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J. Biol. Chem., Vol. 277, Issue 40, 37349-37358, October 4, 2002

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Urea-dependent Signal Transduction by the Virulence Regulator UreR^*

Inessa Gendlina, Delia M. Gutman, Venetta Thomas, and Carleen M. Collins^§

From the Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33101

Received for publication, April 10, 2002, and in revised form, June 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of the environmental triggers involved in the expression of virulence genes is a fundamental objective in studies of bacterial pathogens. For uropathogens, urea, found in the urinary tract at concentrations of up to 500 mM, functions as an environmental signal. Urea freely diffuses into the bacterium Providencia stuartii and activates UreR, a member of the AraC family of transcriptional activators. Active UreR promotes transcription of virulence-associated urease genes and alerts the organisms of its immediate milieu. Thus, the UreR·urea complex has a dual role, acting as both a transcriptional activator as well as an environmental sensor. Here, we describe the molecular events associated with activation of gene expression by urea-bound UreR. The K_d of the urea·UreR binding reaction was measured as 0.2 mM by fluorescence quenching assays, and the shape of the binding curve indicated a single specific urea-binding site on UreR. Histidine residues are critical for urea binding in urease, and therefore to identify the urea-binding site in UreR, five mutant UreR forms were generated with histidine to alanine substitutions. Two of the mutants (UreR^c) exhibited a constitutive phenotype by both activating transcription and binding to DNA with an increased affinity in the absence of urea. The UreR^c bound urea with an affinity similar to that of wild-type UreR. We concluded, therefore, that the mutations resulting in constitutive activity were not involved in the UreR·urea interaction. UreR was activated, then, either by binding urea or by histidine to alanine substitutions at one of two positions. Circular dichroism indicated little change in the structure of UreR when activated, and size-exclusion chromatography demonstrated that both rUreR and rUreR^c were dimers in both the presence and absence of urea. Thus, the structural changes associated with activation are subtle.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial pathogens transcribe genes necessary to establish an infection, proliferate, and overcome host defense mechanisms in response to environmental signals. The most well studied environmental sensing mechanisms involve signal transduction pathways that originate at the bacterial cell envelope and activate cytoplasmic response regulators (1). Environmental signals alerting the organisms of the appropriate host niche include temperature, osmolarity, pH, and metal ions (2). For the majority of virulence-associated genes, however, the sensing and signaling mechanisms and exact in vivo stimuli are not known.

Upon colonization of the urinary tract, uropathogenic bacteria express a specific armament of proteins. These proteins include attachment factors or pili, hemolysin, the iron scavenger aerobactin, and the enzyme urease (3-5). In the urinary tract urease is an important contributor to the virulence of the organism (6-8). Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, and the resulting ammonium elevates urine pH. This results in a more favorable environment for bacterial growth and leads to an increase in bacterial proliferation. In addition, the elevated urine pH facilitates formation of urinary stones, comprised of magnesium ammonium phosphate salts (struvite) (7, 8). The struvite precipitates can be found in the renal pelvis, the bladder, and encrusted on urinary catheters. Stone formation can cause urinary obstruction and interfere with voiding, thereby making it more difficult to clear the infecting organism from the urinary tract (9, 10). Stones can also harbor the infecting bacteria in a protected site (11). In addition to stone formation, the basic urine damages the uroepithelium, inhibits the action of complement, and reduces the efficiency of certain antibiotics.

Providencia stuartii is one of the most frequently isolated ureolytic uropathogens (12, 13). In P. stuartii, the urease genes are found on large plasmids ranging from 82 to 230 kb in size and are termed the plasmid-encoded urease gene cluster (14-16). Similar plasmids are found in ureolytic Escherichia coli and Salmonella species. Seven tandem genes in this plasmid-encoded urease cluster code for urease subunits (ureABC) and accessory polypeptides (ureDEFG), which are needed to form the nickel metallocenter found in the active enzyme (17, 18). An eighth gene, termed ureR, is found 414 bp upstream and divergently transcribed from the ureDABCEFG cluster (19). The ureR gene product, UreR (34.1 kDa), is a member of the AraC family of transcriptional activators and is required for transcription at ureDp, ureGp, and ureRp (19, 20).

In addition to UreR, transcription from each of these promoters is dependent upon the presence of urea. Previous studies indicate UreR binds upstream of ureRp and ureDp, and the affinity of UreR for the DNA-binding sites increases significantly in the presence of urea (21). It is evident that UreR can take on one of two conformations, binding DNA with either high or low affinity. However, the molecular mechanisms responsible for UreR activation have not been determined.

