Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA1
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858, USA2
Author for correspondence: Susan E. H. West. Tel: +1 608 263 7884. Fax: +1 608 263 7884. e-mail: wests{at}svm.vetmed.wisc.edu
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ABSTRACT |
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Keywords: Vfr, catabolite repression control, global regulator, cAMP binding, cAMP receptor protein
Abbreviations: CRP, cAMP receptor protein
a Present address: Department of Microbiology and Immunology, Medical College of Virginia campus of Virginia Commonwealth University, Richmond, VA 23298-0678, USA.
b Present address: Department of Cellular and Molecular Toxicology, Abbott Laboratories, Abbott Park, IL 60064-6104, USA.
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INTRODUCTION |
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A homologue of the Escherichia coli global transcriptional regulator CRP (cAMP receptor protein) was identified in Pseudomonas aeruginosa and it was designated Vfr for virulence factor regulator due to its effect on the production of several virulence factors (West et al., 1994 ). Regulation of virulence factor production by Vfr is partially due to regulation of the las quorum-sensing system by Vfr (Albus et al., 1997
). The las quorum-sensing system in turn induces the rhl quorum-sensing system, and together the two quorum-sensing systems control expression of several genes for virulence factor production in P. aeruginosa (Pesci et al., 1997
). However, Vfr also regulates expression of genes independent of the las and rhl quorum-sensing systems. Vfr binds to the promoter of toxA and regA to regulate the production of exotoxin A, thus demonstrating its direct role in the synthesis of this toxin (Runyen-Janecky et al., 1996
).
P. aeruginosa vfr encodes a 24225 Da polypeptide which is 67% identical and 91% similar to E. coli CRP (West et al., 1994 ). CRP regulates the expression of over 100 genes in E. coli, most notably the genes involved in carbon source utilization whose expression is subject to catabolite repression by glucose (Botsford & Harman, 1992
; Kolb et al., 1993
; Ullmann & Danchin, 1983
). CRP functions by binding cAMP to form an active CRPcAMP complex, which in turn binds to a specific sequence in the promoter of CRP-regulated genes.
Analysis of predicted amino acid sequences between the two proteins demonstrated that the majority of amino acid residues which constitute the structural features of CRP that are associated with cAMP binding, DNA binding and interaction with RNA polymerase are either identical or conserved in Vfr (West et al., 1994 ). The conservation of major functional domains between Vfr and CRP implies that these two proteins have similar functions in their respective organisms and may regulate similar pathways, including catabolite repression control. Previously, we found that the cloned P. aeruginosa vfr gene could partially restore ß-galactosidase activity in an E. coli crp mutant (West et al., 1994
). In an E. coli crp cya mutant, which does not produce cAMP, exogenous cAMP was required for Vfr to restore ß-galactosidase activity. Thus, these observations suggest that Vfr is mechanistically similar to CRP in regard to cAMP binding, recognition of CRP-binding sites and interaction with E. coli RNA polymerase.
In P. aeruginosa, the enzymes of several carbohydrate-transport systems and catabolic pathways are subject to strong repression when acetate or tricarboxylic acid cycle intermediates are present in the growth medium along with the carbohydrate (for a review, see Collier et al., 1996 ). The repressible enzymes include enzymes belonging to the hex regulon (the central pathway of catabolism), the glucose transporter regulon, the mannitol utilization regulon and the fructose regulon. Thus for P. aeruginosa, preferred carbon sources are the tricarboxylic acid cycle intermediates, and non-preferred substrates include glucose, gluconate, glycerate, glycerol, fructose and mannitol. Additionally, intracellular cAMP levels in P. aeruginosa do not fluctuate in response to growth phase or to various carbon sources, including glucose, succinate, acetamide, histidine, lactate, acetate, gluconate and glycerol (Phillips & Mulfinger, 1981
; Siegel et al., 1977
). This observation suggests that cAMP does not play a role in catabolite repression.
A global regulator of catabolite repression control (CRC) in P. aeruginosa has been cloned, designated as crc, and characterized (MacGregor et al., 1991 ). When Crc- mutants were grown in the presence of succinate, production of glucose-6-phosphate dehydrogenase, glucokinase, EntnerDoudoroff dehydratase, aldolase, amidase, and the enzymes involved in glucose and mannitol transport were not repressed (Wolff et al., 1991
). The deduced amino acid sequence of the Crc protein showed that it was similar to a family of DNA repair enzymes composed of apurinic/apyrimidinic endonucleases (MacGregor et al., 1996
). However, MacGregor et al. (1996)
were not able to demonstrate in vitro nuclease or DNA-binding activity with purified Crc protein. Thus, the mechanism by which Crc controls catabolite repression in P. aeruginosa is still unclear.
