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Identification of the Regulators Binding to the Upstream Region of glxR in Corynebacterium glutamicum
Identification of the Regulators Binding to the Upstream Region of glxR in Corynebacterium glutamicum
Journal of Microbiology and Biotechnology. 2015. Sep, 25(8): 1216-1226
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : March 02, 2015
  • Accepted : April 10, 2015
  • Published : September 28, 2015
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About the Authors
Bindu Subhadra
Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea
Durga Ray
Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea
Jong Yun Han
Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea
Kwang-Hee Bae
Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnolgy (KRIBB), Daejeon 305-806, Republic of Korea
Jung-Kee Lee
Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea
leejk@pcu.ac.kr

Abstract
GlxR is considered as a global transcriptional regulator controlling a large number of genes having broad physiological aspects in Corynebacterium glutamicum . However, the expression profile revealing the transcriptional control of glxR has not yet been studied in detail. DNA affinity chromatography experiments revealed the binding of transcriptional regulators SucR, RamB, GlxR, and a GntR-type protein (hereafter denoted as GntR3) to the upstream region of glxR . The binding of different regulators to the glxR promoter was confirmed by EMSA experiments. The expression of glxR was analyzed in detail under various carbon sources in the wild-type and different mutant strains. The sucR and gntR 3 deletion mutants showed decreased glxR promoter activities, when compared with the wild type, irrespective of the carbon sources. The promoter activity of glxR was derepressed in the ramB deletion mutant under all the tested carbon sources. These results indicate that SucR and GntR3 are acting as activators of GlxR, while RamB plays a repressor. As expected, the expression of glxR in the cyaB and glxR deletion mutants was derepressed under different media conditions, indicating that GlxR is autoregulated.
Keywords
Introduction
Corynebacterium glutamicum is a fast-growing, nonmotile, nonpathogenic, high-GC gram-positive soil organism of profound industrial importance, especially for the production of amino acids and nucleic acids [21] . Therefore, extensive genome-wide analyses and strain improvements have been conducted on this industrially important organism [22 , 42] . The genes involved in the carbon metabolism of this organism are controlled by multiple regulators, forming a complex regulatory network, indicating that the expression of genes in different metabolic pathways are finely tuned according to the available carbon sources. Among several regulators identified at the transcriptional level, a cAMPbinding transcriptional regulator of the CRP/FNR family, GlxR, is the potential candidate for the regulatory hub. GlxR shows homology to the well-studied global regulator CRP from E. coli [25] . The complex of cAMP and CRP plays an important role in the transcriptional activation of catabolic operons, and this cAMP-CRP complex is known to relieve glucose catabolite repression in E. coli [9] . The intracellular cAMP level of E. coli is high under glucose starvation via the activation of adenylate cyclase. The synthesis of cAMP occurs in response to the interaction between the PTS for glucose uptake and adenylate cyclase [31] . In E. coli , the cAMP level lowers in the presence of glucose, decreasing the CRP-cAMP complex in cells, leading to carbon catabolite repression (CCR). However, the molecular mechanism of CCR in C. glutamicum seems to be quite different from that of E. coli [3] .
