HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 22, 16476-16491, June 1, 2007

This Article Abstract Full Text (PDF) All Versions of this Article: 282/22/16476    most recent M610838200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lorca, G. L. Articles by Savchenko, A. Search for Related Content PubMed PubMed Citation Articles by Lorca, G. L. Articles by Savchenko, A. Social Bookmarking                      What's this?

Glyoxylate and Pyruvate Are Antagonistic Effectors of the Escherichia coli IclR Transcriptional Regulator^*

Graciela L. Lorca¹, Alexandra Ezersky, Vladimir V. Lunin^¶, John R. Walker^¶||, Svetlana Altamentova^¶, Elena Evdokimova^¶, Masoud Vedadi^||, Alexey Bochkarev^||, and Alexei Savchenko^¶2

From the Banting and Best Department of Medical Research, Toronto, Ontario M5G 1L6, Canada, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, ^¶Ontario Center for Structural Proteomics, Toronto, Ontario M5G 1L6, Canada, and ^||Canada Structural Genomics Consortium, Toronto, Ontario M5G 1L6, Canada

Received for publication, November 22, 2006 , and in revised form, March 28, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Escherichia coli isocitrate lyase regulator (IclR) regulates the expression of the glyoxylate bypass operon (aceBAK). Founding member of a large family of common fold transcriptional regulators, IclR comprises a DNA binding domain that interacts with the operator sequence and a C-terminal domain (C-IclR) that binds a hitherto unknown small molecule. We screened a chemical library of more than 150 metabolic scaffolds using a high-throughput protein stability assay to identify molecules that bind IclR and then tested the active compounds in in vitro assays of operator binding. Glyoxylate and pyruvate, identified by this method, bound the C-IclR domain with K_D values of 0.9 ± 0.2 and 156.2 ± 7.9 µM, as defined by isothermal titration calorimetry. Both compounds altered IclR interactions with operator DNA in electrophoretic mobility shift assays but showed an antagonistic effect. Glyoxylate disrupted the formation of the IclR/operator complex in vitro by favoring the inactive dimeric state of the protein, whereas pyruvate increased the binding of IclR to the aceBAK promoter by stabilizing the active tetrameric form of the protein. Structures of the C-IclR domain alone and in complex with each effector were determined at 2.3 Å, confirming the binding of both molecules in the effector recognition site previously characterized for the other representative of the family, the E. coli AllR regulator. Site-directed mutagenesis demonstrated the importance of hydrophobic patch formed by Met-146, Leu-154, Leu-220, and Leu-143 in interactions with effector molecules. In general, our strategy of combining chemical screens with functional assays and structural studies has uncovered two small molecules with antagonistic effects that regulate the IclR-dependent transcription of the aceBAK operon.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A classical bacterial repressor, termed IclR, participates in the regulation of the aceBAK operon in Escherichia coli (1, 2). The aceBAK operon encodes for the enzymes of the glyoxylate bypass (3) that are required during growth on acetate since it bypasses the two CO₂-evolving steps of the Krebs cycle (4). The expression of these enzymes is induced during growth on minimal medium supplemented with acetate or fatty acids (3) as well as in rich medium as a result of the acetate accumulation during exponential phase. According to early genetic studies (1, 2) IclR represses the expression of aceBAK operon as well as its own gene by binding to specific operator sequences in the promoter region. Two separate operator sequences, termed IclR-boxes, have been identified in the promoter region upstream of the aceB gene (5). The primary operator sequence (IclR box II) has been mapped between -52 and -19 bases of the aceB promoter, and binding of IclR to this site prevents the RNA polymerase-promoter interaction (5). Binding to the second site, IclR box I, which is located between -125 and -99 bases of the aceB promoter, disassembles the open complex through a protein-protein interaction between IclR and the RNA polymerase -subunit (5). A third IclR-box was located between +14 and -21 bases of the iclR promoter (6). All three IclR-boxes identified in the promoters of aceB and iclR genes contain near-perfect palindromes. Although not identical, their sequences share a 15-mer consensus motif, 5'-TGGAAATNATTTCCA, which was identified by in vitro random oligonucleotide selection experiments (7).

In growth media containing acetate, IclR is believed to release the DNA as a result of binding a small molecule. Although this molecule is likely related to a metabolite within the glyoxylate bypass pathway, the precise nature of this IclR effector remains nevertheless in doubt. Phosphoenolpyruvate was reported to alter IclR binding to DNA in vitro (8) but recently was shown to have no effect on IclR activity by Yamamoto and Ishihama (5).

IclR is the founding member of a large family (Pfam 01614, COG1414) of sequence-related microbial transcriptional regulators with more than 500 members identified in bacterial and archaeal genomes. Numerous bacterial genomes such as E. coli K12 (9) and Pseudomonas aeruginosa PA01 (10) contain multiple members of this family (8 and 9, respectively) with the largest number (40 members) identified so far in Bordetella bronchiseptica RB50 (11). The characterized members of this family are involved in the regulation of diverse catabolic pathways ranging from the degradation of plant cell polysaccharides in the plant pathogen Erwinia chrysanthemi (12) to the metabolism of aromatic acids in E. coli, Acinetobacter (13, 14), and Pseudomonas (15).

The characteristic structural features for members of this family, gained primarily from the crystal structure of TM0065 from Thermotoga maritime (PDB code 1MKM), are fully consistent with the role of transcription regulators responding to chemical stimuli (16). The TM0065 structure confirmed the presence of N-terminal classic-winged helix-turn-helix DNA binding domain as well as demonstrated that the effector binding C-terminal domain has significant structural homology to the PAS/GAF domain, a known small molecule binding motif (17, 18). The N- and C-terminal domains in the structure are connected by a linker helix, which together with N-terminal domain participates in protein dimerization. In the crystal lattice TM0065 dimers further oligomerize into tetramers, which appear to be the functional unit common for a number of IclR regulators (16). The C-terminal domain retains its structure and small molecule binding function when expressed without the DNA binding domain and was structurally characterized for three E. coli representatives of IclR family, KdgR (PDB code 1YSP), YiaJ (PDB code 1YSQ), and AllR³ (Ref. 20, PDB code 1TF1). Although sharing relatively low sequence identity between them and with previously characterized TM0065, all four structures were very similar, confirming a common structural framework for most of the IclR family members.

AllR is involved in transcriptional regulation of the allantoin catabolism (19) and shares 42% sequence identity with IclR. The AllR C-terminal domain structure was recently determined in the presence and absence of its chemical effector glyoxylate (20), providing the first detailed view of the molecular interactions of an IclR family representative with a small molecule. According to the AllR-glyoxylate complex structure, the location of the effector binding pocket in IclR regulators corresponded to the co-factor-accommodating cavity in PAS/GAF domains; however, the binding mechanisms were not conserved between these structurally similar domains (17, 18, 20).

Of the hundreds of IclR family members, only a few of the sensor chemicals have been identified, which apart from glyoxylate, include 3-(3-hydroxyphenyl) propionic acid for E. coli MhpR (21), 2-keto-2-deoxyglyconate for E. chrysanthemi KdgR (12), p-hydroxybenzoate and protocatechuate for Acinetobacter PobR and PcaU, respectively (13, 22), N-3-oxo-octanoyl homoserine lactone (3OC8HSL) for Agrobacterium tumefaciens AttJ (23), and 1-naphthol and 2,3-dihydroxynaphthalene for Pseudomonas putida TtgV (24). Thus, the effector specificity of hundreds IclR regulators, including the founding member of the family, remains uncertain.

To address these issues we used IclR as a test case and screened it against a large number of intermediates associated with glyoxylate bypass including phosphoenolpyruvate as part of a library of more than 150 metabolites by a high-throughput protein stability assay (25, 26). This methodology takes advantage of the well documented phenomenon of enhanced thermal stability of the protein in the presence of a specifically bound ligand (27). The molecules that had a stabilizing effect were further assessed for the effector role for IclR using biophysical and functional assays.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
DNA Manipulations and Gene Cloning—Standard methods were used for site-directed mutagenesis, chromosomal DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transformation (28). Plasmids were isolated using spin miniprep kits (Qiagen), and PCR products were purified using Qiaquick purification kits (Qiagen). Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene).