In the urinary tract, urea is found at concentrations of up to 500 mM, and this concentration is at least 50-fold higher than urea concentrations found at other sites in the body. Urea is able to freely diffuse into P. stuartii and other enteric bacteria. Thus, in this system, urea is acting not only as an effector molecule but also as the environmental signal, alerting P. stuartii that it has entered the appropriate site to initiate infection. In fact, the urea·UreR interaction is one of the first signals to the uropathogen that it has entered a urinary tract. Studies reported here are aimed to better understand this signaling mechanism by characterizing the transcriptionally active UreR.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Chemicals were obtained from Sigma-Aldrich and Invitrogen, unless otherwise specified. Restriction enzymes and enzyme buffers were obtained from New England BioLabs, and custom synthesized oligonucleotides were purchased from the Great American Gene Company and Ransom Hill Bioscience, Inc. RNA polymerase was obtained from Amersham Biosciences, and [³²P]dCTP (3000 mCi/mmol and 10 mCi/ml) was purchased from ICN Pharmaceuticals.

Bacterial Strains and Growth Conditions-- Bacterial strains used are listed in Table I. Bacteria were grown in Luria-Bertani (LB) broth with aeration at 37 °C or on LB broth solidified with 1.5% agar. Media were supplemented with carbenicillin at 75 mg/ml, ampicillin at 100 mg/ml, tetracycline at 20 mg/ml, or kanamycin at 25 mg/ml when indicated.

Generation of Point Mutations in ureR-- Mutant ureR were generated using overlap extension PCR with oligonucleotides containing the desired nucleotide substitutions (22). Specifically, two partial ureR products were amplified from the urease encoding pSEF70 (20) with 20-bp oligonucleotides containing the desired mutation. The amplified products were purified and used as template for a second PCR amplification with external flanking primers to yield a full-length, mutated ureR-encoding fragment. The complete double-stranded DNA sequence of each resultant UreR-encoding fragment was determined to ensure that only the single desired mutation was present.

Two plasmid constructs were generated for each ureR mutant. For in vivo studies, ureR and mutant ureR DNA fragments were amplified such that they contained terminal HindIII and BamHI endonuclease recognition sites. The native ureRp was not present on these fragments. The ureR-encoding fragments were inserted in the low copy number pRK415 downstream of the lactose operon promoter lacZp, which controlled transcription of ureR and mutants. For in vitro studies, the ureR-encoding fragments were generated to contain terminal NdeI and BamHI restriction endonuclease recognition sites, to allow insertion into, and expression from, pET15b (Novagen). The complete list of recombinant plasmids used is presented in Table I.

-Galactosidase Assay-- To determine the transcriptional activity of the mutant UreR, UreR-encoding pRK415 derivatives were transformed into Escherichia coli containing a single chromosomal copy of ureRp-lacZ (SF07-1) or containing a single chromosomal copy of ureDp-lacZ (SF15). Cultures were grown with aeration at 37 °C overnight in LB supplemented with tetracycline. Stationary phase cultures were diluted in 1× Christensen broth (0.1% peptone, 0.5% NaCl, 0.1% glucose, 0.2% KH₂PO₄, 10 mM MgCl₂, and 10 nM NiCl₂) supplemented with 1% urea when indicated. Cells were grown to mid-logarithmic phase and assayed for -galactosidase activity as previously described (23). Each sample was assayed in triplicate, a minimum of three independent times.

Western Blot Analysis-- Western blots were preformed by standard protocols. Cell cultures used for -galactosidase activity determination were centrifuged, and cell pellets were resuspended at equal cell densities in 20 µl of water. An equal volume of 2× denaturing protein buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, 10% 2-mercaptoethanol) was added, and samples were boiled for 5 min, loaded onto 12% SDS-PAGE, and resolved at 120 V overnight. Proteins were transferred onto a nitrocellulose membrane using Trans-Blot electrophoretic transfer cell (Bio-Rad). Western blotting was performed according to the ECL Western blotting analysis system protocol (Amersham Biosciences) using a 1:3000 dilution of polyclonal rabbit anti-UreR antibody and 1:5000 dilution of peroxidase-labeled anti-rabbit antibody.