The CRP homolog GlxR was first characterized as a regulator of the glyoxylate bypass genes aceA and aceB [25] . Recently, the global survey of GlxR binding sites in the genome of C. glutamicum using bioinformatic approach followed by in vitro verification by electrophoretic mobility shift assay (EMSA) and the genome-wide identification of in vivo binding sites of GlxR by ChIP-chip and ChIP-seq analyses revealed the GlxR regulon in detail [24 , 26 , 49] . GlxR acts as a transcriptional activator or repressor, controlling hundreds of genes involved in carbohydrate metabolism, cellular stress response, aromatic compound degradation, glutamate and nitrogen metabolism, fatty acid biosynthesis, respiration, and resuscitation [6 , 26 , 27 , 50] . However, the in vivo evaluation of the regulatory effect of GlxR is limited as the construction of a glxR deletion mutant is difficult and the mutants constructed show severe growth defect [25 , 30 , 34 , 48] . Previously, our group reported the GlxR-dependent repression of the glyoxylate bypass and the glutamate uptake system genes using a glxR deletion mutant [34] . In contrast to the case for E. coli , the cAMP level in C. glutamicum is higher when grown on glucose compared with that on acetate [11 , 25] . In addition, it has been noticed that the presence of glucose represses the utilization of ethanol and glutamate [1 , 28 , 29] . The derepression of the glutamate transporter gene in the presence of glucose, in the glxR deletion mutant, confirms the involvement of GlxR in CCR [34] . The repression of genes involved in the ethanol utilization pathway is relieved in glxR and cyaB deletion mutants, indicating the involvement of the GlxR-cAMP complex in the utilization of ethanol [44] . In addition, GlxR controls the TCA cycle genes, including sdhCAB and gltA , whose expression is also controlled by RamA [10 , 32] . Recently, it has been reported that the ramA gene in C. glutamicum is regulated by its own gene product, RamA, and the regulators GlxR and SugR [47] . The succinyl-CoA synthetase gene sucCD is under the control of the transcriptional regulator SucR and RamB, in addition to GlxR in C. glutamicum [12] . SucR is an autoregulator controlling the expression of alcohol dehydrogenase gene adhA of C. glutamicum [5] . GlxR controls the expression of the glyoxylate gene aceB , whose expression is also controlled by RamA and RamB [13 , 18 , 25] . The phosphotransferase system gene for the uptake of glucose, ptsG , is controlled by RamB and SugR, in addition to GlxR [15 , 27] . Therefore, GlxR is likely to co-ordinate different metabolic pathways in the central carbon metabolism in collaboration with other regulators such as SugR, SucR, RamA, and RamB.
cAMP is an important second messenger involved in several intracellular signaling pathways, and regulates many physiological processes such as CCR, sporulation, and the expression of virulence factors in many prokaryotic cells [8 , 35 , 45] . C. glutamicum ATCC 13032 genome has a single AC gene ( cyaB ) that encodes membrane-bound class III AC. The physiological functions of this adenylate cyclase as well as the cAMP regulatory network in C. glutamicum are not yet clearly known. In the case of CCR in C. glutamicum , cAMP binds to GlxR and alters the expression of different carbon metabolic genes depending on the presence of carbon source in the medium [25] . There are many questions to be addressed including the role of cAMP in the cAMPGlxR regulatory network as well the functioning of glxR in the cyaB mutant [11] . Even though GlxR is a global regulator, there is not much information on how the expression of glxR is regulated under different carbon sources and what the signaling system involved is. The current study focuses on the regulation of expression of glxR under different physiological conditions in C. glutamicum . The study identifies three regulators binding to the upstream region of glxR , and these regulators may have a possible role in the fine tuning of central carbon metabolism in C. glutamicum .
Materials and Methods
- Bacterial Strains, Plasmids, and Growth Conditions
The bacterial strains and plasmids used in this study are listed in Table 1 . The E. coli strain was grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, and 10 g/l N aCl) at 37℃, whereas C. glutamicum ATCC 13032 was grown at 30℃ in minimal medium [11] . As the carbon source, glucose or acetate was added to the media at a level of 1% (w/v). BHIS (BHI Sorbitol) medium composed of 3.7% (w/v) Brain Heart Infusion (BHI) medium and 9.1% sorbitol was used for the preparation of competent cells of C. glutamicum [14] . C. glutamicum strains used in this work were precultured in BHI medium overnight in a rotary shaker at 200 rpm. The cells were harvested and washed with a buffer containing 5 g/l (NH 4 ) 2 SO 4 , 5 g/l urea, 0.5 g/l KH 2 PO 4 , 0.5 g/l K 2 HPO 4 , and 21 g/l MOPS. These cells were used to inoculate main cultures of minimal medium containing appropriate carbon sources. When appropriate, kanamycin was added at concentrations of 10 µg/ml. The oligonucleotides used in this study were purchased from Cosmogenetech Co. (Korea).
Bacterial strains and plasmids used in this study.
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Bacterial strains and plasmids used in this study.