For protein expression and purification the iclR gene was amplified from wild type E. coli BW25113 chromosomal DNA by PCR. To identify the boundaries of the ligand binding domain of IclR, a multiple sequence alignment of IclR and its homologues was performed. The link between the DNA and ligand binding domains in E. coli IclR was predicted to lie between amino acids Ser 98 and Leu 101. Two fragments corresponding to the 176 and 173 C-terminal amino acids were amplified and tested for expression of the soluble domain as previously described (20). The expression of a fragment of iclR corresponding to amino acids 98–274 yielded a soluble domain of IclR (C-IclR) that was used for further experiments. All amplified fragments were cloned into the NdeI and BamHI sites of a modified form of pET15b (EMD Biosciences) in which a TEV protease cleavage site replaced the thrombin cleavage site and a double stop codon was introduced downstream from the BamHI site. This construct provides for an N-terminal hexahistidine tag separated from the protein by a tobacco etch virus protease recognition site (ENLYFQGS).

Construction of Deletion Mutants—An IclR deletion mutant was generated by the methods described by Datsenko and Wanner (29). To prepare competent cells for transformation, BW25113 (lacI^q rrnB_T14 lacZ_WJ16 hsdR514 araBAD_AH33 rhaBAD_LD78; 29) containing pKD46 was cultured at 30 °C in SOB broth (28) containing 100 µg of ampicillin/ml. When the A₆₀₀ reached 0.5, the culture was centrifuged at 4000 rpm for 5 min, and the cells were washed 3 times with cold water before being resuspended in 1% of the original culture volume of water. PCR methods were used to amplify the kanamycin resistance gene (km) from pKD4 by using the primers iclRFw (5'-GTTCAACATTAACTCATCGGATCAGTTCAGTAACTATTGCATTAGCTAACGTGTAGGCTGGAGCTGCTTC-3') and iclRrv (5'-GCGATTAACAGACACCCTTATTCTATTGCCACTCAGGTATGATCATATGAATATCCTCCTTA-3'). The PCR products were purified with a Qiagen kit, treated with DpnI, and repurified by electrophoresis. The km gene was transformed into BW25113-competent cells by electroporation (Gene Pulser; pulse controller at 200 ohms, capacitance at 250 microfarads, and voltage at 25 kV). After electroporation, the cells were grown with shaking in 1 ml of SOC (28) medium at 37 °C for 1 h, and the cultures were plated onto Luria-Bertani (LB) agar containing 25 µg of kanamycin/ml. The Km^r transformants were purified on new kanamycin-LB plates. The mutants in which the target genes were replaced by the km gene were verified by PCR using the primers iclRcfw (5'-CTCATCGGATCAGTTCAGTAAC-3') and iclRcrv (5'-CTTATTCTATTGCCACTCAGG-3'). To delete the km gene from the chromosome, pKD46 was removed from the cells by growing the bacteria at 37 °C, and then pCP20, expressing the Flip recombinase, was introduced by transformation. The transformants containing pCP20 were grown overnight with shaking at 42 °C, and the cultures were plated on LB agar without antibiotics. Colonies were tested for sensitivity to kanamycin and ampicillin. crp mutants were constructed by P1 transduction (30) using the strain JW3320 as donor (National BioResource Project, National Institute of Genetics, Mishima, Japan (31)).

Real-time Quantitative PCR Studies—Bacterial cells were cultured in MOPS minimal media (32) with 10 mM pyruvate as carbon source, and when required, 10 mM glyoxylate was added. When the A₆₀₀ reached 0.5, cells were collected by centrifugation at 4 °C. Total RNA was subsequently isolated with RiboPure^TM-Bacteria (Ambion) in accordance with the manufacturer's protocol. cDNAs were synthesized with the superscript first-strand synthesis kit (Invitrogen) in accordance with the manufacturer's instructions and stored at -80 °C before use. Real-time quantitative PCR was carried out on the Applied Biosystems 7300 apparatus (Applied Biosystems) using Platinum® SYBR® Green qPCR SuperMix UDG (Invitrogen) in accordance with the manufacturer's recommended protocol. Primers used for the real-time PCR were as follows: for rrsC, 5'-CAGCCACACTGGAACTGAGA-3' and 5'-GTTAGCCGGTGCTTCTTCTG-3'; for aceB, 5'-CTGCGTGACCATATTGTTGG-3' and 5'-CAGGCGTGAGTAAGCATTCA-3'; for aceK, 5'-TTCGTGCCTGCTATCAACTG-3' and 5'-AGATATTGAGCGGCACCATC-3'.

Protein Purification—The His-tagged fusion proteins were overexpressed in E. coli BL21-Star(DE3) cells (Stratagene) harboring an extra plasmid encoding three rare tRNAs (AGG and AGA for Arg, ATA for Ile). The cells were grown in LB at 37 °C to an A₆₀₀ 0.6, and expression was induced with 0.4 mM isopropyl 1-thio--D-galactopyranoside. After the addition of isopropyl 1-thio--D-galactopyranoside, the cells were incubated with shaking at 15 °C overnight. The cells were harvested, resuspended in binding buffer (500 mM NaCl, 5% glycerol, 50 mM HEPES, pH 7.5, 5 mM imidazole), flash-frozen in liquid N₂, and stored at -70 °C. The thawed cells were lysed by sonication after the addition of 0.5% Nonidet P-40 and 1 mM each of PMSF and benzamidine. The lysate was clarified by centrifugation (30 min at 17,000 rpm) and passed through a DE52 column preequilibrated in binding buffer. The flow-through fraction was then applied to a metal chelate affinity column charged with Ni²⁺. After the column was washed, the protein was eluted from the column in elution buffer (binding buffer with 500 mM imidazole). The hexahistidine tag was then cleaved from the protein by treatment with recombinant His-tagged tobacco etch virus protease. The cleaved protein was then resolved from the cleaved His tag and the His-tagged protease by flowing the mixture through a second Ni²⁺ column.

The purified proteins were dialyzed against 10 mM HEPES, pH 7.5, 500 mM NaCl and concentrated using a BioMax concentrator (Millipore). Before crystallization, any particulate matter was removed from the sample by passage through a 0.2-µm Ultrafree-MC centrifugal filtration device (Millipore). Selenomethionine-labeled proteins were expressed using the same vector and host strain but in supplemented M9 media (33). The sample was prepared under the same conditions as the native protein except for the addition of 5 mM -mercapto-ethanol to the purification buffers.

Size-exclusion Chromatography—100 µl of protein sample contained 10 mM HEPES, pH 7.5, 500 mM NaCl, 25 µM IclR wild type or its mutant derivatives, and when indicated, 1 mM glyoxylate or 10 mM pyruvate. After 20 min of incubation on ice, samples were injected onto a Superose 12 10/300 GL gel filtration column (Amersham Biosciences) installed on anÁ_kta system (Amersham Biosciences) equilibrated with 10 mM HEPES, pH 7.5, 500 mM NaCl. Filtration was performed at 4 °C at a flow rate of 0.5 ml/min, and the protein concentration was monitored by measuring the absorbance at 280 nm. Blue dextran 2000 was used to determine the void volume. A mixture of protein molecular mass standards containing-amylase (200 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa) was applied to the column under similar conditions. The elution volumes and molecular masses of the protein standards were used to generate a standard curve from which the apparent molecular mass was determined.

Crystallization and Data Collection—The primary crystallization condition was determined by a sparse crystallization matrix (Hampton research kits Crystal Screen 1 and PEG/Ion Screen) at room temperature using the sitting drop vapor diffusion technique in 96-well plates. This condition was optimized by varying the pH and the concentration of the solutes. For apoIclR the best crystals were obtained using hanging drops in 0.2 M potassium acetate, pH 7.0, 20% polyethylene glycol 3350 for 2–5 days at room temperature. For diffraction studies, the crystals were flash-frozen with the crystallization buffer plus 30% ethylene glycol as cryoprotectant. For the complex structures the crystals obtained in the crystallization condition described above were soaked in 10 mM solution of either glyoxylate or pyruvate for 1 h. For diffraction studies, the crystals were flash-frozen with the crystallization buffer plus 25% ethylene glycol as the cryoprotectant.