As a part of our continuing effort to characterize the role of Vfr in P. aeruginosa physiology, in this study we addressed whether Vfr is a global regulator of gene expression and whether it has a role in catabolite repression control in this bacterium. We demonstrate that although Vfr is mechanistically similar to CRP of E. coli, its role in P. aeruginosa physiology differs from the role of CRP in E. coli.
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METHODS |
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Two-dimensional electrophoretic analysis.
Cells were grown in TSBD for 10 h (2 h into stationary phase) at 37 °C with aeration, harvested, and resuspended in 10 mM HEPES, pH 7·2, 5 mM EDTA and 1 mM PMSF. Cells were again harvested by centrifugation (15000 g for 5 min), resuspended in SDS boiling buffer (5% SDS, 10%, v/v, glycerol and 60 mM Tris/HCl, pH 6·8), and boiled for 5 min. The insoluble particulate material was removed by centrifugation for 5 min at 15000 g. Protein concentration was determined via the BCA method (Smith et al., 1985 ), and ß-mercaptoethanol was added to the samples at a final concentration of 5%. Approx. 30 µg protein was used to analyse the protein profiles between pI 48·5 and molecular mass 14220 kDa via two-dimensional gel electrophoresis. The electrophoresis was performed by the Kendrick Laboratories, Inc. (Madison, WI), and the protein spots were visualized by silver staining.
Purification of CRP and Vfr.
CRP and Vfr were purified from crude cell lysates of E. coli RZ1330/pRZ1306 or P. aeruginosa PA103/pWNP28, respectively, by affinity chromatography on cAMP-agarose as described by Ghosaini et al. (1988) with the following modifications. To overexpress CRP or Vfr, IPTG (1 mM final concentration) was added to 5-litre mid-exponential-phase cultures of RZ1330/pRZ1306 or PA103/pWNP28 respectively, grown in LB. E. coli RZ1330/pRZ1306 was harvested 15 h after induction and P. aeruginosa PA103/pWNP28 was harvested 6 h after induction by centrifugation for 15 min at 5000 g. To prepare crude cell lysates, the cell pellets were resuspended in 100 mM Tris/HCl, pH 8·0, 50 mM KCl, 2 mM EDTA, 5 mM ß-mercaptoethanol, 1 mM sodium azide and 200 µg lysozyme ml-1 and incubated at 25 °C for 20 min. The solution was adjusted to 25 mM Tris/HCl, pH 8·0, 25 mM MgSO4 and 1% BRIJ 35, and the lysates were incubated at 4 °C for 10 min. To decrease the viscosity due to nucleic acids, the lysate was sonicated at approx. 100 W for three 10 s intervals on ice using a Vibra Cell sonicator (Sonics and Materials). The cell-free extract was obtained by centrifugation at 16000 g for 30 min at 4 °C and was applied to a 5 ml cAMP-agarose column (Sigma) as described by Ghosaini et al. (1988)
.
cAMP-binding assay.
This assay was performed by the ammonium sulfate precipitation method developed by Emmer et al. (1970) with slight modifications. Briefly, the assay was performed in 10 mM potasssium phosphate, pH 7·4, with the protein, [8-3H]adenosine 3',5'-cyclic phosphate (cAMP, 25 Ci mmol-1, 925 GBq mmol-1; Amersham), and a carrier protein, casein (200 µg), in a total volume of 100 µl. After a 5 min incubation at 01 °C, 400 µl ice-cold saturated (NH4)2SO4 was added and the proteincAMP complex was allowed to precipitate for 5 min on ice. The precipitate was collected by centrifugation in a microcentrifuge and the pellet was resuspended in 500 µl double-distilled H2O. The resuspended pellet was transferred to a scintillation vial containing 10 ml Opti-fluor (Packard) and the radioactivity was quantified in a Packard Tri-Carb 4530 scintillation counter. The control reaction contained all of the assay components except for the protein; therefore, the amount of [3H]cAMP bound specifically to the enzyme was determined by subtracting the level of nonspecific binding in the control reaction from the total level of radioactivity in the assay reaction. The saturation in cAMP binding was achieved by increasing the quantity of [3H]cAMP added to the assay. Approx. 0·3 µg CRP and 1·1 µg Vfr was used in the assays to determine the apparent dissociation constants for cAMP.
Gel retardation assays.
A DNA fragment containing the lacZ promoter was isolated from pUCP19 as a 231 bp PvuIIHindIII DNA fragment and was end-labelled with [-32P]dNTP using DNA polymerase Klenow fragment (United States Biochemical). The binding reactions, containing approximately 40 pM DNA and various concentrations of either purified Vfr or CRP, were carried out at room temperature for 20 min in 10 mM Tris/HCl pH 7·4, 100 mM KCl, 1 mM EDTA, 5% (v/v) glycerol, 1 mM DTT, 50 µg BSA ml-1 and 20 µM cAMP as described by DeVault et al. (1991)
, and were then electrophoresed on a 6% native polyacrylamide gel in 10 mM Tris/HCl pH 7·4, 1 mM EDTA and 20 µM cAMP for 1·5 h at 10 V cm-1. The gel was dried and exposed to Kodak X-OMAT AR5 film at -70 °C overnight with an intensifying screen.
