argr–promoter interactions in corynebacterium glutamicum arginine biosynthesis

Biotechnology andApplied Biochemistry

ArgR–promoter interactions inCorynebacterium glutamicumarginine biosynthesis

Grant Theron and Sharon J. Reid∗

Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa

Abstract.Arginine biosynthesis in Corynebacterium glutamicum iscurrently poorly understood. A better understanding of itsregulation will aid in the engineering of overproducing strains.A transcriptional analysis of the argCJBDFRGH genes andconstruction of argR deletion mutant confirmed the role ofArgR as a negative regulator of arginine biosynthesis.Increasing the copy number of the argC promoter regioncaused arginine levels to increase twofold. Electrophoreticmobility shift assays using recombinant ArgR revealed in vitrobinding with dissociation constants between 200 and 250 nMto the putative promoter regions of argC and carAB. In contrast,ArgR did not bind in vitro to the putative argG promoter region.

Binding of ArgR to the argC and carA promoter regions wasprevented by double-stranded competitor oligonucleotidescontaining motifs resembling the universal ARG box consensussequence (two in the case of the argC promoter region and onein the case of the carA promoter region). A single ARG box wasidentified in the carA promoter region. The consensussequence for the three C. glutamicum ARG box motifs was5′-HMT GMA TSW ADW WTW TDY-3′ and the core sequence(underlined) is well conserved throughout the C. glutamicumgenome and located preceding several putative ArgRtargets.

C© 2011 International Union of Biochemistry and Molecular Biology, Inc.Volume 58, Number 2, March/April 2011, Pages 119–127 •E-mail: [email protected]

Keywords: Corynebacterium glutamicum, arginine biosynthesis,ArgR, arg box-binding motifs

1. IntroductionCorynebacterium glutamicum has been widely used for the com-mercial production of various amino acids, including arginineand histidine [1]. Although considered to be a nonessentialamino acid, arginine is widely used in human healthcare andhas considerable commercial potential [2]. Although the reg-ulation of many amino acid biosynthetic pathways has beencomprehensively studied in C. glutamicum, less is known aboutthe complex regulation of the arginine pathway. Reverse engi-neering of the classical high-production strains has providednew opportunities for the rational construction of improved, ro-bust amino acid production strains [3–5]. A better understandingof the genetic regulation of the arginine biosynthetic genes inC. glutamicum can allow for the creation of rationally mutatedstrains capable of overproducing arginine.

Abbreviations: ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobilityshift assay; YT, yeast–tryptone; MM, minimal medium; PCR, polymerase chain reaction;PMSF, phenylmethanesulphonylfluoride; RT, reverse transcriptase.∗Address for correspondence: Professor S. J. Reid, PhD, Department of Molecular and CellBiology, University of Cape Town, Private Bag, Rondebosch 7701, Cape Town, SouthAfrica. Tel.: + 27 21 6503257; Fax: + 27 21 6897573; e-mail: [email protected] article contains supplementary material available from the authors upon request orvia the Internet at http://wileyonlinelibrary.com.Received 1 December 2010; accepted 21 January 2011DOI: 10.1002/bab.15Published online 19 April 2011 in Wiley Online Library(wileyonlinelibrary.com)

The biosynthesis of arginine is similar in most microorgan-isms, wherein glutamate is converted into citrulline via ornithineand various acetylated intermediates and, finally, into arginine.It has been extensively described elsewhere [6].

The genetic organization of the arginine regulon can takethe form of multiple operons, as in the majority of Gram-positivebacteria, wherein the genes responsible for the biosynthesisof citrulline are clustered in an argCJBDFR operon [7],[8]. Incontrast, most Gram-negative bacteria have these genes dis-tributed throughout the genome [9]. In C. glutamicum, the argi-nine biosynthetic genes, composed of argCJBDFGH, are clus-tered on the genome, and the carAB cluster, encoding for thebiosynthesis of carbamoyl phosphate, is not linked to the argi-nine operon [10]. Sakanyan et al. [11] showed that argC and argJwere transcribed together from a putative promoter region pre-ceding argC, and, in a study on the putative global regulator,FarR, the arginine genes were proposed to be transcribed astwo (argCJBDFR and argGH) transcripts [12].