Diffraction data of crystals of C-IclR were collected at 100 K at the 19ID beamline of the Structural Biology Center at the Advanced Photon Source, Argonne National Laboratory. The absorption peak and rising inflection point were determined by calculating and plotting f' and f'' values against energy (34) from the fluorescence spectrum. The three-wavelength anomalous diffraction data were collected from a Se-Met-substituted protein crystal using an inverse beam strategy. All crystallographic data were measured with the custom-built 3 x 3-tiled CCD (charge-coupled device) detector (35). The experiment, data collection, visualization, and processing were controlled with the HKL2000 program package (36). Statistics of data collection and processing are provided in Table 3. Diffraction data for glyoxylate and pyruvate complexes were collected at home source (Rigaku FR-E generator with Raxis4++ detector), and the data were integrated and scaled with ^*TREK (37).

Electrophoretic Mobility Shift Assays (EMSA)—Electrophoretic mobility shift assays for IclR and mutant proteins were performed using proteins purified and concentrated according the procedures described above. A fragment of the aceBAK promoter region, from position -117 to +17 (containing IclR box II), was generated by PCR using biotin-prelabeled (5'-end) primers and purified using QIAquick spin columns (Qiagen). Incubation mixtures for EMSA (20 µl) contained 2.5 nM 5'-labeled DNA fragment, 50 mM Tris-HCl, pH 7.5, 150 mM KCl, 10 mM MgCl₂, 0.01% Triton X-100, 50 ng/µl poly(dI-dC)-nonspecific competitor DNA, purified IclR protein (0–100 nM), and ligand (0–1 mM) where indicated.

After incubation for 20 min at 37 °C, samples were separated on 5% acrylamide-bisacrylamide nondenaturing gels in 0.5x Tris borate-EDTA buffer, pH 8.3 (TBE). Electrophoresis was performed at 100 V using ice-cold 0.5x TBE as a running buffer, and DNA was transferred from the polyacrylamide gel to the Biodyne B Positive Nylon Membrane (Pierce) by electroblotting at 380 mA for 40 min in 0.5x TBE. Transferred DNA was cross-linked for 15 min using UV cross-linker equipped with 312-nm bulbs. Biotin-labeled DNA was detected by horseradish peroxidase/Super Signal detection system (Pierce). Membranes were exposed to Eastman Kodak Co. x-ray film. For quantitative EMSA, the distribution of bound and unbound DNA was quantified using ImageJ (National Institutes of Health).

In Vitro Transcription Runoff Assays—For in vitro transcription runoff the complete aceB promoter was amplified from position -297 to +434 and cloned in a derivative of the pGF-Puv vector (Clontech) in which the gfp gene was replaced with the aceB promotor in the SapI-EcoRI position. The expected specific transcript was 434 bases long. The recombinant plasmids were amplified in E. coli DH5, cut with EcoRI, and gel-purified. The reaction mixtures (20 µl) contained template DNA (5 nM), purified IclR or its mutant forms (100 nM), and glyoxylate (1–1000 µM) in a buffer consisting of 40 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl₂, 0.01% Triton X-100, and 1 unit of RNase inhibitor. The reaction mix was preincubated for 10 min at 37 °C, and 20 nM RNA polymerase (Epicenter) was added. The reaction mix was further incubated for 5 min at 37 °C, and transcription was initiated by adding 2 µl of a mixture containing 2 mM each ATP, GTP, and CTP, 1.5 mM UTP, and 0.5 mM biotin-11-UTP (PerkinElmer Life Sciences). After 20 min at 37 °C, the reactions were terminated with 10 mM EDTA. The ethanol-precipitated transcripts were analyzed on 6% acrylamide, 7 M urea gels. Electrophoresis was performed at 100 V using ice-cold 0.5x TBE as a running buffer, and RNA was transferred from the polyacrylamide gel to the Biodyne B positive nylon membrane (Pierce) by electroblotting at 380 mA for 40 min in 0.5x TBE. Transferred RNA was cross-linked for 15 min using UV cross-linker equipped with 312-nm bulbs. Detection was performed as described above. Preheated biotinylated 2-log DNA ladder (BioLabs) was used as molecular weight marker.

Screening Using Static Light Scattering—Purified C-IclR protein was screened against a library of 160 compounds using static light scattering (StarGazer) (45). Protein samples were diluted to a final concentration of 0.4 mg/ml in 100 mM Hepes, pH 7, 150 mM NaCl (26). 50-µl aliquots of protein solution containing the chemical compounds at 1 mM were placed in duplicate into clear-bottom 384-well plates (Nunc^TM, Nalgene Nunc International) and heated from 27 to 80 °C at the rate of 1 °C/min. The formation of protein aggregates was monitored by static light scattering. Images of scattered light were captured every 30 s, and the light intensities were translated to arbitrary numbers using StarGazer^TM proprietary software. Intensities were plotted against temperature for each sample well, and transition curves were fitted using the Boltzmann equation. The midpoint of each transition was calculated and compared with the one calculated for the reference sample. If the difference between them was greater than 1.5 °C, the corresponding compound was considered to be a "hit," and the experiment was repeated to confirm the effect.

Circular Dichroism (CD) Spectroscopy—CD spectra were collected at 25 °C on a AVIV 62D CD spectrophotometer from 200 to 260 nm using a 0.1-mm path length cell with a scan rate of 100 nm/min, a time constant of 1.0 s, a bandwidth of 1 nm, and a sensitivity of 100 millidegrees. Each spectrum was the average of 10 scans. After background subtraction, the spectrum was expressed in molar ellipticity. The proteins were analyzed at a final concentration of 1 mg/ml, and the assays were performed in 10 mM HEPES, pH 7.5, 0.5 M NaCl. When necessary, glyoxylate or pyruvate was added at 1 mM.

Isothermal Titration Calorimetry—Measurements were performed on a VP-Microcalorimeter (MicroCal, Northampton, MA) at 25 °C. The protein was thoroughly dialyzed against 10 mM HEPES, pH 7.5, and 500 mM NaCl. Solutions of glyoxylate (0.5 mM) and pyruvate (1.5 mM) were directly prepared in dialysis buffer. Each titration involved a series of 4-µl injections of effector molecules into the protein solution. The mean enthalpies measured from injection of the ligand in the buffer were subtracted from raw titration data before data analysis with ORIGIN software (MicroCal). Titration curves were fitted by a nonlinear least squares method to a function for the binding of a ligand to a macromolecule (46). From the curve thus fitted, the parameters H (reaction enthalpy), KA (binding constant, KA = 1/K_D), and n (reaction stoichiometry), were determined. From the values of KA and H, the changes in free energy (G) and in entropy (S) were calculated with the equation G = -RT ln KA =H - TS, where R is the universal molar gas constant, and T is the absolute temperature.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Screening of a Small Molecule Library for Binding the IclR— Significant sequence similarity between IclR and previously characterized AllR regulators, particularly among amino acids involved in glyoxylate binding (20), indicated that the IclR effector may have chemical features similar to glyoxylate rather than phosphoenolpyruvate, which was proposed as the IclR effector by Cortay et al. (8). To clarify the nature of the IclR effector molecule, we decided to perform an unbiased screen of IclR against a variety of glyoxylate-associated compounds along with phosphoenolpyruvate as part of large set of metabolic scaffolds.

View larger version (10K): [in this window] [in a new window]   FIGURE 1. Circular dichroism of C-IclR with (squares) and without (circles) 1 mM glyoxylate. The proteins were used at a final concentration 1 mg/ml, and the assays were performed in 10 mM HEPES, pH 7.5, 0.5 M NaCl.

All five compounds identified for C-IclR contained a common chemical motif in the form of adjacent carbonyl or hydroxyl groups (Table 1) with the strongest thermal stabilizing agent, glyoxylate, being the simplest representation of this chemical scaffold. The intermediate role played by glyoxylate in the pathway regulated by IclR also strengthens its case as a candidate for the native IclR effector molecule. At the same time identification of several metabolites causing IclR thermal stabilization and having a common chemical signature indicates that this sensor protein might be reactive to more than one effector molecule.

Effect of Small Molecule Modulation of IclR Binding in Vitro— The effect of glyoxylate, allantoate, DL-glyceraldehyde-3-phosphate, pyruvate, and phosphoenolpyruvate on IclR activity was tested in vitro by EMSA. The binding of IclR to the DNA fragment corresponding to the promoter region of the aceBAK operon and containing IclR box II (5) was analyzed in the presence and the absence of 100 µM or 1 mM concentrations of each compound. According to EMSA results, glyoxylate caused dissociation of the IclR regulator from DNA, thus increasing the fraction of unbound DNA (Fig. 2). On the contrary, pyruvate and, to a lesser extent, DL-glyceraldehyde 3-phosphate and phosphoenolpyruvate stabilized the IclR-DNA complex and resulted in a decrease of unbound DNA (Fig. 2). Allantoate did not affect the IclR/aceB complex (data not shown).