Enzyme assays.
Cells were disrupted by resuspending frozen cell pellets (from 30 ml cultures) in 2·5 ml 50 mM Tris/HCl pH 8·0 and pressing through a French pressure cell at 16000 p.s.i. (110 MPa). S-180 extracts were obtained after centrifugation at 180000 g for 30 min. The following enzyme assays were done as previously described: glucose-6-phosphate dehydrogenase (Hylemon & Phibbs, 1972 ), mannitol dehydrogenase (Siegel et al., 1977
), urocanase (Lessie & Neidhardt, 1967
), except that urocanate was used at 50 µM, and amidase (Brammar & Clarke, 1964
). Glucose-6-phosphate dehydrogenase and mannitol dehydrogenase were assayed at 25 °C, while urocanase and amidase assays were at 37 °C. Amidase assays were done on cell suspensions; other enzyme assays were done on S-180 extracts. For the determination of specific activities, units refer to µmol product min-1. Protein concentrations were assayed relative to BSA standards by the Lowry method. For cell suspensions, total protein was first precipitated with trichloroacetic acid prior to the protein determination.
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RESULTS |
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Proteome analysis of a vfr101::aacC1 mutant
A proteome analysis was performed to assess whether Vfr is a global regulator of gene expression in P. aeruginosa. As shown in Fig. 1, comparison of the silver-stained two-dimensional polyacrylamide gel electrophoresis patterns between the wild-type strain (PAO1) and the isogenic vfr101::aacC1 null mutant (PAO9002) revealed that a null mutation in vfr resulted in altered production of numerous proteins. We found that at least 43 protein spots were either absent or significantly decreased in the proteome pattern of the vfr mutant when compared to that of the parent strain (Fig. 1
, upper panel). This confirmed our hypothesis that Vfr acts as a positive regulator of gene expression in P. aeruginosa and that Vfr induces the expression of many genes. In contrast, we also found that production of 17 protein spots appeared to be repressed by the presence of Vfr (Fig. 1
, lower panel). Thus, Vfr is a global regulator of gene expression in P. aeruginosa that has either a positive or negative effect on expression of at least 60 genes.
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Vfr is not required for catabolite repression control in P. aeruginosa
Since Vfr can function as a positive regulator of catabolite repression by substituting for the cAMP receptor in E. coli, Vfr may fulfil a similar role in catabolite repression control in P. aeruginosa. To examine this possibility, we determined whether catabolite repression control occurred in two vfr null mutants of PAO1. The parental strain PAO1 and the vfr null mutants, PAO9001 and PAO9002, were grown in basal salts medium that was supplemented with either succinate, a strong repressor of inducible catabolic operons, or lactate, a weak repressor of inducible catabolic operons, as sole carbon sources. Metabolic enzyme synthesis was induced by the addition of lactamide for amidase, mannitol for mannitol dehydrogenase and glucose-6-phosphate dehydrogenase, and histidine for urocanase. In both PAO1 and the vfr null mutants PAO9001 and PAO9002, the presence of succinate in the growth medium repressed the synthesis of amidase, mannitol dehydrogenase, glucose-6-phosphate dehydrogenase and urocanase (Table 2). Based on these results, Vfr does not appear to be required for catabolite repression control in P. aeruginosa.
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DISCUSSION |
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We are also interested in elucidating the mechanism of Vfr function in P. aeruginosa. The requirement of cAMP for Vfr binding to the E. coli lac promoter further enhances our hypothesis that Vfr uses a similar molecular mechanism as CRP of E. coli (i.e. formation of a complex with cAMP and binding to a specific CRP-binding sequence) to activate transcription in E. coli. We found that Vfr bound cAMP with an apparent dissociation constant (Kd) of 1·6 µM, which was similar to the apparent Kd of CRP. The similar binding constant implies that, like CRP, Vfr activity should be sensitive to fluctuations in the cAMP levels. We previously demonstrated that Vfr-dependent activation of ß-galactosidase production in E. coli could be repressed in the presence of glucose (West et al., 1994 ). Based on our data showing that Vfr binds cAMP with a similar dissociation constant as CRP, we propose that, as with CRP, glucose repression of Vfr-dependent ß-galactosidase production in E. coli is a direct result of decreased cAMP levels. Thus, our data suggest that in E. coli Vfr functions in a manner mechanistically similar to CRP. However, in P. aeruginosa, it is not clear that cAMP is the only ligand which binds to and activates Vfr. cAMP levels remain constant and do not change in response to various carbon sources (Phillips & Mulfinger, 1981
; Siegel et al., 1977
), suggesting that it may not function as a modulator of Vfr activity. A role for cAMP has not been established in P. aeruginosa. Additionally, even though there is a high degree of similarity between Vfr and CRP, the greatest variability between these two proteins occurs in the cAMP-binding domain (West et al., 1994
). Additional work is needed to determine if cAMP is the only ligand required for Vfr activity.