Despite significant differences in their organization, theregulation of the arginine biosynthetic genes is similar acrossa range of archaea, Gram-positive, and Gram-negative bacteria[13–15]. Detailed studies in Escherichia coli and Bacillus subtilishave shown that regulation is exerted by the binding of the argi-nine repressor, ArgR, in conjunction with arginine, to specificoperator sites known as ARG boxes [13]. ARG boxes are gener-ally found in pairs and consist of an imperfect 18 bp palindromicsequence overlapping the promoter region of the target gene

119

and separated by 2–3 bp [16]. When ArgR binds to an ARG box,it sterically hinders transcription initiation by RNA polymerase,resulting in repression of the target genes. Recently, evidenceof a broader regulatory role for ArgR has emerged in a varietyof bacteria. The Lactococcus lactis and E. coli ArgR proteins,for example, bind in vitro to the promoters of genes encodingamino acid export systems [17],[18], and in E. coli, ArgR acts asa repressor for the glutamate synthase operon gltBD [19]. Leeet al. [20] have demonstrated, using chromatin immunoprecipi-tation (ChIP) assays, that the C. glutamicum ArgR binds in vitroto the region upstream of argC, argB, argF, and argG, althoughthe interaction was weak with respect to the latter two genes.Interestingly, this binding was unaffected by the presence or ab-sence of arginine, and proline was shown to reduce the bindingof ArgR to the argB region.

In this study, we sought to elucidate the structure ofthe promoter region upstream of the first gene of the argininebiosynthetic operon. We also confirm that ArgR downregulatesthe C. glutamicum arginine operon by binding to DNA motifswithin this region. Furthermore, we characterized the effect ofhigh copy numbers of the argC promoter region on transcrip-tion. In order to further characterize the DNA motifs to whichArgR binds, we studied the in vitro DNA-binding activity ofhexahistidine-tagged ArgR to the argC and carAB promoters us-ing electrophoretic mobility shift assays (EMSAs). This allowedus to arrive at a putative consensus C. glutamicum ARG box se-quence that was subsequently used for the in silico screeningof the C. glutamium genome to reveal potential targets of ArgRbinding.

2. Materials and methods2.1. Bacterial strains and mediaEscherichia coli strains (Table 1) were grown in Luria–Bertanimedium or 2 × yeast–tryptone (2 × YT) medium accordingto standard methods [21]. All C. glutamicum media containednalidixic acid (30 μg mL−1) and were incubated for growth at30◦C. CGXII minimal medium (MM) was prepared according toKeilhauer et al. [22]. Where appropriate, ampicillin (100μg mL−1)or kanamycin (30 μg mL−1) was used.

2.2. DNA manipulation and amplificationDNA was prepared and isolated according to standard methods[21],[28]. Polymerase chain reactions (PCRs) were performedwith 2 × KAPATaq ReadyMix [10 mM Tris–HCl (pH 8.6), 50 mMKCl, 0.05% (v/v) Tween 20, 0.5 mM dithiothreitol (DTT), 5% (v/v)glycerol, 1.5 mM MgCl2, 1.25 U KAPATaq, and 0.2 mM of each de-oxyribonucleotide triphosphate (dNTP) (Kapa Biosystems, CapeTown, South Africa), using the conditions and primers describedin Table S1.

2.3. Construction of strains and plasmidsA C. glutamicum argR deletion mutant was constructed using a�argR suicide plasmid. The knockout plasmid was constructedas follows: a 1.8 kb region of the C. glutamicum genome span-ning the argR gene was amplified using the argR–MS primer pair

Table 1Strains and plasmids

Strains or plasmids Description Source

E. coli DH5α F− �80dlacZ �M15�(lacZYA-argF)U169 deoRrecA1 endA1 hsdR17 (rK−,mK+ ) phoA supE44 λ− thi-1gyrA96 relA1

ref. [23]

E. coli BL21 (DE3) ompT hsdSB(rB−, mB

−) gal dcm(DE3)

ref. [24]

C. glutamicum ATCC13032

Type strain; nalidixic acidR ref. [25]

CG�argR C. glutamicum ATCC 13032�argR mutant

This study

pK19mobsacB sacB lacZα oriE.coli oriT);kanamycinR

ref. [26]

pK19mobsacB::�argR

pK19mobsacB containing anin-frame deletion of argR

This study

pEKEX2 E. coli–C. glutamicum shuttlevector containing anIPTG-inducible tac promoter(Ptac lacIq oriE.coli oriCglu)kanamycinR

ref. [27]

pArgC-P pEKEX2 derivative containingthe CAF21404–argCintergenic DNA and flankingregions