View larger version (36K): [in this window] [in a new window]   FIGURE 2. Effect of different ligands on the binding of IclR to the aceB promoter. EMSA results using 2.5 nM biotin-labeled aceB promoter and 25 nM (A) or 50 nM (B) IclR with different inducer molecules. The full binding conditions are described under "Experimental procedures." Lanes 1 and 11 show the migration of the target DNA fragment; lanes 2–10, IclR 25 nM; lanes 12–14, IclR 50 nM. Glyoxylate (lanes 3, 4, and 13), pyruvate (lanes 5, 6, and 14), phosphoenolpyruvate (lanes 7 and 8), and glyceraldehyde 3-phosphate (lanes 9 and 10) were added at a concentration of 0.1 mM (3, 5, 7, 9, and 13) or 1 mM (4, 6, 8, 10, and 14).

View larger version (52K): [in this window] [in a new window]   FIGURE 3. In vitro transcription runoff experiments using the aceB promoter, IclR, and different concentrations of glyoxylate. A, in vitro transcription assays were performed as described under "Experimental Procedures." The assays were performed at 37 °C for 30 min in the absence (lane 1) or in the presence of 100 nM IclR added before 20 nM RNA polymerase (lanes 2–6). Glyoxylate was added at 1 µM (lane 3), 5 µM (lane 4), 10 µM (lane 5), and 50 µM (lane 6). The arrows denote the aceB (434 nucleotides) and the bla (860 nucleotides) transcripts. B, quantitative analysis of the in vitro transcription runoff experiment presented on A. For each lane the amount of aceB transcript was normalized (average of three experiments) to the expression level of the bla transcript. Expression of aceB in the absence of IclR represented 10% of the expression of bla transcript (internal control). Each bar represents the percentage of the aceB transcript relative to the transcription level in the absence of IclR protein (lane 1).

View larger version (43K): [in this window] [in a new window]   FIGURE 4. Effect of glyoxylate and pyruvate on the binding of IclR to its operator site. A, EMSA results using 2.5 nM biotin-label aceB promoter, 50 nM of IclR, and increasing concentrations of glyoxylate (0–500 µM) as shown on the top of the picture. Pyruvate 500 µM was added to a second set of samples that also contained increasing concentrations of glyoxylate as shown on the top of the panel. No protein was added to the first lane. B, EMSA results using 25 nM IclR and increasing concentrations of pyruvate (0–500 µM) as shown on the top of the picture. Glyoxylate 500 µM was added to a second set of samples that also contained increasing concentrations of pyruvate as shown on the top of the panel. C and D, EMSA results from A and B, respectively, quantified by image analysis. Each graph represents the percentage (average of three experiments) of the bound DNA in the presence of the ligand relative to the amount of the DNA bound to the same protein in the absence of the ligand.

Phosphoenolpyruvate and DL-glyceraldehyde 3-phosphate were able to stabilize the IclR-DNA complex (Fig. 2); however, the concentrations required to obtain this effect (over 10 mM, data not shown) were out of the physiological range for these compounds (47–50). Thus, phosphoenolpyruvate and DL-glyceraldehyde 3-phosphate were not further considered for an IclR effector role.

Because glyoxylate and pyruvate had distinctive and antagonist effects on the formation of the IclR-DNA complex, in the next series of EMSA experiments we analyzed the combined effect of glyoxylate and pyruvate on the stability of the IclR-DNA complex. In this setting one of compounds was added in increasing amounts, whereas the concentration of the other was kept constant. The results obtained (Fig. 4A) demonstrated that the glyoxylate-induced dissociation of the IclR-operator complex was overturned by a 50-fold excess of pyruvate (500 µM pyruvate with 10 µM glyoxylate). These results were corroborated by doing the reciprocal experiment (Fig. 4B). The common chemical features of these compounds suggested that this effect might be due to the competition for the same binding site in IclR, with glyoxylate having a significantly higher binding affinity than pyruvate.

To further characterize IclR interactions with potential effector molecules, the thermodynamic properties of IclR interactions with glyoxylate and pyruvate were determined using isothermal titration calorimetry. The titration of C-IclR with each compound followed an exothermal heat change profile, giving rise to a sigmoidal binding curve with glyoxylate or hyperbolic with pyruvate (Fig. 5). The calculated thermodynamic parameters are summarized in Table 2. The stoichiometry of the reaction, 0.5, is consistent with the binding of one ligand molecule per IclR dimer. In accordance with EMSA results, the C-IclR dissociation constant (K_D) for glyoxylate (0.9 ± 0.2 µM) was significantly lower (150 times) than that for pyruvate (156.2 ± 7.9 µM). Although the K_D values obtained for binding of glyoxylate and pyruvate to the full-length IclR were higher (14.2 ± 1.2 and 321.7 ± 20 µM, respectivelytively), their relative ratio was within the same range as obtained for C-IclR.

Modulation of IclR Binding by Glyoxylate in Vivo—To assess the effect of glyoxylate on IclR activity in vivo, the expression level of aceBAK operon was measured during growth on minimal media in the presence and absence of this compound. Although early genetic studies identified IclR as the main regulator of aceBAK operon (51), recent E. coli transcriptome analysis indicated that this operon is also under control of the global catabolite regulation protein, Crp. The Crp potential binding sequence was found overlapping the IclR box II (52). In light of this new data, the IclR activity was monitored in the presence and the absence of a functional Crp regulator.

Single iclR and crp as well as double (iclRcrp) deletion strains were prepared in the E. coli BW25113 strain background. Deletion strains were grown to exponential phase on MOPS minimal medium with pyruvate (10 mM) as the carbon source in the presence and in the absence of 5 mM glyoxylate. The expression levels of aceB and aceK genes were measured using real-time quantitative PCR. The expression profiles of both genes followed the same trend; thus, only the results for aceB gene will be discussed further on.

View larger version (32K): [in this window] [in a new window]   FIGURE 5. Isothermal titration calorimetry data for the binding of glyoxylate (A) or pyruvate (B) to C-IclR. Heat changes (upper panel) and integrated peak areas (lower panel) for the injection of a series of 4-µl aliquots of 500 µM glyoxylate or 1.5 mM pyruvate in a solution of 50 µM protein. Data were fitted with ORIGIN.

The substantial derepression effect of the Crp deletion confirmed the major role played by this global regulator on the expression of the aceBAK operon. The effect of the IclR deletion was significantly smaller yet consistent with its role as a specific regulator of this operon. The derepression effect in the presence of glyoxylate observed in the crp strain appears to be specific to the presence of functional IclR, indicating that glyoxylate can affect the activity of the IclR regulator in a manner expected from the native effector molecule. Unfortunately we were unable to study the corepressor effect of pyruvate in vivo since the crp deletion strain is unable to grow on the variety of carbon sources (52) required for these kinds of studies.

View larger version (13K): [in this window] [in a new window]   FIGURE 6. Effect of glyoxylate on the expression of aceB on different strains measured by quantitative real-time PCR. The strains BW25113 (wild type), iclR, crp, or iclRcrp were grown under aerobic conditions in MOPS with 10 mM pyruvate as carbon source with (light gray bars) or without 5 mM glyoxylate (dark gray bars). RNA extractions and real-time quantitative PCR was performed as described under "Experimental Procedures." The amplification values obtained were corrected for those obtained using rrsC as internal control. The values shown are relative to those observed for the same gene in the wild type strain grown with pyruvate.

The C-IclR domain displays a similar architecture to the two other IclR family members whose structures have been determined, TM0065 (16) and AllR (20). The C-IclR structure comprises a centrally located six-stranded anti-parallel -sheet surrounded on one side by two long -helices (5 and 9) and on the other by three shorter -helices (6–8). The -sheet is strongly curved, with the shortest helix (6) fitting on the inside of the half-barrel (Fig. 7).

