factors in the Pseudomonas aeruginosa strain PAO1. Mol Microbiol 61, 782–794.[CrossRef][Medline]
Colmer, J. A. & Hamood, A. N. (1998). Characterization of ptxS, a Pseudomonas aeruginosa gene which interferes with the effect of the exotoxin A positive regulatory gene, ptxR. Mol Gen Genet 258, 250–259.[CrossRef][Medline]
Colmer-Hamood, J. A., Aramaki, H., Gaines, J. M. & Hamood, A. N. (2006). Transcriptional analysis of the Pseudomonas aeruginosa toxA regulatory gene ptxR. Can J Microbiol 52, 343–356.[CrossRef][Medline]
Dasgupta, N., Ferrell, E. P., Kanack, K. J., West, S. E. H. & Ramphal, R. (2002). fleQ, the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma70 dependent and is downregulated by Vfr, a homolog of Escherichia coli cyclic AMP receptor protein. J Bacteriol 184, 5240–5250.
de Kievit, T. R. & Iglewski, B. H. (2000). Bacterial quorum sensing in pathogenic relationships. Infect Immun 68, 4839–4849.
DeVault, J. D., Hendrickson, W., Kato, J. & Chakrabarty, A. M. (1991). Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli. Mol Microbiol 5, 2503–2509.[CrossRef][Medline]
Frank, D. W., Storey, D. G., Hindahl, M. S. & Iglewski, B. H. (1989). Differential regulation by iron of regA and toxA transcript accumulation in Pseudomonas aeruginosa. J Bacteriol 171, 5304–5313.
Gaines, J. M., Carty, N. L., Colmer-Hamood, J. A. & Hamood, A. N. (2005). Effect of static growth and different levels of environmental oxygen on toxA and ptxR expression in the Pseudomonas aeruginosa strain PAO1. Microbiology 151, 2263–2275.
Gaines, J. M., Carty, N. L., Tiburzi, F., Davinic, M., Visca, P., Colmer-Hamood, J. A. & Hamood, A. N. (2007). Regulation of the Pseudomonas aeruginosa toxA, regA, and ptxR genes by the iron-starvation sigma factor PvdS under reduced levels of oxygen. Microbiology 153, 4219–4233.
Hamood, A. N., Colmer, J. A., Ochsner, U. A. & Vasil, M. L. (1996). Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production. Mol Microbiol 21, 97–110.[CrossRef][Medline]
Hamood, A. N., Colmer-Hamood, J. A. & Carty, N. L. (2004). Regulation of Pseudomonas aeruginosa exotoxin A synthesis. In Pseudomonas: Virulence and Gene Regulation, vol. 2, pp. 389–423. Edited by J.-L. Ramos. New York: Kluwer/Plenum.
Hindahl, M. S., Frank, D. W. & Iglewski, B. H. (1987). Molecular studies of a positive regulator of toxin A synthesis in Pseudomonas aeruginosa. Antibiot Chemother 39, 279–289.[Medline]
Holloway, B. W., Krishnapillai, V. & Morgan, A. F. (1979). Chromosomal genetics of Pseudomonas. Microbiol Rev 43, 73–102.
Kanack, K. J., Runyen-Janecky, L. J., Ferrell, E. P., Suh, S.-J. & West, S. E. H. (2006). Characterizaion of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein. Microbiology 152, 3485–3486.
Kolb, A., Busby, S., Buc, H., Garges, S. & Adhya, S. (1993). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62, 749–795.[CrossRef][Medline]
Liu, P. V. (1973). Exotoxin A of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A. J Infect Dis 128, 506–513.[Medline]
Lory, S., Wolfgang, M., Lee, V. & Smith, R. (2004). The multi-talented bacterial adenylate cyclases. Int J Med Microbiol 293, 479–482.[CrossRef][Medline]
Maxam, A. M. & Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65, 499–560.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
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.[CrossRef][Medline]
Ohman, D. E., Sadoff, J. C. & Iglewski, B. H. (1980). Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization. Infect Immun 28, 899–908.