This study

pET22b( + ) IPTG-inducible E. coli proteinexpression vector; encodes aC-terminal hexahistadyl tag(PT7 lacIq oriE.coli); ampicillinR

Novagen

pArgR–His ArgRHis expression vector This study

(Table S1). The PCR product was digested with EcoRV and BsrBI,and the three resulting fragments were separated by agarosegel electrophoresis. The 5′- and 3′-fragments were excised andligated together, resulting in an in-frame 318 bp deletion withinargR. This fragment was digested with HindIII and BamHI and lig-ated into pK19mobsacB [26] to give pK19mobsacB::�argR. Thisconstruct was electroporated into C. glutamicum [29]. Doublecross-over (DCO) mutants were selected according to Eggelingand Reyes [28]. The mutant, CG�argR, was confirmed by PCRand Southern blotting, using a 522-bp digoxigenin (DIG)-labeledDNA probe internal to argR according to the DIG ApplicationManual (Roche, Indianapolis, IN, USA).

pArgC-P was constructed by ligating a 491-bp fragmentcontaining the 115-bp intergenic region preceding the argC gene,amplified via PCR using the CAF21404–argC fragment primers,into pTZ57R/T (Fermentas, Glen Burnie, MD, USA). The 491 bpfragment was excised with BamHI and EcoRI and cloned into theC. glutamicum plasmid, pEKEX2 [27].

To create a construct for the overexpression of ArgR (witha C-terminal hexahistidine fusion), argR was amplified by PCRusing the argR–His primers. The fragment was ligated intothe BamHI and EcoRI sites of pET22b( + ) (Novagen, Madison,WI, USA) and transformed into E. coli BL21 (DE3) cells to givepArgR–His.

120 Biotechnology and Applied Biochemistry

2.4. RNA preparation, reverse-transcriptase PCR,and primer extensionTotal RNA from mid-log phase (optical density at wavelength of600 nm (OD600) ≈ 7) C. glutamicum cells grown in MM brothwas extracted using an RNeasy Mini Kit (Qiagen) with the fol-lowing modifications: cells were harvested and incubated in100 μL of Tris-EDTA (TE) buffer (10 mM Tris–Cl, 1 mM EDTA,pH 8.0) containing lysozyme (Sigma–Aldrich) (15 mg mL−1) andproteinase K (Fermentas) (1 mg mL−1) for 10 Min. Buffer RLT(Qiagen, Valencia, CA, USA) (350 μL), containing 1% (v/v)β-mercaptoethanol (Sigma–Aldrich), was added and the cellswere mechanically disrupted using a Mini-Beadbeater (BioSpec,Bartlesville, OK, USA). RNA was extracted according to the man-ufacturer’s instructions, followed by a 16 H DNAse digestionat 37◦C using RNAse-free DNAse I (Roche). RNA samples werestored in diethylpyrocarbonate-treated dH2O at −80◦C.

Reverse transcriptase (RT) reactions were performed us-ing the FirstStrand cDNA Synthesis Kit (Fermentas) with 1 μgpurified RNA. Complementary DNA (cDNA) fragments were am-plified via PCR KAPATaq ReadyMix. The oligonucleotide primersused for each RT-PCR are listed in Table S1.

Primer extension was performed as described by Pateket al. [30] using a 5′-Cy5-labeled primer (5′-GGC TCC TGC GATTGC AAC CT-3′) (University of Cape Town, South Africa). The RTreaction was performed using the Fermentas RT kit with 1.5 Mbetaine [31] and products were analyzed using an ALFexpressAutomated DNA Sequencer (Pharmacia, Freiburg, Germany) asrecommended by the manufacturer.

2.5. Arginine assaysCorynebacterium glutamicum containing pArgC-P or pEKEX2was grown in 50 mL 2 × YT broth to OD600 1. Cells werewashed twice and incubated in 20 mL MM broth at 30◦C withshaking. Volumes were withdrawn for dry cell mass measure-ments (0.5 mL) and arginine quantitation (1.5 mL) along a timecourse. Cells were mechanically disrupted (10 Min) using a Vir-sonic Digital 600 sonicator (Virtis, Gardiner, NY, USA). Cell de-bris was removed by centrifugation (15,000g, 5 Min, 4◦C) andtrichloroacetic acid treatment [21]. The arginine concentrationin each cell-free extract sample was quantitated colorimetricallyusing the method of Sakaguchi [32] as modified by Tomlinsonand Viswanatha [33].