Purification of rUreR-- The Novagen E. coli expression system was used to generate recombinant UreR (rUreR).¹ UreR-encoding sequences were inserted in-frame into pET15b, an expression plasmid that codes for an N-terminal leader peptide containing six histidine residues and a thrombin cleavage site. Expression from the plasmid and initial purification were as described by the manufacturer. E. coli BL21(DE3) containing wild-type and mutant ureR were grown to mid-exponential phase in LB broth supplemented with 1 mM isopropyl-1-thio--D-galactopyranoside. Cells were pelleted by centrifugation and lysed by passage through a French pressure cell. rUreR was separated from the cell lysate by affinity chromatography over the His-Bind affinity resin (Novagen). rUreR was eluted from the column with an imidazole gradient, and rUreR containing fractions were collected and dialyzed into a buffer of 50 mM Bis-Tris, pH 6.5, 100 mM EDTA, 300 mM NaCl, and 15% glycerol.

A MBP-UreR fusion proteins was also purified. Wild-type UreR-encoding sequence was inserted in-frame into pMAL-c2E (New England BioLabs) expression plasmid, thereby generating a translational MBP-UreR fusion. Protein expression and purification were performed according to the manufacturer's protocol. E. coli DH5 (pIG1.11) were grown in LB supplemented with glucose and 1 mM isopropyl-1-thio--D-galactopyranoside, and cells were pelleted and lysed as above. MBP-UreR was purified by affinity chromatography over Amylose resin (New England BioLabs) and eluted with buffer containing 10 mM maltose, 20 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA.

Fraction purity was assessed by SDS-PAGE and Coomassie Blue staining. Total protein concentration was determined using the BCA Protein Assay kit (Pierce) or Bradford protein assay. rUreR refers to the wild-type protein with an N-terminal histidine tag, and MBP-UreR refers to the N-terminal maltose binding protein fusion. Proteins were kept at 4 °C for immediate use and kept at 20 °C for long term storage.

Radiolabeling of DNA Fragments-- Plasmid DNA (30-50 µg) was linearized with either SpeI (pIG0.2, for ureDp fragment) or XhoI (pVJT9, containing a 147-bp intergenic region) and radiolabeled by incubating with the Klenow fragment of DNA polymerase I (1.5 µl of 5 units/ml)(New England BioLabs), 2 mM cold NTPs (G, A, T), and 5 µl of [³²P]dCTP (10 mCi/ml) in a final volume of 50 µl at 37 °C for 30 min. Enzymes were inactivated at 75-90 °C for 10 min. Fragments were released by digestion with either EcoRI (pIG0.1 and pIG0.2) or HindIII (pVJT9) and were separated on 4% native polyacrylamide gel (38:2 acrylamide:bisacrylamide) in TBE buffer (100 mM Tris, pH 8.0, 100 mM boric acid, 5 mM EDTA) for 3-4 h at 120 V. Labeled fragments were localized by brief exposure to radiographic film, excised from the gel, and eluted overnight at 37 °C in two volumes of elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, pH 8.0, 0.1% SDS). Eluted DNA was precipitated in two volumes of 95% ethanol and resuspended in 50 ml of 0.1× TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Typically, 5-10 ng of DNA was recovered, ranging from 1,000 to 50,000 cpm.

Electrophoretic Mobility Shift Assay-- Radiolabeled DNA (2000-5000 cpm or 0.5-1 ng) was incubated with varying concentration of rUreR in 25 µl of 10 mM Tris HCl (pH 8.0), 100 mM NaCl, 90 mM KCl, 0.125 mM dithiothreitol, 2 mM EDTA, 12.5 mg/ml bovine serum albumin, 12.5% glycerol, and 150-200 ng of poly(dI-dC) (Fluka BioChemika) for 30 min at room temperature. When indicated, 50 mM urea was present. Reactions were then loaded onto 5% native polyacrylamide gel and resolved in Tris-glycine buffer (50 mM Tris, 100 mM glycine, and 2 mM EDTA) for 3 h at 150 V. Gel was vacuum-dried and exposed to radiographic film overnight at 70 °C.

Dissociation Rate Constant Determination-- rUreR (28 nM final concentration) was incubated with radiolabeled 88-bp ureDp fragment (1.6 nM, from pIG0.2) in the presence or absence of 50 mM urea for 30 min at room temperature, using the conditions described for EMSA. Total reaction volume was adjusted to accommodate the desired number of time points. All samples were loaded onto 5% native acrylamide gel under current to minimize rUreR·DNA dissociation in the well. Sample (25 µl) was loaded at the zero time point. Then 100-fold molar excess of plasmid containing both ureRp and ureDp (pVJT9, 220 mM) was added to the reaction mixture, and samples were loaded onto the gel at 15-, 30-, and 60-s intervals. For calculations, 90 s were added to each time point to correct for the time required for the sample to migrate into the gel (24). Gel was analyzed as described for EMSA. Percentage of label remaining in the complex was determined by densitometry. The fraction of the DNA remaining in the complex was termed F. In a plot, where natural log of F was plotted as a function of time, the slope is equal to k₁, where k₁ is the dissociation rate constant (25).