- DNA Treatment and Analysis
Standard molecular cloning procedures were followed in this study [36] . The chromosomal DNA from the C. glutamicum cells was isolated using a genomic DNA purification kit (SolGent, Korea). The plasmids were introduced into C. glutamicum by either electroporation [46] or conjugation [37] . The restriction enzymes were obtained from New England Biolabs (Frankfurt, Germany). The PCR products were purified using the PCR purification kit from Qiagen (Hilden, Germany). The DNA fragments from agarose gel were eluted using a Qiagen gel extraction kit (Hilden, Germany). The nucleotide sequence of all the cloned DNA fragments was determined by sequencing (Cosmogenetech Co. Korea).
- Cloning of glxR Promoter Fragment
The promoter probe transcriptional fusion plasmids were constructed using the vector pSK1CAT [33] , which harbors a promoterless cat (chloramphenicol acetyl transferase) gene encoding CAT enzyme. The promoter fragment of the glxR gene was generated by PCR using primers tagged with Bam HI and cloned in front of the cat gene of pSK1CAT. The promoter fragment containing 520 bp upstream of the translational start site (400 bp from the transcriptional start site) and 14 bp in the glxR gene was amplified to construct the recombinant plasmid, pglxR . The primers used to amplify the promoter sequences are listed in Table S1. All the constructs were checked by sequencing.
- Construction ofsucRandgntR3Deletion Mutants
To construct the deletion mutant of sucR , two DNA fragments were generated by PCR using the following two oligonucleotide pairs: suc dA/ suc dB and suc dC/ suc dD. Fragment 1 covers 375 bp upstream of the sucR gene and 91 bp of the 5’ end of sucR , and fragment 2 covers 254 bp of the 3’ end of the sucR gene and 206 bp downstream of the sucR gene. The two fragments were annealed together yielding a DNA fragment having deletion of 504 bp in the sucR gene. The construct was ligated into pK19 mobsacB to obtain pKSCD. For the construction of gntR3 deletion mutant, two oligonucleotide pairs ( gnt dA/ gnt dB and gnt dC/ gnt dD) were used, generating two DNA fragments. The two DNA fragments were annealed together yielding a DNA fragment having deletion of 211 bp in the gntR3 gene. The first fragment covers 78 bp upstream of the gntR3 gene and 219 bp of the 5’ end of gntR3 , and the second fragment covers 293 bp of the 3’ end of gntR3 and 6 bp downstream of the gntR3 gene. The two fragments were annealed together, resulting in a DNA fragment having deletion of 211 bp in the gntR3 gene. This construct was cloned into pK19 mobsacB yielding pKGCD. The plasmids pKSCD and pKGCD were transformed into C. glutamicum by conjugation. The integration of pKSCD or pKGCD into the chromosome was confirmed by selection of colonies on a BHI plate containing kanamycin. The chromosomal deletion within the sucR or gntR3 gene was obtained by double crossover according to a protocol described by Schäfer et al . [38] . The deletion in the resultant strain was confirmed by PCR and sequencing.
- DNA Affinity Purification
The enrichment of proteins interacting with the upstream region of glxR was carried out as described previously [4] . Primers glxR F and glxR R generated a 451 bp DNA fragment, including 420 bp upstream of the glxR gene and 31 bp in the glxR gene. The primer glxR F was tagged with biotin (Cosmogenetech Co., Korea), and 100 pmol of biotin-labeled PCR product was immobilized with 3 mg of streptavidin-coated magnetic beads, Dynabeads M280-strepavidin (Invitrogen) and uncoupled DNA was removed by magnetic separation following the manufacturer’s protocol. Before incubation with C. glutamicum crude extracts, the coupled Dynabeads were equilibrated with 300 µl of binding buffer (20 mM Tris–HCl (pH 7.8), 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 10% (v/v) glycerol, and 0.05% (v/v) Triton X-100) for 10 min.