2.6. Overexpression and purification ofhexahistidine-tagged ArgRPurified hexahistidine-tagged ArgR (ArgRHis) overproductionwas induced according to the pET Manual (Novagen) with var-ious modifications to limit insoluble inclusion body formation.A 100 mL E. coli BL21 (DE3) culture was grown to OD600 0.4–0.6, 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) wasadded, and the culture was shaken at 30◦C for 2 H. Cells weredisrupted by 2.5 Min of sonication in 1.5 mL 50 mM Tris–HCl (pH7.5) containing 1 mM phenylmethanesulphonylfluoride (PMSF)(Merck, Darmstadt, Germany) as above. Cell lysates were cen-trifuged (15,000g, 2 Min, 4◦C) and the soluble fraction elutedthrough a HIS-Select agarose column (Sigma–Aldrich). Afterwashing three times with buffer [20 mM imidazole, 0.4 mM

NaCl (pH 8), 50 mM NaPO4, 1 mM PMSF, 0.1% (w/v) Triton X-100], the protein was eluted using buffer containing 250 mMimidazole, 0.4 M NaCl, 100 mM NaPO4 (pH 8.0), and 25% (w/v)glycerol. Imidazole was removed using an Amicon Ultra-15 filterunit (Millipore, Bedford, MA, USA) and buffer containing 0.2 MNaCl, 30 mM Tris–HCl (pH 8.0), 25% (w/v) glycerol, 2 mM DTT,and 1 mM PMSF using the manufacturer’s standard protocol.

Total protein was quantitated using Coomassie Bril-liant Blue G-250 dye (Bio-Rad, Richmond, CA, USA). Proteinswere analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) as described by Laemmli [34].Hexahistidine-tagged proteins were detected by standard West-ern blot methods [21].

2.7. Electrophoretic mobility shift assaysThe argC promoter, argG promoter, carA promoter, andnarKGHJI PCR product fragments were purified by agarose gelelectrophoresis, DIG-labeled, and bound to purified ArgRHis

using the DIG 2nd Generation Gel Shift Kit (Roche) as recom-mended. Arginine (10 mM) was included in the binding re-action and polyacrylamide solution unless otherwise stated.Typically, 20 fmol of DIG-labeled target DNA was included inthe reaction with 5 pmol of purified ArgRHis protein, unless oth-erwise stated. Apparent dissociation constants were calculatedaccording to Sekino et al. [35]. Protein–DNA complexes wereresolved by electrophoresis through a native 8% (w/v) poly-acrylamide gel (29:1; Sigma–Aldrich) in 0.25 × TBE (80 V, 4◦C,3 H). DNA was transferred to a Hybond-N + nylon membrane(Amersham Biosciences, Buckinghamshire, England) and visu-alized as in the DIG Application Manual (Roche). Band intensi-ties were quantified using ImageJ (version 1.40g; US NationalInstitutes of Health, Bethesda, MD, USA). For binding reactionsinvolving competitor oligonucleotides, unlabeled, heat dena-tured (95◦C, 5 Min) double-stranded DNA fragments (Table S2)(Inqaba Biotechnical Industries, Pretoria, South Africa) were in-cluded at a 1,500-fold molar excess relative to the DIG-labeledtarget fragment.

3. Results3.1. ArgR-mediated repression of thearginine operonThe in-frame argR deletion mutation, CG�argR, was confirmedby PCR, sequencing, and Southern blotting (results not shown).Transcriptional analysis of C. glutamicum ATCC 13032 andCG�argR was performed using RT-PCR. Oligonucleotide primers(Table S1) spanning intergenic regions, shown in Fig. 1a, wereused to amplify cDNA synthesized from RNA extracted from cellsgrown in MM. When the argC–argJ, argJ–argB, argB–argD, argD–argF, argF–argR, and argG–argH primers were used with cDNAfrom WT cells grown without arginine, fragments of the expectedsize were observed, whereas no amplified DNA was detected inWT cells grown with arginine (results not shown). In contrast,amplicons for all primer pairs were obtained from CG�argR,irrespective of whether arginine was included in the growthmedium. When CG�argR was grown in MM, significantly higherlevels of arginine after 4 and 6 H [21.04 ± 3.36 and 23.07 ±1.33 μmol (mg dry cell mass)−1] relative to the WT [7.58 ± 1.10

ArgR binding in C. glutamicum arginine biosynthesis 121

Fig. 1. Genetic organization of the C. glutamicum arginine biosynthetic genes (a) and the proposed structure of the argCpromoter region as determined by primer extension (b). The proposed ARG boxes argC1 and argC2, the putative argCtranscriptional start site (indicated by an arrow), and the putative −10 and −35 hexameric sequences are shown.

and 7.49 ± arginine (mg dry cell mass)−1] (P < 0.05 for bothtime points) were accumulated.