Fluorescence Quenching Measurements-- The intrinsic fluorescence of rUreR, rUreR^c, and MBP-UreR was measured using a PerkinElmer Life Sciences LS 50 luminescence spectrophotometer. Protein (5-20 mM) was excited at 280 nm, and emission from 310 to 400 nm was recorded. Each sample was scanned 8 to 10 times, and the emission signal was summed and averaged.

Circular Dichroism Measurements-- Near and far CD spectra of UreR and mutants were measured with a JASCO J-710/720 spectropolarimeter. Each sample was scanned 40 times at a speed of 50 nm/min, and the signals were summed and averaged. Near UV spectra (240-320 nm) were determined using a cell path length of 1.0 cm, and far UV spectra (200-320 nm) were determined using a path length of 0.1 cm. Protein samples used were 0.5 mg/ml for near UV and 0.25 mg/ml for far UV spectra.

Size Exclusion Chromatography-- The relative molecular weights of rUreR and rUreR^c were predicted by size-exclusion chromatography using a Sephadex G-75 column with buffers either with or without 50 mM urea. Size standards were purchased from Sigma Chemical Co. The presence of protein was determined by reading absorbance of the fractions at 280 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

UreR Contains a Specific Urea-binding Site-- In the presence of urea, UreR obtains a transcriptionally active conformation with a high DNA binding affinity, whereas in the absence of urea, it is not an efficient activator of transcription and has a lower DNA binding affinity (21). Because the urea·UreR interaction is critical for urease gene activation, we asked whether there is a specific urea-binding site on UreR necessary for a low to high DNA affinity conformational change. This binding site would differ from the relatively nonspecific binding responsible for urea denaturation of proteins.

The UreR·urea interaction was assessed by measuring alterations in the intrinsic fluorescence of the protein as the consequence of ligand (urea) binding. UreR contains a number of fluorescent aromatic amino acids, including a single tryptophan residue at position 185 (Trp-185). Recombinant UreR (rUreR, UreR with an N-terminal 6-histidine tag attached, see "Experimental Procedures") was excited at 280 nm, a wavelength that excites tryptophans, and to a lesser extent tyrosines, and the emission spectrum from 310 to 400 nm was recorded (Fig. 1A). This spectrum peaked at 338.5 nm. In the presence of urea, the relative fluorescence of the sample decreased as a function of the urea concentration, while the emission peak remained at 338.5 nm. The fractional change of the decrease in fluorescence as a function of urea concentration is shown in Fig. 1B. The shape of the plot indicates that the observed quenching was due to urea binding UreR at a single site. Total fluorescence for each peak was summed, and the change in the mean of this total fluorescence was analyzed using a curve-fitting algorithm (SigmaPlot). From this algorithm, a dissociation constant (K_d) of the interaction was determined to be 0.23 mM (Table II).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A, relative fluorescence of rUreR with increasing concentrations of urea. Protein (20 µM) was excited at 280 nm, and emission from 310 to 400 nm recorded. Urea concentrations are as indicated in the inset. B, fractional change in the decrease of relative fluorescence as a function of urea concentration.

In an initial attempt to map the urea-binding site on UreR, an N-terminal 22-kDa truncate of the proteins was generated. Similar to other members of the AraC family, UreR contains two C-terminal helix-turn-helix (HTH) motifs that presumably mediate binding to DNA. rUreR22.0 is the N-terminal 22.0 kDa of the protein that lacks the second half of the predicted N-terminal HTH, the second C-terminal HTH, and the remainder of the protein. rUreR22.0 contains Trp-185, the chromophore likely responsible for the fluorescence change observed. The affinity of this truncate for urea was essentially identical to that of wild-type full-length rUreR (Table II). Therefore, the N-terminal 22 kDa of UreR contains a functional urea-binding site.

Both rUreR and rUreR22.0 contain N-terminal histidine tag sequences. Because we postulate that histidines might be involved in urea binding to UreR (see below), it was necessary to determine that the protein, and not the histidine tag, was responsible for the observed high affinity urea binding. To do this a maltose binding protein UreR fusion protein was generated. A decrease in fluorescence was observed when urea was added to the MBP-UreR fusion, in a similar fashion as observed with rUreR (not shown). An approximate 0.5 mM K_d value was predicted, indicating that the urea binding observed is not due to the recombinant N-terminal histidine tag sequence.