C. glutamicum cultures were grown in glucose or acetate minimal medium, harvested at an OD 600 of 4.5, and washed with 1 volume of 20 mM Tris–HCl buffer (pH 7.6), followed by resuspension in 5 ml of disruption buffer (50 mM Tris–HCl (pH 7.6), 1 mM DTT, 10 mM MgCl 2 , 1 mM EDTA, and 10% (v/v) glycerol). Aliquots (1 ml) of the cells were dirupted using a MiniBeadbeater (Biospec Products, USA) with intermittent cooling on ice for 1 min. After centrifugation, the supernatant was concentrated by incubation on solid polyethylene glycol 20000 in Visking dialysis tubes (Serva, Heidelberg, Germany) with a pore size of 25 Å. The crude extract was incubated with the equilibrated and coupled Dynabeads for 2 h at room temperature while being shaken at 350 rpm using a SLRM-2M Intelli Mixer (My Lab, Korea). The unbound proteins were removed by magnetic separation with a magnet particle concentrator (DynaI, Invitrogen, Hamburg, Germany), followed by two washing steps with 300 µl of binding buffer. The bound proteins were eluted with 15 µl of each elution buffer (binding buffer containing 0.2, 0.3, 0.5, and 1 M N aCl) and magnetic separation was carried out after each step. The eluted fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gels were subsequently stained with Coomassie Brilliant Blue. The selected protein bands were excised from the gel and subjected to tryptic digestion.
- Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS)
For peptide mass fingerprinting, the protein bands of interest were excised from the gel and in-gel tryptic digestion was carried out as described earlier [18] . The mass spectrometric analyses were done using a PerSeptive Biosystems MALDI-TOF Voyager DE-RP mass spectrometer (Framingham, MA, USA) operated in the delayed extraction and reflector mode at Korea Research Institute of Bioscience and Biotechnology, Korea.
- Overproduction and Purification of SucR, RamB, and GntR3
For the overexpression of hexahistidyl (His)-tagged SucR and GntR3 fusion proteins, the sucR and gntR3 genes of C. glutamicum were amplified by PCR using the primers pET- sucR F/pET- sucR R and pET- gntR3 F/pET- gntR3 R respectively. The PCR products were digested with Nde I and Xho I (for SucR) or Nde I and Hin dIII (for GntR3) and ligated into the corresponding restriction enzyme-digested pET28a, and transformed into E. coli BL21 (DE3). The E. coli BL21 (DE3) cells harboring the recombinant plasmids pET_ sucR , pET_ ramB [18] , or pET_ gntR3 were grown at 37℃ until the OD 600 reached 0.5 to 0.7, before 1 mM IPTG was added. After cultivation for another 3 h at 30℃, cells were harvested, resuspended in lysis buffer, and disrupted mechanically using a Mini-Beadbeater (Biospec Products). The purification of fusion proteins was carried out with Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography according to the protocol given by Qiagen (Hilden, Germany). For desalting the SucR fusion protein, the purified protein was dialyzed overnight against 30% (w/v) glycerol in water. The RamB protein was desalted against a buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 10% (w/v) glycerol. The concentration of glycerol in the buffer was adjusted to 30% after desalting, before storing at -20℃. The purified GntR3 fusion protein was dialyzed overnight against a buffer containing 30 mM Tris-HCl (pH 7.5), 30 mM NaCl, and 30% (w/v) glycerol in water. All the proteins were then used directly for the promoter binding assay.
- DNA Binding Assays with His-Tagged Proteins
Electrophoretic mobility shift assay was carried out to check the binding of SucR, RamB, and GntR3 proteins to the promoter region of glxR . The promoter fragments that were generated by PCR are given in Fig. 2 A. For the binding assays with SucR protein, the PCR-generated DNA fragments were end-labeled with γ - 32 P. Various amounts of SucR protein (0-1 µg) in 20 µl of the reaction buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10% (w/v) glycerol, and 1 µg Poly[d(I-C)] were incubated with 50 ng of the labeled DNA fragments for 30 min at room temperature. A control was also taken with bovine serum albumin (BSA) instead of the purified protein. Subsequently, the reaction mixture was separated on a 4% prerun acrylamide gel (prerunning for 30 min at 100 V) in 0.5× TBE buffer (100 V for 1 h at RT). The gels were removed, dried using a gel dryer, and exposed to a plate in a BAS cassette for 40 min, and imaging was done in a phosphorimager (Bio-Rad, USA). In the case of RamB and GntR3 proteins, 300 ng of the PCRgenerated probe was incubated with RamB and GntR3 proteins ranging from 0-1 µg, in the same reaction buffer mentioned above, for 30 min at room temperature. A control was also taken with BSA instead of the purified protein. The reaction mixture was subsequently separated on a 4% acrylamide gel in 0.5× TBE buffer and stained with ethidium bromide.