In contrast to the ability of Vfr to mediate catabolite repression control in E. coli, Vfr does not function in catabolite repression control in P. aeruginosa. Based on the differences in metabolic capabilities of P. aeruginosa and E. coli, it was not surprising that the P. aeruginosa catabolite repression control system did not require Vfr. First, the carbon source preference is very different between the two bacteria: while E. coli prefers a carbohydrate such as glucose P. aeruginosa prefers an organic acid such as succinate. Second, many of the genes that are catabolite repressed in P. aeruginosa, such as those encoding amidase and mannitol dehydrogenase, are not found in E. coli, and therefore it is reasonable to suppose that a catabolite repression system other than a CRPcAMP-like mechanism may control their expression. In addition, Vfr does not appear to regulate genes in P. aeruginosa that are regulated by CRP in enterobacteria. For example, CRP is required for the production of succinate dehydrogenase in E. coli (Wood et al., 1984 ) and urocanase in Klebsiella aerogenes (K. pneumoniae) (Nieuwkoop et al., 1984
). These enzymes permit growth of these bacteria on succinate and histidine, respectively, as sole carbon sources. However, the P. aeruginosa PAO1 vfr insertion mutant PAO9002 was able to grow with either succinate or histidine as a sole carbon source (L. J. Runyen-Janecky, unpublished results). Furthermore, the vfr null mutants PAO9001 and PAO9002 produced a similar amount of urocanase as the parent strain. Finally, a VfrcAMP complex would be a poor candidate for mediating catabolite repression in response to various carbon sources, since cAMP levels are constant in P. aeruginosa (Phillips & Mulfinger, 1981
; Siegel et al., 1977
).
In addition to P. aeruginosa Vfr, homologues of the E. coli CRP protein have been identified in numerous physiologically and ecologically distinct members of the gamma subdivision of the purple eubacteria including Salmonella enterica Serovar Typhimurium, Vibrio fischeri, V. harveyi and Shigella flexneri, members of the Enterobacteriaceae family; Haemophilus influenzae, a member of the Pasturellaceae family; and Xanthomonas campestris, a member of the Pseudomonadaceae family (Chandler, 1992 ; Cossart et al., 1986
; DeCrecy-Lagard et al., 1990
). Among these species, the members of the Enterobacteriaceae family (E. coli, Salmonella enterica and Shigella flexneri) are the most closely related, while X. campestris and P. aeruginosa are more distantly related to the Enterobacteriaceae family and also only distantly related to each other (Woese et al., 1985
). The presence of CRP in such a diverse group of bacteria suggests that CRP was present in the common ancestor of these species. Although the structure of a CRP homologue remained conserved as members of the gamma subdivision of the purple eubacteria evolved from this common ancestor, the cellular role of this protein did not. For instance, Vfr does not appear to play a role in catabolite repression in P. aeruginosa, and the genes identified thus far that are members of the Vfr regulon are secreted virulence determinants or regulators of secreted virulence determinants (Albus et al., 1997
; Maleniak et al., 1996
; West et al., 1994
). Likewise, the X. campestris CRP homologue, CLP, does not regulate metabolism but instead regulates phytopathogenicity (DeCrecy-Lagard et al., 1990
). In H. influenzae, CRP regulates development of competence for transformation (Chandler, 1992
). The evolution of diverse roles of the CRP homologues in various species may reflect the different physiological needs of each species.
In conclusion, this report demonstrates that although Vfr, the P. aeruginosa CRP homologue, can function as a component of catabolite repression control in E. coli, Vfr is not required for catabolite repression in P. aeruginosa. Although Vfr and CRP are both global transcriptional activators, their specific roles appear to have evolved to suit the particular needs of each species that are influenced by their natural niches and physiologies. Thus, it is not surprising that different sets of genes have evolved as members of the CRP/Vfr regulon in E. coli and P. aeruginosa. The corollary to this is that functions that are under the control of the CRP/Vfr regulon in one species may be part of a different regulon in another species, as seems to be the case for catabolite repression. Future study of identification of Vfr-regulated genes will help elucidate the function of this regulator on the physiology of P. aeruginosa.
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ACKNOWLEDGEMENTS |
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Received 2 November 2001;
revised 2 January 2002;
accepted 31 January 2002.