Conformation of rUreR as Predicted by Circular Dichroism-- To determine the conformational changes associated with urea binding and subsequent high DNA binding affinity, circular dichroism spectra of rUreR in the presence and absence of urea were obtained. Both far- and near-ultraviolet spectra for wild-type rUreR demonstrated little change when urea was added (Fig. 2). The far-ultraviolet spectra predicted the secondary structure of the protein as 56% -helix, 10%  sheet, and 33% random coil (Table III). There was no significant difference in these percentages when the spectra were obtained in the presence of urea. Thus, as determined by CD, there was not a detectable conformational change occurring upon binding urea. This suggests that the structural differences between activated and urea-free rUreR are subtle.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Circular dichroism spectra of wild-type rUreR in the presence and absence of urea. Each spectrum is the average of 40 scans. A, far-ultraviolet spectra; B, near-ultraviolet spectra. Dashed or dotted lines are for no urea samples, and solid lines are for plus urea samples.

View this table: [in this window] [in a new window]   Table III Estimates of percent -helix in UreR and UreRc Estimates were determined from CD spectra as described.

Generation of Mutant UreR-- Mutant UreR forms were generated to identify amino acids comprising the urea-binding site. The urea-binding site on the enzyme urease is composed of four histidines, one arginine, and one lysine residue. To test the possibility that UreR contains a similar urea-binding site, UreR with alanine residues substituted at positions that normally contain histidine residues were produced.

UreR has seven histidine residues found at positions 73, 102, 107, 175, 186, 205, and 240 (Fig. 3). These residues are conserved in the primary amino acid sequence of P. mirabilis UreR (27), which is functionally interchangeable with the P. stuartii UreR (20). By site-directed mutagenesis, the ureR codons for His-73, His-102, His-107, His-175, and His-240 were changed to code for alanines. His-186 and His-205 are found in the predicted N-terminal helix-turn-helix (HTH) motif and thus were not mutated. Two constructs of each mutant ureR were generated. To determine the transcriptional activity of each mutant form, the altered ureR were inserted downstream of the lactose operon promoter, lacZp, on the low copy number plasmid pRK415. For in vitro studies, each mutated gene was inserted into the pET15b expression plasmid, and the mutated rUreR were expressed and purified as described.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Amino acid sequence of UreR. The asterisks highlight positions of histidine residues. Underlined amino acids are in the predicted helix-turn-helix motifs.

There are four possible phenotypes for the mutant proteins: 1) mutant UreR could appear wild-type and dependent on urea for activity, 2) mutant protein might have little or no activity, due to loss of a critical urea binding residue, 3) the protein could be inactive due to loss of a DNA binding residue or a residue integral to overall stability and tertiary structure, or 4) the mutant UreR could be active independent of urea.

In Vivo Activity of Mutant UreR-- The mutated UreR were tested for their ability to activate transcription at the ureD and ureR promoters (ureDp and ureRp). Mutant constructs in pRK415 were placed in trans to a single copy chromosomal ureDp-lacZ and ureRp-lacZ transcriptional fusions (Fig. 4) (19), and the activity of each mutant was determined by assaying for -galactosidase (Fig. 5). UreR-His73Ala and UreR-His107Ala exhibited similar activity to that of wild-type UreR. There was no -galactosidase detected in either the presence or absence of urea with UreR-His240Ala. UreR-His102Ala and UreR-His175Ala exhibited activity similar to that of wild-type UreR in the presence of urea, but were almost as active in the absence of urea. UreR-His102Ala and UreR-His175Ala were considered to have a constitutive phenotype (UreR^c). Therefore, a single substitution at one of two positions can result in mutant UreR with active urea-independent conformations. In vivo activity of recombinant UreR was also measured. This protein has somewhat elevated activity in the absence of urea but is as active in the presence of urea, when compared with wild-type UreR. We address this elevated activity under "Discussion" below.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   The plasmid-encoded urease genes. A, open boxes represent the urease genes. The horizontal arrows show the direction of transcription. ureRp, ureDp, and ureGp are UreR- and urea-dependent promoters. B, the sequence of the 147-bp fragment containing ureRp and ureDp. The black line above the sequence indicates the 79-bp ureRp fragment. The black line below the sequence indicates the 88-bp ureDp sequence. Highlighted sequences are the base pairs protected by rUreR in DNase I protection assays.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Transcriptional activity of wild-type and mutant UreR. Plasmids expressing the indicated UreR were placed in trans to single-copy chromosomal fusions of either (A) ureDp-lacZ or (B) ureRp-lacZ. All constructs were under control of lacZp in pRK415, with the exception of ureRp-UreR, which is ureR under control of the ureRp in pRK415. Vector Control is the low copy plasmid pRK415 with no insert.