To test whether ArgR binds to DNA motifs upstream ofthe argC gene, the multicopy pArgC-P plasmid (with 115 bp ofthe CAF21404–argC intergenic region) was introduced into C.glutamicum WT cells. C. glutamicum (ArgC-P) grown in MM dis-played significantly elevated levels of total arginine relative toC. glutamicum (pEKEX2) cells, showing levels of 20 ± 0.03(P < 0.005), 5.03 ± 0.13 (P < 0.0005), and 7.21 ± 0.99 μmol

arginine (mg dry cell mass)−1 (P < 0.05) after 6, 9, and 24 Hof incubation. In contrast, arginine levels for the control cellsremained unchanged throughout the 24 H incubation period[between 2.61 ± 0.13 and 3.40 ± 0.28 μmol (mg dry cellmass)−1]. This clearly suggests that the CAF21404–argC frag-ment in pArgC-P, representing nucleotides from + 1 to −115upstream of argC, is capable of alleviating repression of thearginine operon through the titration of ArgR molecules awayfrom the promoter region.

Fig. 2. EMSAs showing ArgRHis–argC promoter binding. (a) The binding of crude cell extracts to the argC promoter region areshown. Lane 1 contains a negative control binding reaction performed with 20 fmol of DIG-labeled argC promoter fragmentsand no protein; lanes 2 and 3 contain the products of binding reactions using 2.0 mg total protein extracted from E. coliBL21cells containing pArgR–His or pET22b. Arginine (10 mM) was included in binding reactions and the polyacrylamide gelmatrix. (b) The binding of different amounts of ArgRHis to the argC promoter region is shown. Binding was performed in thepresence or absence of arginine in the binding reaction, although arginine was included in the gel matrix. ArgRHis was used inthe following amounts (pmol): lanes 1 and 6, 0; lanes 2 and 7, 1.09; lanes 3 and 8, 2.19; lanes 4 and 9, 4.1; lanes 5 and 10, 5.47.(c) The impact of different effector molecules [citrulline and canavanine (each 10 mM)] on ArgRHis–argC promoter binding isshown. Each binding reaction included 20 fmol of labeled DNA and 5.47 pmol of ArgRHis. Lane 1, negative control using noprotein or effector; lane 2, ArgRHis and no effector; lane 3, ArgRHis and the relevant effector molecule. (d) The molecularstructures of arginine and canavanine are shown.


Together, these results confirm that ArgR is a negativeregulator of the C. glutamicum arginine operon in the presenceof arginine and that deletion of this gene results in increasedtranscription and a consequent increase in arginine production,regardless of the presence or absence of arginine.

3.2. Analysis of the transcriptional start site ofthe argC genePrimer extension analysis of mRNA encoding ArgC revealed thepresence of a transcriptional start site 109 bp upstream of theargC start codon (Fig. 1b). Preceding this initiation site, a se-quence (5′-TGG CTA CTA AAA A-3′) (core hexamer underlined)closely resembling the extended C. glutamicum −10 consensussequences (5′-TgtGsTAtAATGG-3′) [36] was detected. A hexam-eric sequence (5′-GTG GTG-3′), resembling several of the knownpoorly conserved C. glutamicum −35 motifs was identified (Fig.1b) [36]. A transcriptional start site corresponding to the argGHgenes could not be detected, despite the use of a variety ofprimers and experimental conditions.

3.3. In vitro DNA-binding activity of ArgRHis

Purified hexahistidine-tagged ArgR (ArgRHis) was confirmed tohave the expected molecular weight of approximately 21 kDavia SDS-PAGE. EMSAs using crude cell extracts from E.coliBL21(pArgR–His) cells showed a distinct reduction in the elec-trophoretic mobility of the labeled-argC promoter fragment ver-sus those performed using extracts from E.coli BL21(pET22b)cells (Fig. 2a). This shift increased with increasing amounts ofArgRHis (Fig. 2b).