Pollack, M. (2000). Pseudomonas aeruginosa. In Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases, pp. 2310–2327. Edited by G. L. Mandell, J. E. Bennett & R. Dolin. Philadelphia, PA: Churchill Livingstone.
Ross, W., Thompson, J. F., Newlands, J. T. & Gourse, R. L. (1990). E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J 9, 3733–3742.[Medline]
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning, a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schaber, J. A., Carty, N. L., McDonald, N. A., Graham, E. D., Cheluvappa, R., Griswold, J. A. & Hamood, A. N. (2004). Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa. J Med Microbiol 53, 841–853.
Spaink, H. P., Okker, R. H., Wijffelman, C. A., Pees, E. & Lugtenberg, B. J. (1987). Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1J1. Plant Mol Biol 9, 27–39.[Medline]
Stachel, S. E., An, G., Flores, C. & Nester, E. W. (1985). A Tn3 lacZ transposon for the random generation of β-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J 4, 891–898.[Medline]
Stintzi, A., Cornelis, P., Hohnadel, D., Meyer, J. M., Dean, C., Poole, K., Kourambas, S. & Krishnapillai, V. (1996). Novel pyoverdine biosynthesis gene(s) of Pseudomonas aeruginosa PAO. Microbiology 142, 1181–1190.
Storey, D. G., Frank, D. W., Farinha, M. A., Kropinski, A. M. & Iglewski, B. H. (1990). Multiple promoters control the regulation of the Pseudomonas aeruginosa regA gene. Mol Microbiol 4, 499–503.[CrossRef][Medline]
Swanson, B. L., Colmer, J. A. & Hamood, A. N. (1999). The Pseudomonas aeruginosa exotoxin A regulatory gene, ptxS: evidence for negative autoregulation. J Bacteriol 181, 4890–4895.
van Delden, C. (2004). Virulence factors in Pseudomonas aeruginosa. In Pseudomonas: Virulence and Gene Regulation, vol. 2, pp. 3–45. Edited by J.-L. Ramos. New York: Kluwer/Plenum.
Vasil, M. L., Ochsner, U. A., Johnson, Z., Colmer, J. A. & Hamood, A. N. (1998). The Fur-regulated gene encoding the alternative sigma factor PvdS is required for iron-dependent expression of the LysR-type regulator PtxR in Pseudomonas aeruginosa. J Bacteriol 180, 6784–6788.
West, S. E. H., Kaye, S. A., Hamood, A. N. & Iglewski, B. H. (1994a). Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A. Infect Immun 62, 897–903.
West, S. E. H., Sample, A. K. & Runyen-Janecky, L. J. (1994b). The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. J Bacteriol 176, 7532–7542.
Wolfgang, M. C., Lee, V. T., Gilmore, M. E. & Lory, S. (2003). Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. Dev Cell 4, 253–263.[CrossRef][Medline]
Received 12 July 2007;
revised 28 October 2007;
accepted 29 October 2007.
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1000 c.p.m.) plus 10 ng purified r-Vfr in EMSA binding buffer were incubated for 20 min at room temperature. Samples were separated on 5 % polyacrylamide gels, with 20 µM cAMP added to the electrophoresis buffer in the upper chamber to promote Vfr binding. (a) Autoradiogram of Vfr binding to the 354 bp intergenic probe. Lanes: 1, 354 bp probe alone; 2, probe plus 10 ng Vfr; 3, probe plus 10 ng Vfr plus fivefold (5x) unlabelled probe; 4, probe plus 10 ng Vfr plus tenfold (10x) unlabelled probe. (b) Autoradiogram of Vfr binding to smaller fragments of the intergenic region. Lanes: 1, 134 bp probe alone; 2, 134 bp probe plus 10 ng Vfr; 3, 49 bp probe alone; 4, 49 bp probe plus 10 ng Vfr. Free probe size is indicated to the left of the autoradiograms. Arrows indicate DNA–protein complexes.
) and PAO9001 (
) carrying pJAC24 (ptxR P1 and P2) (a) or pJH2 (ptxR P2 only) (b) were grown as described in Methods, and the level of β-galactosidase activity was determined. Values represent the mean of three independent experiments (±SEM); *P<0.001