The relative amounts of mutant UreR produced from the pRK415 constructs were determined by Western analysis. The cultures used above for the -galactosidase assays were lysed, and the proteins were separated by SDS-PAGE and immunoblotted with rabbit antibody recognizing rUreR (Fig. 6). Western analysis indicated that approximately equal amounts of all the mutant proteins were produced, with the exception of UreR-His240Ala. There was no immunoreactive material seen in the pRK415-UreR-His240Ala lysates, suggesting UreR-His240Ala was not stable. We attributed the lack of transcriptional activity found in these lysates to the absence of this protein.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Expression levels of mutant UreR as determined by Western analysis. Plasmids are as described in the Fig. 5 legend.

rUreR^c Have Increased Affinity for DNA in the Absence of Urea-- Wild-type rUreR has increased affinity for the DNA-binding site when in the presence of urea. To determine if the rUreR^c exhibited increased DNA affinity in the absence of urea, electrophoretic mobility shift assays (EMSA) were preformed. The divergently transcribed plasmid-encoded ureRp and ureDp map to a 147-bp fragment from the approximate middle of the 414-bp ureR-ureD intergenic region (Fig. 4). Previously, we demonstrated that there are two independent rUreR-binding sites on this 147-bp segment (20, 21). In EMSA with the 147-bp fragment and rUreR, the mobility of this DNA piece changes (Fig. 7). At low concentrations of rUreR only one slower migrating band was seen, whereas at the higher concentrations of rUreR two shifted bands were observed. This change in migration is sensitive to the presence of urea, and the shifted bands were seen with lower rUreR concentrations when urea was present.

View larger version (58K): [in this window] [in a new window]   Fig. 7.   Binding of rUreR and rUreRc to ureRp-ureDp. Gel shift assay using the 147-bp ureRp-Dp fragment. Urea (50 mM) was added to lanes 11-20. C, control lane with no rUreR added.

rUreR-His102Ala and rUreR-His175Ala were generated and used in EMSA (Fig. 7). Both mutant forms were able to bind to the 147 plasmid-encoded ureRp-ureDp containing fragment. When compared with the interaction of wild-type UreR with the 147-bp fragment, both rUreR-His102Ala and rUreR-His175Ala showed increased binding in the absence of urea, indicating the rUreR^c had increased affinity for DNA independent of urea.

Affinity of Wild-type and Mutant rUreR for ureDp-- Previously we measured the dissociation rate constant (k₁) of the rUreR·ureDp and rUreR·ureRp complexes, and found that the half-life of the complexes increased substantially in the presence of urea (21). Similar experiments were preformed here to measure the k₁ of the rUreR^c·DNA complexes.

rUreR and mutant rUreR were bound to radiolabeled ureDp as above for EMSA, 100-fold excess cold DNA was added, and the mix was loaded quickly onto a 5% acrylamide gel under current (Fig. 8). The loss of radioactivity from the rUreR- and rUreR^c-ureDp band was measured to determine the dissociation rate constant (Table IV). As demonstrated previously, in the absence of urea the rUreR·ureDp complex dissociated with a rate too rapid to measure, whereas in the presence of urea the rUreR·ureDp complex had a dissociation half-life of greater than 250 s. In contrast, in the absence of urea the rUreR·His102Ala·ureDp complex half-life was ~130 s and the rUreR·His175Ala·ureDp complex half-life was ~200 s (Table IV). Therefore, affinity of the mutant rUreR for the DNA-binding site in the absence of urea was increased and similar to the affinity of wild-type rUreR for the DNA in the presence of urea. When urea was added there was no measurable dissociation of the UreR^c·DNA complexes during the course of the experiment, indicating the half-life of the interaction was greater than 60 min.

View larger version (55K): [in this window] [in a new window]   Fig. 8.   Dissociation of rUreR and rUreRc from ureDp. rUreR or rUreRc were bound to the radiolabeled ureDp fragment, in the presence and absence of urea, and the dissociation was measured after the addition of 100-fold excess unlabeled fragment. Lanes indicated consecutive time points as described under "Experimental Procedures" with (A) lanes 1, 5, 9, and 15 time zero, and (B) lanes 1, 5, 11, and 15 time zero.