In complex structures both the glyoxylate and pyruvate molecules occupied the cavity corresponding to the effector binding pocket in the AllR structure (20). The map quality was assessed by constructing an omit F_o - F_c electron density map. The map was calculated after 10 cycles of refinement without the ligand in the model (Fig. 8). Similar to the C-AllR-glyoxylate complex structure, no significant conformational changes were observed in C-IclR complexes with pyruvate or glyoxylate when compared with C-IclR apo structure. The ligands were bound in the same orientation with almost identical positions for atoms C1, C2, O1, O2, and O3 (Fig. 8). Oxygen atoms of glyoxylate and pyruvate were coordinated by an extensive network of hydrogen bonds with the side chains and backbone of the protein. For each ligand the O1 atom is hydrogen-bound to the Asp-212O2 and Gly-160N, the O2 atom to the Ser-239O and Ser-241O, and the O3 to the Ala-161N. The C1-Sg distance varies from 1.86 to 2.09 Å for glyoxylate complex and from 1.90 to 1.97 Å for the pyruvate complex. Both complex models were refined with the same restraints with no covalent bond between ligand C1 and Cys-222S in the dictionary. In agreement with previous sequence analysis, all but two amino acids, Ile-134 and Leu-143, in the effector binding area are conserved between IclR and AllR (20). The corresponding positions in AllR are represented by leucine (Leu-129) and methionine (Met-138), respectively (Fig. 9).

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Overall structure of C-IclR. A, schematic diagram of a monomer of the IclR ligand binding domain in complex with glyoxylate (PDB code 2O99). B, C-IclR interdomain interface between two monomers in the (PDB code 2O99) structure. Loops are colored green, helices are red, and -strands are yellow. Glyoxylate is represented as a stick figure in cyan. The N and C termini are labeled.

Taking into account the almost identical position of glyoxylate and pyruvate in the C-IclR structures, the interactions involving the methyl group of pyruvate, which is absent in glyoxylate, become critical in understanding the different effects of these compounds on IclR activity. The methyl group is well defined in the pyruvate electron density map (Fig. 8B) and is oriented toward the side chain of the Leu-143 residue belonging to the other molecule of the C-IclR dimer. The distances between C3 of the pyruvate and C2 atom of Leu-143 are on average 4.24 ± 0.4 Å for the four molecules in the C-IclR asymmetric unit. Leu-143 makes part of the hydrophobic patch formed by side chains of Met-146, Leu-154, His-216, and Leu-220, which are in the range of van de Waals interactions with the pyruvate methyl group. The presence of methionine instead of leucine in the corresponding position in AllR would explain why pyruvate had no stabilization effect (Table 1).

In C-IclR the interdomain interactions between C-IclR monomers are similar to those observed in the C-AllR and TM0065 structures. The C-IclR and C-AllR dimers are superimposable to a root mean square deviation (r.m.s.d.) of 1.75 Å, compared with an r.m.s.d. of 1.01 Å for individual subunits. This common arrangement of the C-terminal domains of three different IclR family members in three different unit cells supports the physiological relevance of these interactions for tetramerization of IclR regulators (Fig. 7B). The additional intermolecular interactions between the pyruvate methyl group and hydrophobic patch observed in the C-IclR-pyruvate complex structure would, thus, stabilize the tetramerization state of IclR protein.

Effect of Glyoxylate and Pyruvate on the Oligomerization of IclR—To establish the effect of glyoxylate or pyruvate on the oligomeric state of IclR protein, this protein was preincubated with 1 mM glyoxylate or 10 mM pyruvate and analyzed by gel filtration. The untreated IclR protein was used as the control.

In the absence of effector molecules IclR was eluted in two peaks corresponding to the dimeric (60 ± 5 kDa) or tetrameric (120 ± 12 kDa) states. The abundance of both fractions (54/46% relative percentage, respectively) indicated that under our experimental conditions the IclR protein is in a dynamic equilibrium between these two oligomeric states (Fig. 10, Table 4). After preincubation with glyoxylate, IclR was eluted predominantly in the form of the lower (60 kDa) molecular mass fraction, indicating a dramatic shift of equilibrium toward the dimeric state. On the other hand, preincubation with 10 mM pyruvate resulted in elution of IclR in the form of the higher (120 kDa) molecular mass fraction corresponding to the tetramer. Similar results were obtained if glyoxylate was included in the elution buffer; however, the tetrameric state of IclR was more favorable if pyruvate was included in the elution buffer. These results can be explained by the difference in affinity between the ligands (Table 2). Overall, size exclusion experiments demonstrated that glyoxylate and pyruvate have a dramatic effect on the oligomeric state of the IclR regulator. This suggests that the glyoxylate effect on IclR activity is due to the destabilization of tetramers, which is required for efficient binding to the operator DNA, whereas the co-repressor effect observed for pyruvate might be due to the stabilization of the IclR tetramer on the DNA.

View larger version (55K): [in this window] [in a new window]   FIGURE 8. Molecular details of C-IclR interactions with glyoxylate (A and C) or pyruvate (B and D). In A and B the side chains of amino acids within a distance of 8 Å are shown and labeled in the figure. Protein residues (yellow), glyoxylate (pink), and pyruvate (green) are represented as stick figures. The backbone of the protein is shown as a thin yellow line. Leu-143 from another monomer is represented in cyan.In C and D a3 Fo - Fc omit map was built using the REFMAC5 program (43, 44) for the glyoxylate and pyruvate complex structures, respectively. The map was calculated after 10 cycles of refinement without the ligand in the model. The omit Fo - Fc electron density map covering the ligands is shown in stick representation (blue for C or cyan for D). Neighboring residues are also shown in stick representation (cyan in C or yellow in D).

View larger version (45K): [in this window] [in a new window]   FIGURE 9. Ligand binding pocket superimposition of AllR and IclR. Superimposition of the active site of C-IclR with that of C-AllR. Protein residues and glyoxylate are represented as stick figures. Components of C-AllR are represented in green and light pink, whereas components of IclR are colored in yellow and cyan.

The alanine substitution of Leu-143 did not significantly affect the gel filtration profile of IclR (Fig. 11). However, according to the EMSA experiments a higher concentration (100 nM) of the L143A mutant protein was required to achieve the DNA binding comparable with the wild type IclR (Table 4). The addition of pyruvate stabilized complex of this mutant with operator DNA similarly to the wild type IclR, whereas the glyoxylate had a significantly lower effect on the L143A mutant (Fig. 12). In the presence of the latter ligand an additional band was observed on the EMSA gels. This additional band migrated faster than the one corresponding to the wild type IclR-DNA complex but slower that the free DNA (Fig. 12). This band would correspond to an IclR-DNA complex with a lower molecular weight than in case of the wild type IclR. Taking into account that IclR might bind DNA as a tetramer (5), this lower molecular weight complex would correspond to the association of operator DNA with the dimers of L143A as an intermediate in the releasing of IclR from the protein-DNA complex.

The gel filtration profiles of the alanine substitutions of Met-146, Leu-154, and Leu-220 showed them predominantly as dimers in solution (70/30 relative percentage, respectively, Table 4); however, they were still able to tetramerize over DNA as evidenced by a high molecular weight migrating complex (Fig. 12). The mutant proteins showed a significant variation of responses to glyoxylate and pyruvate (Table 4). Although the M146A mutant response to these ligands was similar to the wild type IclR protein, the L220A mutant showed no response to glyoxylate but was stabilized by pyruvate. The L154A mutant, to the contrary, responded to glyoxylate but not to pyruvate. These results put in evidence the different involvement of these amino acids in ligand binding.

View larger version (24K): [in this window] [in a new window]   FIGURE 10. Gel filtration analysis of IclR oligomerization in presence of glyoxylate or pyruvate. IclR (thick line), IclR preincubated with pyruvate (continuous line), or with glyoxylate (dotted line) was passed through a Superose 12 column as described under "Experimental Procedures." The inset shows the calibration curve. The standards used were -amylase (200 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12.4 kDa). mAU, milliabsorbance units.

EMSA experiments on M146D showed low and high molecular weight protein-DNA complexes (Fig. 12). In agreement with the mutations to alanine, M146D does not interfere in the binding of glyoxylate or pyruvate. Moreover, glyoxylate disrupted the formation of the high molecular weight protein-DNA complex, increasing the amount of the lower molecular weight complex (Fig. 12). The presence of pyruvate, on the contrary, increases the formation of the high molecular weight complex. These results show that although the Met-146 is important for the stabilization of tetramers in solution, it is not directly involved in the binding of the ligands, although it might contribute to the overall hydrophobicity of the area.