- Enzyme Assay
To determine the CAT activity, the strains were cultivated in LB or minimal medium containing either glucose or acetate (1% (w/v) each). The cells were harvested in the exponential phase, washed twice in 50 mM Tris-HCl (pH 7.0), and resuspended in the same buffer containing 10 mM MgCl 2 , 1 mM EDTA, and 30% (v/v) glycerol. The cell suspension was mixed with glass beads (BioSpec Products, Inc., USA), subjected to mechanical disruption, and centrifuged, and the supernatant was used for the assay. The protein concentration was measured using the Bradford method (Bio-Rad, Hercules, USA) using BSA as the standard. CAT activity was assayed as described by Shaw [40] with modifications outlined by Schreiner et al . [39] . Briefly, the assay mixture (1 ml) contained 100 mM Tris-HCl (pH 7.8), 0.1 mM acetyl-CoA, 1 mM 5,5-dithio bis -2-nitrobenzoic acid, 0.25 mM chloramphenicol, and crude extract. The formation of free 5-thio-2-nitrobenzoate was measured photometrically at 412 nm. One unit of CAT activity corresponds to 1 µmol of chloramphenicol acetylated per minute at 37℃.
Results
- Identification of Transcriptional Regulators Binding to the Upstream Region ofglxR
To identify the possible transcriptional regulators involved in the control of the glxR gene, DNA affinity purification was performed to enrich the regulatory proteins binding specifically to the upstream region of glxR . A 451 bp promoter fragment of the glxR gene was amplified by PCR and was linked to streptavidin-coated magnetic beads and incubated with crude extracts of C. glutamicum grown on minimal medium containing glucose or acetate. After the removal of the unbound proteins (with low-salt washing steps), the specifically bound proteins were eluted with buffers containing high salt concentrations. The eluted proteins were analyzed by SDS-PAGE. As shown in Fig. 1 , several proteins were highly enriched from the extracts of both acetate- and glucose-grown cells, and the protein bands of interest were excised from the gel and subjected to MALDI-TOF/MS and peptide mass fingerprint analysis. The comparison of the data obtained by peptide mass fingerprint analysis with the non-redundant NCBI database resulted in the identification of various protein bands. The majority of the bands were corresponding to ribosome and DNA-binding proteins, including DNA polymerase, exonucleases, stress-sensitive restriction system proteins, ribonuclease E, transcription termination factors, and polynucleotide phosphorylase. Among the four regulator proteins selected, the prominent band eluted with both the acetate- and glucose-grown cells was identified as RamB (cg0444). The two other regulator proteins identified with glucose-grown cells were GlxR (cg0350) and SucR (cg0146). Finally, the protein band of about 26 kDa from the acetategrown cells was determined as GntR (cg2544). It was obvious that this protein was eluted only with a high concentration of NaCl and there is not much information on this GntR-type protein (designated as GntR3), encoded by cg2544 in C. glutamicum ( http://www.coryneregnet.de/ ). The gene encoding GntR3 is shown as a repressor for the operon that includes genes for probable glycolate oxidase (cg2543), predicted permease (cg2542), and putative protein (cg2540).
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SDS-PAGE of C. glutamicum proteins eluted from a DNA affinity chromatography experiment using glxR promoter probe. (A) Extracts from cells grown on glucose and (B) on acetate. The lanes 1, 2, 3, and 4 are elutions with a buffer containing 0.2, 0.3, 0.5, and 1 M NaCl, respectively. The protein bands were identified by MALDI-TOF/MS and peptide mass fingerprint analysis. The molecular mass standard (lane M) is given to the left in kDa.