Thus, by in vivo transcriptional activity and in vitro DNA binding measurements, the histidine to alanine substitutions at position 102 and position 175 result in UreR forms with a constitutive urea-independent phenotype. These mutant proteins appear to have the active conformation in the absence of urea.

rUreR^c Have Functional Urea Binding Sites-- The constitutively active rUreR-His102Ala and rUreR-His175Ala were examined for the ability to bind urea using the fluorescence quenching assay described above (data not shown). The mutant forms bound urea with an affinity similar to the affinity of wild-type rUreR for urea (Table II). The histidine to alanine substitutions that conferred a constitutive, urea-independent phenotype did not affect urea binding, and these residues were not likely to be found in the urea-binding site.

Conformation of rUreR^c-- Circular dichroism spectra for the two UreR^c were obtained with and without urea. Similar to the observations with wild-type rUreR, there was little change in predicted structure when urea was added to the protein sample. However, the predicted -helical content of the mutants was decreased, because both rUreR^c had ~40% -helical structure, as compared with 56% for wild-type rUreR (Table III).

Some AraC family members act as dimers (28). As determined by gel filtration chromatography, rUreR, 34 kDa (Fig. 9) and the two rUreR^cs (not shown) were dimers in both the presence and absence of urea. Each protein migrated with the bovine serum albumin standard of 66 kDa. Therefore, activation resulting from either urea binding or loss of the histidine residues was not associated with the formation of a dimer.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Relative molecular weight of rUreR. rUreR (0.5 mg/ml) was run on a Sephadex G-75 column in buffers (A) without urea or (B) with 50 mM urea. Molecular weight standards were also run with and without urea as appropriate. Dashed line, rUreR; solid line, standards: 1, bovine serum albumin, 66 kDa; 2, carbonic anhydrase, 29 kDa; 3, cytochrome C, 12.4 kDa; 4, aprotinin 6.5 kDa.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The AraC family of transcriptional activators contains over 100 members and is named after the regulator of the L-arabinose operon (28). AraC was the first transcriptional activator identified and was the first member of this family to be purified and characterized biochemically. Proteins in this family are characterized by a 17-residue conserved motif and an HTH motif located in a 100-residue stretch of amino acids (28). A small number of the proteins in this family also have known signal molecules and signal receptor domains within the polypeptide. These include AraC, XylS, MelR, RhaR, and UreR. Although many of the AraC family members are involved in expression of virulence determinants, of the virulence associated regulators, only UreR has a known effector molecule and contains the signal receptor and activator domains on the same molecule.

Transcription of P. stuartii urease genes is dependent on the presence of the substrate of urease, urea, and UreR. The half-life of the rUreR·DNA association increases when urea is present, and we believe this increase in affinity is responsible, at least in part, for the activity of the protein. The relatively modest affinity of rUreR for urea (K_d of 0.2 mM) correlates with the levels of urea in environments where urease genes are expressed. Urea is found at concentrations of up to 10 mM in the blood and 500 mM in the urine, whereas P. stuartii requires urea concentrations of 5 mM and greater to initiate transcription of urease genes. The observed low affinity of urea for UreR assures that the organism will initiate transcription of urease genes only in appropriate and biologically relevant environments that contain a relatively high concentration of urea.

The fluorescence emission profiles of urea-bound and urea-free rUreR differed, indicating that the environment of an aromatic chromophore was altered by urea binding. The chromophore is the tryptophane at position 185.² Unfortunately, the involvement of this chromophore does not identify the position of the urea-binding site, because we cannot conclude with certainty that this residue is in the urea-binding pocket. To map the urea-binding site, histidine to alanine substitutions were generated in UreR. Histidine residues were chosen based on the composition of the urea-binding site in urease, where histidines coordinate the binding of a nickel ion, which in turn, interacts with urea. In urease, specific histidine mutations led to loss of nickel binding and loss of activity (26). Here, we found the opposite to be true. Only one histidine residue substitution (UreR-His240Ala) resulted in loss of activity, and this loss can be attributed to the instability of the mutant protein. None of the other histidine substitutions led to loss of urea binding. Thus, the histidine to alanine substitutions did not specifically localize the urea-binding site. We can state, however, that the urea-binding site lies on the N-terminal 22.0 kDa of the protein, because rUreR22.0 bound urea with the same affinity as wild-type rUreR. This binding site is not on the recombinant N-terminal histidine tag, because the MBP-UreR derivative, which does not contain a histidine tag, was able to bind urea with an affinity similar to that of rUreR.