In the case of L154D, the EMSA profile was similar to that of the wild type IclR; however, the response to glyoxylate was weaker (Fig. 12). Pyruvate had no stabilization effect on this mutant protein (Fig. 12), confirming the Leu-154 role in the specific interactions with pyruvate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The IclR family of transcriptional regulators shares common structural features with their C-terminal domains representing small molecule binding modules adapted to recognize a wide variety of cellular metabolites as effector molecules. The identity of the effector molecules remains unknown for a vast majority of these proteins. This reflects a general situation within the large transcriptional factor families such as LysR (53), LuxR (54), TetR (55), and GntR (56), accounting for thousands of transcriptional regulators that respond to unidentified chemical stimuli which need to be addressed.

View larger version (21K): [in this window] [in a new window]   FIGURE 11. Analysis of IclR L143A and M146A oligomerization in presence of glyoxylate and pyruvate. L143A (A) and M146A (B)(thick line) were preincubated with pyruvate (continuous line), or with glyoxylate (dotted line) and passed through a Superose 12 column as described under "Experimental Procedures." mAU, milliabsorbance units.

View larger version (66K): [in this window] [in a new window]   FIGURE 12. Functionalanalysisofkeyaminoacidsinvolvedininterdomaininteractionsandeffectorbinding. The glyoxylate and pyruvate binding on mutant IclR proteins was tested by EMSA. The name of the corresponding mutant protein and its concentration used for binding is indicated above each image. Glyoxylate and pyruvate were tested at 1 mM. The full binding conditions are described under "Experimental Procedures."

Taking into account the physiological data, we could hypothesize that the cell could maximize the efficiency of expression/repression of the aceBAK operon by using two antagonistic effectors on the same transcription factor. By EMSA and biophysical tests we were able to establish that IclR binds glyoxylate and pyruvate. Pyruvate is the first non-phosphorylated intermediate in glycolysis and a key intermediate in catabolic and biosynthetic reaction pathways. There are several pathways that deliver this compound (48, 72, 73). Siddiquee et al. (48, 49) have shown that the intracellular concentration of pyruvate can be from 0.15 to 0.4 mM depending if samples were taken from batch or continuous cultures. These values are within the affinity constants defined for IclR binding. Glyoxylate, on the other hand, is an important intermediate of the central microbial metabolism in the glyoxylate bypass. Glyoxylate is also generated from glycolate or purine degradation (74, 75). The intracellular concentration for this metabolite in E. coli has not been reported; however, there are studies in the closely related bacterium Salmonella typhimurium (76) which suggest that the concentration of glyoxylate should be very low (10 µM). Thus, the big difference in affinity between glyoxylate and pyruvate binding to IclR might be linked to their intracellular concentrations.

The posttranscriptional regulation of isocitrate dehydrogenase, termed the branch point effect, has been the focus of numerous studies (77, 78). This effect is a consequence of the difference in the affinities of isocitrate dehydrogenase and isocitrate lyase for isocitrate (K_m of 8 and 600 mM, respectively) (77, 78). As a result, the flux of the glyoxylate bypass is strikingly sensitive to the phosphorylation state of isocitrate dehydrogenase. It has been proposed that the presence of pyruvate during the acetate metabolism alters the isocitrate dehydrogenase activity, resulting in inhibited growth due to reduced carbon flux through the glyoxylate shunt (79). Our results clearly show that the effect observed by El-Mansi et al. (79) could be explained by the binding of pyruvate to IclR, which would quickly repress the expression of aceK (encoding for isocitrate dehydrogenase kinase/phosphatase) and would be reflected as a decrease in isocitrate dehydrogenase activity.

According to the data on IclR presented in this study, a mechanism of dual regulation by two effectors can be proposed. When cells grow on complex media or if glucose is added to the media, Crp is the primary transcription regulator (52). The DNA binding sequences for IclR (box II) and CRP in the ace-BAK operon have been predicted in silico to overlap (52). However, when the levels of cAMP increase and CRP is inactive (i.e. growth on acetate) the regulation due to IclR becomes predominant. Under these conditions the concentration of glyoxylate increases due to an increase in the flux through the shunt and is now able to compete with pyruvate for binding of IclR. We suggest that glyoxylate is the primary ligand for IclR, which will trigger IclR dissociation from the DNA. However, when the concentrations of pyruvate are increased due to gluconeogenesis, the complex aceB-IclR-pyruvate might be formed as a safe mechanism to quickly repress the glyoxylate shunt. This mechanistic hypothesis is supported by the fact that both ligands bind to the same site in IclR.

According to the structural analysis and mutagenesis studies presented in this work, the hydrophobic patch formed by Leu-143, Met-146, Leu-154, and Leu-220 plays a critical role in IclR interactions with pyruvate. Moreover, mutagenesis studies demonstrated that Leu-154 substitution by alanine would abolish the pyruvate binding by IclR while leaving the binding of glyoxylate unaffected.

Glyoxylate interactions with IclR strongly resemble those of glyoxylate with another E. coli member of this protein family, the allantoin utilization regulatory protein (AllR). The two proteins share 42% sequence identity, and all but two amino acids, Ile-134 and Leu-143, are conserved in the effector binding area (20). The binding of glyoxylate causes conformational changes in both IclR and AllR proteins, resulting in their dissociation from corresponding DNA binding sites. Nevertheless, in the case of IclR, the glyoxylate-bound conformation causes the shift from tetramer to dimer state, whereas in case of AllR the binding of glyoxylate appeared not to affect its dimer to tetramer ratio (20). In the AllR-glyoxylate complex structure Met-138 (corresponding to the Leu143 in IclR) is involved in interactions with the glyoxylate bound to the adjacent AllR molecule. This interaction would strengthen the tetramerization interface in the glyoxylate-bound conformation of AllR; however, it is absent in the IclR-glyoxylate complex structure. Full understanding of the differences between IclR and AllR interactions with glyoxylate will have to wait for the structural information on the full-length IclR and AllR in complex with this ligand.

The presence of two similar regulators, AllR and IclR, responsive to the same effector in E. coli, might be conditional upon different physiological roles played by these regulators. The pathways regulated by these transcriptional factors share glyoxylate as a common intermediate; however, the physiological conditions required for their expression are quite different. AllR regulates the utilization of allantoin as the sole nitrogen source. The genes coding the enzymes involved in this metabolic process are organized in three transcriptional units (allA, gcl-hyi-glxR-o484-allB-o433-glxK, and allD-f411-f261) that constitute the allantoin regulon. These three units are induced by growth on allantoin or glyoxylate and only seem to be regulated by the action of AllR and AllS (80). On the other hand, IclR represses the expression of the genes that code for the enzymes of the glyoxylate shunt, and the regulation of this operon is extremely complex. Isothermal titration calorimetry experiments indicated that AllR has a 10-fold lower affinity for glyoxylate than does IclR. It may be possible that the regulators are tuned to launch their transcription programs as graded responses to the presence of glyoxylate and that the intracellular concentrations of glyoxylate integrate different metabolic inputs, i.e. acetate metabolism for IclR and allantoin utilization for AllR. Oh et al. (81) characterized the transcript profile of E. coli in acetate cultures using microarrays. The authors observed that not only the genes involved in glyoxylate shunt pathway (ace and glc operons) but also those involved in other glyoxylate-related metabolic pathways such us glycolate and allantoin metabolism were all up-regulated. In a similar manner, Pellicer et al. (82) observed cross-induction of glcDEF and aceBAK operons attributable to pathway intersection at the glyoxylate level. However, in this case the signal molecule for each transcription factor is different.

In summary, we present the binding of two small molecule effectors to a single transcription factor (IclR) with antagonistic functional effects in E. coli. The two ligands recognized by the IclR transcriptional regulator act as corepressor (pyruvate) or activator (glyoxylate) on the transcription of the aceBAK operon, likely in response to intracellular concentrations of these compounds in the bacterial cell.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2O99 and 2O9A) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant GM62414-01, the Ontario Research and Development Challenge Fund, and by the Canadian Institutes of Health Research Grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address and to whom correspondence may be addressed: Dept. of Microbiology and Cell Science, UF Genetics Institute, University of Florida, P. O. Box 103610, Gainesville, FL 32610-3610. Tel.: 352-273-8090; E-mail: glorca{at}ufl.edu. ² To whom correspondence may be addressed: Banting and Best Dept. of Medical Research, 112 College St., Toronto, Ontario M5G 1L6, Canada. Tel.: 416-978-4013; Fax: 416-946-0078; E-mail: alexei.savchenko{at}utoronto.ca.