- Binding of Purified SucR, RamB, and GntR3 to the Upstream Region ofglxR
Bioinformatic analysis revealed the presence of SucR, RamB, and GntR3 binding sites in the upstream region of glxR . In order to prove the direct binding of the SucR, RamB, and GntR3 proteins to the promoter region, hexahistidyl fusion proteins were overexpressed in E. coli BL21(DE3) and purified by nickel affinity chromatography. The glxR promoter fragments glxR p1 to glxR p7 ( Fig. 2 A) were amplified by PCR and incubated with varying amounts of SucR proteins. Promoter fragments glxR p1 to glxR p4 were used for the binding assays with RamB protein. The binding assays with GntR3 protein was carried out using the promoter fragments glxR p1 to glxR p3 . In order to find out the specific binding motifs, EMSAs were carried out with different mutated oligonucleotides. The EMSA results show that the SucR binds to the promoter fragments glxR p1 , glxR p3 to glxR p6 . The whole promoter fragment used for EMSA was denoted as glxR p1 . The EMSA data imply that SucR binds to the glxR promoter region to at least four different motifs ( Figs. 2 A- 2 D and 3 ). The SucR motifs were located at positions -91 to -100 ( CT GC CA G CAA ), -29 to -37 ( CTAACAT GG), +54 to +63 ( CTAACA C C CC), and +59 to +68 ( AC C C C AGG T G ) relative to the transcriptional start site [23] , according to the consensus sequences reported earlier [5 , 12] . The consensus bases matching with those in the already reported binding motifs are underlined. The sequence analysis indicated that at least 7 out of 10 bases are shared with the reported binding motifs. The RamB protein was binding to the position at -203 to -215 (T AAAGTT A GCAAA ) with respect to the transcriptional start site ( Figs. 2 A, 2 E, and 3 ). The underlined sequences are the consensus binding motifs matching with the already reported RamB binding motifs ( http://www.coryneregnet.de ). The binding assays with GntR3 protein showed that GntR3 binds to the glxR promoter region at -49 to -65 ( TAAAGT G T G AC A TTT ) with respect to the transcriptional start site ( Figs. 2 A, 2 F, and 3 ), according to the consensus sequences (underlined) reported earlier [16] . The sequence analysis indicated that at least 12 out of 15 bases are shared with the reported binding motif. The results of the DNA affinity chromatography together with the EMSA results indicate that SucR, RamB, and GntR3 proteins specifically bind to the glxR promoter and thus suggest an involvement in glxR expression control.
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Binding of purified SucR, RamB, and GntR3 to the upstream region of glxR. (A) The promoter fragments used for EMSA. (B) EMSA with promoter fragments glxRp1, glxRp4a and 0, 0.25, 0.5, and 1 µg of SucR protein (lanes 1-4, respectively). Lane 5 shows a control with 1 µg of bovine serum albumin (BSA) instead of SucR. (C) Representative binding assays of glxRp4b to glxRp7 with 0, 0.5, or 1 µg (lanes 1-3, respectively) of SucR protein. Lane 4 is a control with 1 µg of BSA instead of SucR. (D) Binding assays with mutated promoter fragments and 0 to 1 µg (lanes 1-2, respectively) of SucR protein. (E) EMSAs using RamB protein and glxR promoter fragments. Promoter fragments, including the mutated oligonucleotides, were mixed with 0, 0.5, or 1 µg (lanes 1-3, respectively) of RamB protein. Lane 4 shows a control with 1 µg of BSA instead of RamB. (F) Representative binding assays of glxR promoter region with GntR3 protein. The GntR3 protein of varying concentrations of 0, 0.2, 0.3, 0.5, 0.7, or 1 µg (lanes 1-6, respectively) were mixed with promoter fragments including the mutated oligonucleotides. Binding of the respective fragments to SucR, RamB, and GntR3 is indicated on the right side, where +, -, and n.d. indicate binding, no binding, and not determined, respectively.