The histidine substitutions did help address another question. It has been suggested that, similar to the urea-binding site of urease, the UreR urea-binding site may contain nickel. We found no evidence that this is true. With the exception of rUreR-His240Ala, which is rapidly unstable, loss of the histidines residues did not result in lost of activity. Therefore, if nickel is present, its binding is not dependent on histidine residues. Other evidence against Ni-UreR interaction includes that wild-type UreR did not bind to nickel affinity columns, nor did it bind to nickel-coated 96-well plates. The positive control of rUreR, which contains the N-terminal histidine tag, bound both the column resin and the 96-well nickel-coated plates. Furthermore, the MBP-UreR fusion protein, which binds urea, was determined to be metal ion-free by plasma emission analysis using a Jarrell-Ash ICP plasma emission spectrophotometer. Thus, we conclude that nickel does not have a physiologic role in the activity of UreR.

Instead of localizing the urea-binding pocket, the histidine substitutions led to the discovery of two independent UreR^c. These rUreR^c have increased affinity for the DNA-binding site and activate transcription in the absence of urea. These mutations are not in the urea-binding pocket, because both mutants bound urea with an affinity equal to the wild-type UreR·urea interaction. These UreR^c indicate that UreR can be activated either by urea binding or by amino acid residue substitutions at one of two positions. To understand the conformation changes resulting in UreR activation, circular dichroism spectra were obtained. These data predicted that the activation-associated structural changes are subtle. rUreR and the two rUreR^c are dimers in both the presence and absence of urea. (Recently, P. mirabilis UreR was demonstrated to be a dimer (29).) Therefore, activation does not result from the formation of a dimer. A more detailed analysis, such as an x-ray structure, is required to determine differences between the active and inactive conformations.

The in vitro studies performed here used UreR with the six-histidine tag fused at the N terminus. This protein had elevated activity in the absence of urea when compared with UreR without the histidine tag. The reason for this elevated activity is unclear and could result from increased affinity for urea, increased or decreased kinetics of dimer formation, or changes in DNA binding affinity. The MBP-UreR shows comparable affinity for urea as rUreR, eliminating urea binding as a cause. It is demonstrated here that rUreR is a dimer in the presence and absence of urea, and similar results were reported with P. mirabilis UreR (29), suggesting the histidine tag does not affect dimer formation. Thus, this increase in activity probably results from elevated DNA affinity. We conclude, therefore, that the DNA binding data presented in Table IV are valid as relative measurements of the affinities of the recombinant proteins but might reflect affinities higher than those found in the in vivo situation. Current experiments are underway to examine the role of the N terminus of UreR in DNA binding activity.

A key question in the study of microbial pathogens is to identify the environmental signals triggering the expression of virulence genes. For the P. stuartii urease genes, that signal is urea. In the infected urinary tract, urea freely diffuses into the bacteria, where it binds to UreR. This interaction activates UreR and alerts the organism of its immediate environment. Thus the UreR·urea complex has a dual role, acting as both a transcriptional activator as well as an environmental sensor. Future experiments are needed to determine whether UreR is a specific regulator of urease genes or if it regulates the expression, and possible repression, of other plasmid-encoded and/or chromosomal virulence-associated genes.

	ACKNOWLEDGEMENTS

We are grateful to Keith Brew for assistance with the fluorescence and CD experiments and for many helpful discussions. We thank R. Hausinger for helpful discussions.

	FOOTNOTES

^* This work was funded by Public Health Service Grants DK50495 and DK60163 (to C. M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Dept. of Internal Medicine, Section of Rheumatology, P. O. Box 208031, New Haven, CT 06520.

^§ To whom correspondence should be addressed (current address): Dept. of Microbiology, University of Washington School of Medicine, Box 357242, Seattle, WA 98195. Tel.: 206-616-0581; Fax: 206-543-8297; E-mail: carleen@u.washington.edu.

Published, JBC Papers in Press, July 29, 2002, DOI 10.1074/jbc.M203462200

² M. C. Parra and C. M. Collins, unpublished.

	ABBREVIATIONS

The abbreviations used are: rUreR, recombinant UreR; UreR^c, constitutive UreR, UreR that acts in the absence of urea; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MBP, maltose binding protein; EMSA, electrophoretic mobility shift assay; HTH, helix-turn-helix.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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