³ The abbreviations used are: AllR, allantoin utilization regulatory protein; MOPS, 4-morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assays; TBE, Tris-buffered EDTA.

	ACKNOWLEDGMENTS

 
We thank all members of the Structural Biology Centre at Argonne National Laboratory and Greg Brown at the University of Toronto for help in conducting experiments and to Aled Edwards, Alexander Iakounine, and Claudio Gonzalez for critical reading of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Kornberg, H. L. (1967) Bull. Soc. Chim. Biol. 49, 1479-1490[Medline] [Order article via Infotrieve]
2. Maloy, S. R., and Nunn, W. D. (1982) J. Bacteriol. 149, 173-180[Abstract/Free Full Text]
3. Chung, T., Klumpp, D. J., and LaPorte, D. C. (1988) J. Bacteriol. 170, 386-392[Abstract/Free Full Text]
4. Kornberg, H. L., and Madsen, N. B. (1957) Biochim. Biophys. Acta 24, 651-653[Medline] [Order article via Infotrieve]
5. Yamamoto, K., and Ishihama, A. (2003) Mol. Microbiol. 47, 183-194[CrossRef][Medline] [Order article via Infotrieve]
6. Gui, L., Sunnarborg, A., Pan, B., and LaPorte, D. C. (1996) J. Bacteriol. 178, 321-324[Abstract/Free Full Text]
7. Pan, B., Unnikrishnan, I., and LaPorte, D. C. (1996) J. Bacteriol. 178, 3982-3984[Abstract/Free Full Text]
8. Cortay, J. C., Negre, D., Galinier, A., Duclos, B., Perriere, G., and Cozzone, A. J. (1991) EMBO J. 10, 675-679[Medline] [Order article via Infotrieve]
9. Blattner, F. R., Plunkett, G., III, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., and Shao, Y. (1997) Science 277, 1453-1474[Abstract/Free Full Text]
10. Stover, C. K., Pham, X.-Q. T., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S. L., Hufnagle, W. O., Kowalik, D. J., Lagrou, M., Garber, R. L., Goltry, L., Tolentino, E., Westbrook-Wadman, S., Yuan, Y., Brody, L. L., Coulter, S. N., Folger, K. R., Kas, A., Larbig, K., Lim, R. M., Smith, K. A., Spencer, D. H., Wong, G. K.-S., Wu, Z., Paulsen, I. T., Reizer, J., Saier, M. H., Hancock, R. E. W., Lory, S., and Olson, M. V. (2000) Nature 406, 959-964[CrossRef][Medline] [Order article via Infotrieve]
11. Sebaihia, M., Preston, A., Maskell, D. J., Kuzmiak, H., Connell, T. D., King N. D., Orndorff, P. E., Miyamoto, D. M., Thomson, N. R., Harris, D., Goble, A., Lord, A., Murphy, L., Quail, M. A., Rutter, S., Squares, R., Squares, S., Woodward, J., Parkhill, J., and Temple, L. M. (2006) J. Bacteriol. 188, 6002-6015[Abstract/Free Full Text]
12. Reverchon, S., Nasser, W., and Robert-Baudouy, J. (1991) Mol. Microbiol. 5, 2203-2216[Medline] [Order article via Infotrieve]
13. DiMarco, A. A., Averhoff, B., and Ornston, L. N. (1993) J. Bacteriol. 175, 4499-4506[Abstract/Free Full Text]
14. Gerischer, U., Segura, A., and Ornston, L. N. (1998) J. Bacteriol. 180, 1512-1524[Abstract/Free Full Text]
15. Arias-Barrau, E., Olivera, E. R., Luengo, J. M., Fernandez, C., Galan, B., Garcia, J. L., Diaz, E., and Minambres, B. (2004) J. Bacteriol. 186, 5062-5077[Abstract/Free Full Text]
16. Zhang, R. G., Kim, Y., Skarina, T., Beasley, S., Laskowski, R., Arrowsmith, C., Edwards, A., Joachimiak, A., and Savchenko, A. (2002) J. Biol. Chem. 277, 19183-19190[Abstract/Free Full Text]
17. Pellequer, J. L., Wager-Smith, K. A., Kay, S. A., and Getzoff, E. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5884-5890[Abstract/Free Full Text]
18. Repik, A., Rebbapragada, A., Johnson, M. S., Haznedar, J. O., Zhulin, I. B., and Taylor, B. L. (2000) Mol. Microbiol. 36, 806-816[CrossRef][Medline] [Order article via Infotrieve]
19. Cusa, E., Obradors, N., Baldoma, L., Badia, J., and Aguilar, J. (1999) J. Bacteriol. 181, 7479-7484[Abstract/Free Full Text]
20. Walker, J. R., Altamentova, S., Ezersky, A., Lorca, G., Skarina, T., Kudritska, M., Ball, L. J., Bochkarev, A., and Savchenko, A. (2006) J. Mol. Biol. 358, 810-828[CrossRef][Medline] [Order article via Infotrieve]
21. Torres, B., Porras, G., Garcia, J. L., and Diaz, E. (2003) J. Biol. Chem. 278, 27575-27585[Abstract/Free Full Text]
22. Popp, R., Kohl, T., Patz, P., Trautwein, G, and Gerischer, U. (2002) J. Bacteriol. 184, 1988-1997[Abstract/Free Full Text]
23. Zhang, H. B., Wang, L. H., and Zhang, L. H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 4638-4643[Abstract/Free Full Text]
24. Guazzaroni, M. E., Krell, T., Felipe, A., Ruiz, R., Meng, C., Zhang, X., Gallegos, M. T., and Ramos, J. L. (2005) J. Biol. Chem. 280, 20887-20893[Abstract/Free Full Text]
25. Senisterra, G. A., Markin, E., Yamazaki, K., Hui, R., Vedadi, M., and Awrey, D. E. (2006) J. Biomol. Screen 11, 940-948[Abstract/Free Full Text]
26. Vedadi, M., Niesen, F. H., Allali-Hassani, A., Fedorov, O. Y., Finerty, P. J., Jr., Wasney, G. A., Yeung, R., Arrowsmith, C., Ball, L. J., Berglund, H., Hui, R., Marsden, B. D., Nordlund, P., Sundstrom, M., Weigelt, J., and Edwards, A. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 15835-15840[Abstract/Free Full Text]
27. Shrake, A., and Ross, P. D. (1990) J. Biol. Chem. 265, 5055-5059[Abstract/Free Full Text]
28. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
29. Datsenko, K. A., and Wanner, B. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6640-6645[Abstract/Free Full Text]
30. Silhavy, T. J., Berman, M. L., and Enquist, L. W. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
31. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L., and Mori, H. (2006) Mol. Syst. Biol. 2, 1-11
32. Neidhardt, F. C., Bloch, P. L., and Smith, D. F. (1974) J. Bacteriol. 119, 736-747[Abstract/Free Full Text]
33. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1993) J. Mol. Biol. 229, 105-124[CrossRef][Medline] [Order article via Infotrieve]
34. Evans, G., and Pettifer, R. F. (2001) J. Appl. Crystallogr. 34, 82-86[CrossRef]
35. Westbrook, E. M., and Naday, I. (1997) Methods Enzymol. 276, 244-268[Medline] [Order article via Infotrieve]
36. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326[CrossRef]
37. Pflugrath, J. W. (1999) Acta Crystallogr. Sect. D 55, 1718-1725[CrossRef][Medline] [Order article via Infotrieve]
38. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
39. Terwilliger, T. C. (2003) Acta Crystallogr. Sect. D 59, 45-49[CrossRef][Medline] [Order article via Infotrieve]
40. Terwilliger, T. C. (2002) Acta Crystallogr. Sect. D 58, 1937-1940[CrossRef][Medline] [Order article via Infotrieve]
41. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
42. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