The presence of arginine increased the apparent affinityof ArgRHis for argC promoter DNA. The inclusion of 10 mM argi-nine within the polyacrylamide gel matrix and the binding reac-tion was required for the stable in vitro formation of the argCpromoter–ArgRHis complex. Under these conditions, a strongreduction in the mobility of the argC promoter fragment wasobserved (Fig. 2b, lanes 9 and 10). When arginine was absentfrom the binding reaction but still present in the matrix, weakerbinding and a smaller shift in the DNA (lanes 4 and 5) wasseen. A variety of amino acids and other metabolites (N-acetylglutamate, citrulline, ornithine, lysine, glutamate, uracil, andcanavanine) that form part of the arginine biosynthetic path-way were individually included as effector molecules in boththe binding reactions and gel matrices to detect the activationof in vitro ArgRHis–argC promoter binding. With the exception ofthe arginine analogue, canavanine, none of these compoundsresulted in a stable ArgRHis–argC promoter complex (Fig. 2c).

ArgRHis bound strongly to the argC and carA promoterregions, with approximately 4.1 pmol of ArgRHis sufficient toshift 20 fmol of fragment (Figs. 3a and 3c). ArgRHis did not bindto the argG promoter or to the negative control, the narKGHJIpromoter (Fig. 3d), even when relatively high amounts of proteinwere used (Fig. 3b) [37].

The change in electrophoretic mobility of the labeled argCand carA promoter fragments in response to increasing con-centrations of ArgRHis was used to calculate the apparent Kd

Fig. 3. EMSAs showing binding between ArgRHis and C.glutamicum promoter regions for the following genes oroperons: argC (a), argG (b), carA (c), and narKGHJI (d). Eachlane contains the products of the binding reaction. Eachreaction used 20 fmol of the relevant promoter region andthe following amounts of ArgRHis (pmol): lane 1, 0; lane 2,1.09; lane 3, 2.19; lane 4, 4.1; and lane 5, 5.47. All bindingreactions and gels were supplemented with 10 mM arginine.

value. Several EMSAs with various ArgRHis concentration gra-dients were performed on 20 fmol of each DNA fragment inthe presence of 10 mM arginine. For the argC promoter–ArgRHis

interaction, an apparent Kd value of 247.90 ± 46.80 nM (formonomers of ArgRHis) was calculated from six independent EM-SAs, whereas a value of 205.26 ± 114.29 nM was calculated forthe carA promoter (three independent EMSAs). These Kd valuesare not significantly different; however, they nevertheless aresimilar to those reported for E. coli, L. lactis, and Thermatogamaritima (100–600 nM) [14],[17],[38].

3.4. Elucidation of the ArgRHis–argC andArgRHis–carA promoter binding sitesIn order to locate ArgRHis-binding motifs, competitive EM-SAs were conducted using unlabeled double-stranded oligonu-cleotides homologous to specific portions of the argC and carApromoter regions (Table S2). The largest amount of unshiftedargC target was observed with competitor oligonucleotide 4,which is complementary to the −27 to + 18 region (Fig. 4).An analysis of this region using the Virtual Footprint software(http://prodoric.tu-bs.de/vfp/index2.php) [39] detected a pairof 14 bp ARG box sequence motifs (argC1: 5′-TGA ATC AAA AATTT-3′ and argC2: 5′-TGC ATG AAT AAT TT-3′) separated by 1 bp


Fig. 4. Identification of ArgRHis-binding motifs within the argC promoter region using competitive EMSAs. (a) Detailed view ofthe CAF21404–argC intergenic region showing the expected binding sites of double-stranded competitor oligonucleotides(labeled by the circled numbers). The coordinates for each fragment are relative to the argC start codon ( + 1). The presence ofan observed ArgRHis-meditated mobility shift is indicated. The bracketed sequence contains ArgRHis-binding motifs(highlighted). (b) Mobility shift assay performed using 20 fmol argC promoter DNA and 5.47 pmol of ArgRHis. The presence orabsence of ArgRHis, in addition to a competitor fragment (30 pmol) indicated in (a), is indicated above each lane.

and similar to the E. coli K12 ARG box core consensus sequence(Fig. 5a) [13]. Competitive EMSAs were also performed on thecarA promoter region (Fig. 6). Both oligonucleotides 8 and 9were able to prevent binding of ArgRHis, with oligonucleotide 8being the most effective (Fig. 6b). This oligonucleotide includesa 14 bp putative ARG box core sequence motif (−61 to −88)(5′-TGA ATG TAG TTT AT-3′; designated carA1) detected by VirtualFootprint software. In this case, the shorter oligonucleotide 9was less effective than the longer oligonucleotide 8, despitealso containing the putative carA1 ARG box sequence.