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Analysis of the upstream region of the glxR gene. (A) Schematic representation and (B) nucleotide sequence of the upstream region of the glxR gene. The gene including the promoter region with known or presumed binding sites for the transcriptional regulators RamB, SucR, GlxR, and GntR3 is shown. The numbers beneath the indicated binding sites of regulators indicate the position of the first nucleotide in the binding motif relative to the transcriptional start site.
- Inactivation ofsucR,ramB, andgntR3 inC. glutamicumand Effect on Expression ofglxR
The binding studies revealed an interaction of SucR, RamB, and GntR3 with the glxR promoter region. To functionally analyze the effect of these regulator proteins on glxR expression, deletion mutants were constructed and promoter fusion experiments were carried out. For this purpose, the wild-type and mutant strains were transformed with the plasmid p glxR . The recombinants were grown in minimal medium containing glucose or acetate (1% each) and specific CAT activities were determined. As shown in Fig. 4 , the glxR promoter activity was high in glucose medium compared with acetate. The specific CAT activity (promoter activity) in ∆ sucR (p glxR ) was repressed compared with that in the wild type (p glxR ). This result shows that SucR is an activator of glxR irrespective of the carbon source. The specific CAT activities were derepressed in glucose and acetate media in ∆ ramB , indicating that RamB has a repressive effect on the expression of glxR ( Fig. 4 ). However, it is better to conclude that these two regulators, SucR and RamB, might be needed for the fine tuning of the expression of glxR , as the difference in activity in the deletion mutants compared with the wild type was not that significant. In the gntR3 deletion mutant, the glxR promoter activity was decreased in both glucose and acetate media compared with that of wild type, indicating that GntR3 is an activator of glxR .
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Specific CAT activities of C. glutamicum wild type (WT) and mutant strains. The WT, sucR, ramB and gntR3 deletion mutants (∆sucR, ∆ramB, ∆gntR3) were harboring the glxR promoter fragment in the transcriptional fusion vector pSK1CAT. The cells were harvested in the early exponential phase, after growing in minimal medium containing 1% glucose or 1% acetate. All the values are from at least three independent cultivations and two determinations per experiment with standard deviations.
- Effect ofcyaBInactivation onglxRPromoter Activity
In vitro studies have reported the binding of GlxR on the promoter region of glxR , acting as a repressor of its own gene [23] . To find out the physiological relevance of binding of GlxR to the promoter region of glxR , specific CAT activities were determined in the wild type, cyaB deletion mutant (∆ cyaB ), and glxR deletion mutant (∆ glxR ). It has been reported by in vitro experiments that, in most of the cases, cAMP is needed for the binding of GlxR to the promoter region of target genes. The strains were grown in LB containing glucose or acetate (1% each) and the cells were harvested in the early exponential phase and specific CAT activity was measured. As shown in Table 2 , the promoter activity was derepressed in ∆ cyaB and ∆ glxR , in both the carbon sources tested. This result shows that GlxR is an autoregulator, repressing the expression of its own gene, as reported earlier [23] .
Specific CAT activity ofC. glutamicumwild type (WT),cyaBdeletion mutant (∆cyaB), andglxRdeletion mutant (∆glxR) harboring theglxRpromoter fragment in the transcriptional fusion vector pSK1CAT.
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aThe strains were cultivated in LB medium containing glucose or acetate (1% each) and the enzyme activity was assayed in cell extracts from the early exponential phase. All the values are from at least three independent cultivations and two determinations per experiment with standard deviations.