43. Skubak, P., Murshudov, G. N., and Pannu, N. S. (2004) Acta Crystallogr. Sect. D 60, 2196-2201[CrossRef][Medline] [Order article via Infotrieve]
44. Vagin, A. A., Steiner, R. A., Lebedev, A. A., Potterton, L., McNicholas, S., Long, F., and Murshudov, G. N. (2004) Acta Crystallogr. Sect. D 60, 2184-2195[CrossRef][Medline] [Order article via Infotrieve]
45. Senisterra, G., Markin, E., Yamazaki, K., and Hui, R. (August 28, 2003) Canada, U. S. Patent application WO 03/071269 AZ
46. Wiseman, T., Williston, S., Brandts, J. F., and Lin, L. N. (1989) Anal. Biochem. 179, 131-137[CrossRef][Medline] [Order article via Infotrieve]
47. Nobeli, I., and Thornton, J. M. (2006) BioEssays 28, 534-545[CrossRef][Medline] [Order article via Infotrieve]
48. Siddiquee, K. A., Arauzo-Bravo, M. J., and Shimizu, K. (2004) FEMS Microbiol. Lett. 235, 25-33[CrossRef][Medline] [Order article via Infotrieve]
49. Siddiquee, K. A. Z., Marcos, M. J., and Shimizu, K. (2004) Appl. Microbiol. Biotechnol. 63, 407-417[CrossRef][Medline] [Order article via Infotrieve]
50. Peng, L., Arauzo-Bravo, M. J., and Shimizu, K. (2004) FEMS Microbiol. Lett. 235, 17-23[CrossRef][Medline] [Order article via Infotrieve]
51. Kornberg, H. L. (1966) Biochem. J. 99, 1-11[Medline] [Order article via Infotrieve]
52. Zhang, Z., Gosset, G., Barabote, R., Gonzalez, C. S., Cuevas, W. A., and Saier, M. H., Jr. (2005) J. Bacteriol. 187, 980-990[Abstract/Free Full Text]
53. Schell, M. A. (1993) Annu. Rev. Microbiol. 47, 597-626[CrossRef][Medline] [Order article via Infotrieve]
54. Nasser, W., and Reverchon, S. (2007) Anal. Bioanal. Chem. 387, 381-390[CrossRef][Medline] [Order article via Infotrieve]
55. Ramos, J. L., Martinez-Bueno, M., Molina-Henares, A. J., Teran, W., Watanabe, K., Zhang, X., Gallegos, M. T., Brennan, R., and Tobes, R. (2005) Microbiol. Mol. Biol. Rev. 69, 326-356[Abstract/Free Full Text]
56. Gorelik, M., Lunin, V. V., Skarina, T., and Savchenko, A. (2006) Protein Sci. 15, 1506-1511[CrossRef][Medline] [Order article via Infotrieve]
57. Brandts, J. F., and Lin, L. N. (1990) Biochemistry 29, 6927-6940[CrossRef][Medline] [Order article via Infotrieve]
58. Weber, P. C., Pantoliano, M. W., and Salemme, F. R. (1995) Acta Crystallogr. Sect. D 51, 590-596[CrossRef][Medline] [Order article via Infotrieve]
59. Gloss, L. M., and Matthews, C. R. (1997) Biochemistry 36, 5612-5623[CrossRef][Medline] [Order article via Infotrieve]
60. Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) J. Biomol. Screen 6, 429-440[Abstract/Free Full Text]
61. Carver, T. E., Bordeau, B., Cummings, M. D., Petrella, E. C., Pucci, M. J., Zawadzke, L. E., Dougherty, B. A., Tredup, J. A., Bryson, J. W., Yanchunas, J., Jr., Doyle, M. L., Witmer, M. R., Nelen, M. I., DesJarlais, R. L., Jaeger, E. P., Devine, H., Asel, E. D., Springer, B. A., Bone, R., Salemme, F. R., and Todd, M. J. (2005) J. Biol. Chem. 280, 11704-11712[Abstract/Free Full Text]
62. Gui, L., Sunnarborg, A., and LaPorte, D. C. (1996) J. Bacteriol. 178, 4704-4709[Abstract/Free Full Text]
63. Spiro, S., and Guest, J. R. (1991) Trends Biochem. Sci. 16, 310-314[CrossRef][Medline] [Order article via Infotrieve]
64. Resnik, E., Pan, B., Ramani, N., Freundlich, M., and LaPorte, D. C. (1996) J. Bacteriol. 178, 2715-2717[Abstract/Free Full Text]
65. Ramseier, T. M., Negre, D., Cortay, J. C., Scarabel, M., Cozzone, A. J., and Saier, M. H., Jr. (1993) J. Mol. Biol. 234, 28-44[CrossRef][Medline] [Order article via Infotrieve]
66. Cortay, J. C., Negre, D., Scarabel, M., Ramseier, T. M., Vartak, N. B., Reizer, J., Saier, M. H., Jr., and Cozzone, A. J. (1994) J. Biol. Chem. 269, 14885-14891[Abstract/Free Full Text]
67. Thorsness, P. E., and Koshland, D. E., Jr. (1987) J. Biol. Chem. 262, 10422-10425[Abstract/Free Full Text]
68. LaPorte, D. C., and Koshland, D. E., Jr. (1982) Nature 300, 458-460[CrossRef][Medline] [Order article via Infotrieve]
69. Kao, K. C., Tran, L. M., and Liao, J. C. (2005) J. Biol. Chem. 280, 36079-36087[Abstract/Free Full Text]
70. Krebs, A., and Bridger, W. A. (1980) Can. J. Biochem. 58, 309-318[Medline] [Order article via Infotrieve]
71. Phue, J. N., Noronha, S. B., Hattacharyya, R., Wolfe, A. J., and Shiloach, J. (2005) Biotechnol. Bioeng. 90, 805-820[CrossRef][Medline] [Order article via Infotrieve]
72. Ponce, E., Martinez, A., Bolivar, F., and Valle, F. (1998) Biotechnol. Bioeng. 58, 292-295[CrossRef][Medline] [Order article via Infotrieve]
73. Steiner, P., Fussenegger, M., Bailey, J. E., and Sauer, U. (1998) Gene (Amst.) 220, 31-38[CrossRef][Medline] [Order article via Infotrieve]
74. Ornston, L. N., and Ornston, M. K. (1969) J. Bacteriol. 98, 1098-1108[Abstract/Free Full Text]
75. Vogels, G. D., and Van der Drift, C. (1976) Bacteriol. Rev. 40, 403-468[Free Full Text]
76. Epelbaum, S., LaRossa, R. A., VanDyk, T. K., Elkayam, T., Chipman, D. M., and Barak, Z. (1998) J. Bacteriol. 180, 4056-4067[Abstract/Free Full Text]
77. LaPorte, D. C., Walsh, K., and Koshland, D. E., Jr. (1984) J. Biol. Chem. 259, 14068-14075[Abstract/Free Full Text]
78. Walsh, K., and Koshland, D. E., Jr. (1985) J. Biol. Chem. 260, 8430-8437[Abstract/Free Full Text]
79. el-Mansi, E. M., Nimmo, H. G., and Holms, W. H. (1986) J. Gen. Microbiol. 132, 797-806[Abstract/Free Full Text]
80. Rintoul, M. R., Cusa, E., Baldoma, L., Badia, J., Reitzer, L., and Aguilar, J. (2002) J. Mol. Biol. 324, 599-610[CrossRef][Medline] [Order article via Infotrieve]
81. Oh, M. K., Rohlin, L., Kao, K. C., and Liao, J. C. (2002) J. Biol. Chem. 277, 13175-13183[Abstract/Free Full Text]
82. Pellicer, M. T., Fernandez, C., Badia, J., Aguilar, J., Lin, E. C., and Baldoma, L. (1999) J. Biol. Chem. 274, 1745-1752[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. Hasegawa, H. Ogasawara, A. Kori, J. Teramoto, and A. Ishihama The transcription regulator AllR senses both allantoin and glyoxylate and controls a set of genes for degradation and reutilization of purines Microbiology, November 1, 2008; 154(11): 3366 - 3378. [Abstract] [Full Text] [PDF]

				T. Stratmann, S. Madhusudan, and K. Schnetz Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO J. Bacteriol., February 1, 2008; 190(3): 926 - 935. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 282/22/16476    most recent M610838200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lorca, G. L. Articles by Savchenko, A. Search for Related Content PubMed PubMed Citation Articles by Lorca, G. L. Articles by Savchenko, A. Social Bookmarking                      What's this?