An analysis of the ARG box motifs in the argC and carA pro-moter regions identified a 14 bp ARG box consensus sequencefor C. glutamicum (5′-HMT GMA TSW ADW WTW TDY-3′) (Fig. 5).This consensus sequence consists of a core (positions 3–16) withseveral highly conserved residues at positions 3, 4, 7, 14, and16 that are similar to the universal core consensus sequenceproposed by Makarova et al. [16] (Fig. 5b). This sequence wasused to screen the C. glutamicum genome using Virtual Foot-print software. More than 350 putative ARG box motifs weredetected either within intergenic DNA or within 250 bp after anATG start codon. Some of these genes could be identified us-ing the Genbank database (http://www.ncbi.nlm.nih.gov), andincluded genes involved in biosynthesis of amino acids such as

methionine, cysteine, isoleucine, and glutamate, as well as thelysine/arginine exporter, lysE [40]. Several genes involved inthe glycolytic cycle, purine biosynthesis, fatty acid metabolism,and TCA cycle were also detected (Fig. 5c).

4. DiscussionBoth deletion of argR and increased copy number of the argCpromoter fragment resulted in constitutive expression of thearginine operon and elevated arginine production, thereby con-firming the role of ArgR as a negative regulator in C. glutamicum.In a transcriptomic study of the C. glutamicum arginine biosyn-thetic gene cluster, inactivation of argR was also shown to causesignificant derepression (approximately 20-fold) of all argininebiosynthetic genes, although this was not linked to arginineproduction levels [41]. Interestingly, Hanßler et al. [12] foundusing RT-PCR that deletion of the C. glutamicum global regu-lator, FarR, caused derepression of the arginine biosyntheticgene cluster, and that these genes were transcribed as two sep-arate transcripts (argCJBDFR and argGH). In other Gram-positivebacteria, such as Streptomyces clavuligerus and Lactobacillusplantarum, the argGH genes are separately transcribed [8],[42].


Fig. 5. Identification of an ArgRHis-binding motif within the carA promoter region by competitive EMSAs. (a) Detailed view of thepyrC–carA intergenic region showing the binding sites of double-stranded competitor oligonucleotides (labeled by the circlednumbers). The coordinates for each fragment are supplied relative to the carA start codon ( + 1). The presence of an observedArgRHis-meditated mobility shift is indicated. The bracketed sequence contains a putative ArgRHis-binding motif (highlighted).(b) Mobility shift assay performed using 20 fmol DIG-labeled carA promoter DNA and 5.47 pmol ArgRHis in each reaction. Thepresence or absence of ArgRHis, in addition to a competitor fragment (30 pmol) indicated in (a), is indicated above each lane.

Examination of the intergenic sequences of the C. glutamicumarginine gene cluster revealed a terminator structure within theargR–argG intergenic region, suggesting the presence of a pu-tative promoter upstream of the argG gene. Despite this, wewere unable to demonstrate a transcriptional start site in thisputative promoter region. In addition, we could not show stablebinding of ArgRHis to this region in vitro and were unable toidentify putative in silico ARG box motifs in the sequence of thisintergenic region.

Using ChIP assays, Lee et al. [20] have shown ArgR to bindto several intergenic regions within the arginine operon. Strongbinding was observed for argC and argB, and weaker bindingwas seen for argF and argG. This binding was independent ofthe presence of arginine. Hanßler et al. [12] used EMSAs to showthat FarR does bind to the intergenic region upstream of argG,although the deletion of farR did not affect intracellular argininelevels. FarR clearly plays a regulatory role in the arginine operon,and transcription of the argGH gene cluster may therefore bedifferentially regulated by FarR, ArgR (under different conditionsused in this study), and/or other as yet unknown transcriptionalregulators.

We have demonstrated via EMSAs that ArgR affinity for thepromoter region of the first gene in the C. glutamicum arginine

biosynthetic operon, argC, is increased in the presence of argi-nine. This is similar to E. coli, L. plantarum, and S. clavuligerus[8],[13],[42], although it differs from that found in T. maritima[14]. In addition, we showed that, similar to that seen in L. plan-tarum and L. lactis, ArgR binds to the carA promoter region inC. glutamicum, [38],[42], wherein it would presumably functionto limit the supply of the carbamoyl phosphate and exert a neg-ative regulation on pyrimidine biosynthesis. In C. glutamicum,purified ArgRHis was able to bind DNA in vitro in the absence ofaccessory proteins, distinguishing it from L. plantarum, whereinArgR is formed as a heterohexamer from two separate geneproducts [42].