Discussion
Even though GlxR is a global regulator controlling the expression of a large number of genes involved in the central carbon metabolism of C. glutamicum [26 , 27 , 49] , there is not much information on the transcriptional regulation of the glxR gene under different carbon sources and the signaling system involved. In this study, DNA affinity experiments revealed that the regulators SucR, RamB, GlxR, and GntR3 bind to the upstream region of glxR . The GntR regulator is found in diverse bacterial groups and was characterized first in Bacillus subtilis [17] . In C. glutamicum , 11 genes encode GntR-type regulators, and among them GntR1 and GntR2 co-coordinately control gluconate catabolism and glucose uptake [16] . However, there is not much information known about the GntR (GntR3) that binds to the promoter region of glxR . GntR3 is a 240-amino-acid protein having a molecular mass of 26.95 kDa and it possesses a conserved N-terminal helix-turn-helix DNA binding domain and a C-terminal effector binding domain. The data from the current study reveals that GntR3 is an activator of glxR . In accordance with previous studies ( http://www.coryneregnet.de/ ), we noticed that GntR3 binds to the promoter region of glcD , encoding probable glycolate oxidase (FAD-linked subunit) (data not shown). As mentioned above, GntR1 and GntR2 co-coordinately control ptsG in C. glutamicum . However, DNA binding assay showed that GntR3 does not bind to the promoter region of ptsG (data not shown). Moreover, further studies are needed in order to understand the functional aspects of this regulator in detail.
SucR was first reported as a transcriptional repressor, controlling the expression of the sucCD gene encoding succinyl-CoA synthetase of C. glutamicum [12] . In addition, SucR acts as a repressor of the alcohol dehydrogenase gene adhA and also controls the expression of its own gene [5] . The transcriptional fusion experiments revealed that SucR acts as an activator of gene glxR irrespective of the carbon source in the medium. It has been noticed that expression of SucR is high in glucose medium compared with that in acetate [12] . Another study suggested a strong derepression of gene sucR in the presence of ethanol and it was speculated that the expression of the ethanol catabolic gene, ald , synthesizing acetaldehyde dehydrogenase, is derepressed in ∆ sucR [5] . However, they could not observe the binding of SucR protein to the promoter region of ald , indicating an indirect effect of SucR on ald expression. It is known that GlxR acts as repressor of ald in C. glutamicum [26 , 44] . The results from our study reveal that SucR acts as an activator of the glxR gene, and this suggests the GlxRmediated control of SucR on ald expression.
RamB was first reported as a repressor protein regulating the genes aceA and aceB involved in the acetate metabolism of C. glutamicum [18] . It plays a role in the transcriptional regulation of the gene mctC , involved in acetate/propionate/ pyruvate transport, acetate and ethanol catabolic genes ( ack, pta, adhA, ald encoding acetate kinase, phosphotransacetylase, alcohol dehydrogenase, and acetataldehyde dehydrogenase, respectively), and aceE encoding E1p subunit of the pyruvate dehydrogenase complex involved in the TCA cycle [2 , 4 , 7 , 18] . Transcriptional fusion experiments with the glxR promoter shows that RamB acts as a repressor of glxR in glucose and acetate media, thereby removing the repressive effect of GlxR over glycolytic and acetate metabolic genes.
In the current study, we have identified four regulators, SucR, RamB, GlxR, and GntR3, which bind to the promoter region of glxR . In addition, the data from the current study give insights into the fine tuning of the TCA cycle and the glyoxylate pathway by these regulators. It has been reported that the aceA gene, encoding isocitrate lyase, the first enzyme in the glyoxylate cycle, is under the control of the regulators GlxR and RamB [18 , 25 , 34] . In addition, SucR, RamB, and GlxR act as repressors of the sucCD gene, encoding succinyl Co-A synthetase, which converts succinate to succinyl-CoA [12 , 18 , 19 , 27] . The current study points out the involvement of regulators SucR, RamB, GlxR, and GntR3 in the fine tuning of the expression of glxR . According to the availability of different carbon sources in the medium, these regulators might play an important role in coordinating the central carbon metabolism of C. glutamicum .
Acknowledgements
This research was supported by the Basic Science Research Programme through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (Grant No. 2012006648). I would like to extend my sincere gratitude to Prof. Kyu-Ho Lee and Jeong-A Kim of the Department of Life Science, Sogang University, Korea for their timely help in conducting the EMSA studies and Prof. Bernhard J. Eikmanns of the Institute of Microbiology and Biotechnology, University of Ulm, Germany for kindly providing theramBdeletion mutant.
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