Our finding that ArgR binding was stimulated by arginineis in contrast to that found by Lee et al. [20] who reportedthat the presence of arginine was not required for the bindingof ArgR to target DNA. In most prokaryotes, such as Bacillusstearothermophilus, the binding of arginine to the repressorstabilizes the structure [15] and enhances ARG box affinity [43].It is possible, however, that, by virtue of the more sensitivedetection method, Lee et al. [20] was able to detect looselybound nonactivated ArgR.

The C. glutamicum ArgRHis–argC promoter interaction alsooccurred in the presence of canavanine (Fig. 2c). This molecule


Fig. 6. Overview of putative ArgRHis-binding sites in C. glutamicum. (a) The extended C. glutamicum ArgRHis-binding siteswithin the argC and carA promoter regions (identified via competitor EMSAs and the Virtual Footprint tool) are shown alignedwith the proposed ARG box universal core consequence sequence of Makarova et al. [16] and the extended ARG box E. coli K12consensus sequence of Maas [13]. Where relevant, the position weight matrix score of each core sequence (positions 3–16)relative to the E. coli K12 core consensus sequence is indicated. The position of the first nucleotide of each motif is indicatedrelative to the first nucleotide of the start codon of the relevant gene. The convergent arrows indicate dyad symmetry. (b) Theproposed C. glutamicum extended ARG box consensus sequence is shown in a sequence logo diagram constructed usingWebLogo (http://weblogo.berkeley.edu). (c) Selected results from an in silico analysis of intergenic C. glutamicum ATCC13032 genomic motifs similar to the C. glutamicum ARG box core consensus sequence are shown.

differs from arginine by the shifting of a double bond from theprimary to the secondary amine and the substitution of a carbonwith an oxygen atom. In Geobacillus stearothermophilus, it hasbeen proposed that the activation of ArgR DNA binding is relianton the formation of a salt bridge between the guanidino groupof each arginine molecule and conserved aspartic acid residueslocated on either side of the ArgR trimer–trimer interface [15].The guanidino group remains intact in canavanine, and thus thesalt bridge-mediated stabilization and activation of each ArgRtrimer remains feasible. The failure of ornithine and citrulline tostimulate ArgRHis binding is in agreement with results seen forthe E. coli argECBH operon [44].

The ArgR-binding sites identified via competitive EMSAsare highly similar to both the 18 bp ARG box motifs of E.coli K12 and the 14 bp ARG box universal core consensus se-quence [13],[16]. A preliminary ARG box consensus sequence(5′-HMT GTA TSW ADW WTW TDY-3′ (Fig. 5a) was constructedfor C. glutamicum. The putative core ARG box sequences de-tected either in silico or in vitro via competitive EMSAs are all inpossession of a conserved T nucleotide at position 11 (Fig. 5b). Incontrast, the nucleotide at this position in the ARG boxes of otherbacteria, such as E. coli K12 and various Streptomyces spp., is

less conserved [8],[13]. It is expected that highly conserved basepairs detected within the core ARG box sequences would be es-pecially important for ArgR DNA binding in C. glutamicum. Forexample, the conserved G residue and the semiconserved Aresidue (positions 4 and 10 in Fig. 5b) have been shown in B.subtilis to play an important role in establishing specific con-tacts with ArgR amino acids that are critical for formation of theprotein–DNA complex.

In order to investigate whether ArgR plays a broader regu-latory role in C. glutamicum as it does in E. coli, a transcriptome-based analysis of the �argR mutant should reveal novel tar-gets of ArgR, in the same way that this strategy was used forAmtR, the master nitrogen regulator in C. glutamicum [45]. Inaddition, in silico screening of the C. glutamicum genome withthe ARG box core consensus sequence has revealed putativesites of ArgR interaction preceding particular genes, indicatinga broader regulatory role for ArgR in this bacterium. This addi-tional information of the promoter region of the arginine operonand the ARG box motif will aid future work on the further char-acterization of the role of ArgR in C. glutamicum, which couldreveal potentially novel targets for mutagenesis to improve thebiosynthesis of arginine.


AcknowledgementsThe authors are thankful to the National Research Foundationof South Africa for funding.

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argr–promoter interactions in corynebacterium glutamicum arginine biosynthesis

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