JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M408271200 on October 19, 2004

J. Biol. Chem., Vol. 280, Issue 1, 585-595, January 7, 2005
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Identification of AcnR, a TetR-type Repressor of the Aconitase Gene acn in Corynebacterium glutamicum*

Andreas Krug, Volker F. Wendisch, and Michael Bott{ddagger}

From the Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany

Received for publication, July 21, 2004 , and in revised form, October 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Corynebacterium glutamicum, the activity of aconitase is 2.5-4-fold higher on propionate, citrate, or acetate than on glucose. Here we show that this variation is caused by transcriptional regulation. In search for putative regulators, a gene (acnR) encoding a TetR-type transcriptional regulator was found to be encoded immediately downstream of the aconitase gene (acn) in C. glutamicum. Deletion of the acnR gene led to a 5-fold increased acn-mRNA level and a 5-fold increased aconitase activity, suggesting that AcnR functions as repressor of acn expression. DNA microarray analyses indicated that acn is the primary target gene of AcnR in the C. glutamicum genome. Purified AcnR was shown to be a homodimer, which binds to the acn promoter in the region from -11 to -28 relative to the transcription start. It thus presumably acts by interfering with the binding of RNA polymerase. The acn-acnR organization is conserved in all corynebacteria and mycobacteria with known genome sequence and a putative AcnR consensus binding motif (CAGNACnnncGTACTG) was identified in the corresponding acn upstream regions. Mutations within this motif inhibited AcnR binding. Because the activities of citrate synthase and isocitrate dehydrogenase were previously reported not to be increased during growth on acetate, our data indicate that aconitase is a major control point of tricarboxylic acid cycle activity in C. glutamicum, and they identify AcnR as the first transcriptional regulator of a tricarboxylic acid cycle gene in the Corynebacterianeae.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Corynebacterium glutamicum is a non-pathogenic, aerobic Gram-positive soil bacterium that was described in 1957 (1) as an L-glutamate-excreting bacterium. It has gained considerable interest because of its use in the large scale biotechnological production of L-glutamate (>1 million tons per year) and L-lysine (~0.6 million tons per year) (2-5) and because of its emerging role as a model organism for the Corynebacterineae, a suborder of the actinomycetes, which also includes the genus Mycobacterium (6).

The tricarboxylic acid cycle is of central importance for the metabolism of C. glutamicum because it provides energy and biosynthetic precursors and therefore the flux through this cycle is an important aspect for the production of amino acids of the aspartate and glutamate family. Thus, it is not surprising that several tricarboxylic acid cycle enzymes of C. glutamicum have been studied biochemically and/or genetically in the past, i.e. citrate synthase (gltA, Ref. 7), isocitrate dehydrogenase (icd, Ref. 8), 2-oxoglutarate dehydrogenase (9), malate:quinone oxidoreductase (mqo, Ref. 10), and malate dehydrogenase (mdh, Ref. 11). However, no information is available hitherto on the genetic regulation of this pathway in C. glutamicum, although there is evidence that it differs from that of the model bacteria Escherichia coli and Bacillus subtilis, since e.g. glucose and acetate are consumed in parallel rather than successively (12).

No studies have been performed yet in C. glutamicum on aconitase (EC 4.2.1.3 [EC] ), which catalyzes the stereospecific and reversible isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle and in the glyoxylate cycle. It is an unusual enzyme in that it contains a [4Fe-4S] cluster, which is not involved in electron transfer, but in binding of the substrate (13). Besides their catalytic function, a certain class of aconitases can also have a regulatory function by binding to certain mRNAs and inhibiting or increasing their translation into protein (14-18). This regulatory function is carried out by a catalytically inactive form of aconitase, which is formed under conditions of iron starvation or oxidative stress, when the iron-sulfur cluster is disassembled and the apoprotein is formed.

In many bacteria, expression of the aconitase genes is controlled by transcriptional regulators (see "Discussion"). Here, we provide evidence that this is also the case for the C. glutamicum aconitase gene (acn)1 and describe the identification and characterization of the TetR-type repressor protein AcnR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Culture Conditions—All strains and plasmids used in this work are listed in Table I. C. glutamicum strain ATCC13032 was used as wild type in this study. Strain {Delta}acnR is a derivative containing an in-frame deletion of the acnR gene. For cultivation of C. glutamicum in liquid media, 5 ml of CGIII medium (19) or brain heart infusion (BHI) medium (Difco Laboratories, Detroit, MI), both supplemented with 2% (w/v) glucose, were inoculated with colonies from a fresh Luria-Bertani (LB) agar plate (20) and incubated overnight at 30 °C and 170 rpm. This first preculture was used to inoculate 60 ml of CGXII minimal medium (21) in a 500-ml shake flask with two baffles. The second preculture was incubated overnight at 30 °C and 120 rpm and then used to inoculate the main culture, for which the same conditions were applied as for the second preculture. The CGXII minimal medium contained as a carbon source either 222 mM glucose, 244 mM sodium acetate, 50 mM sodium citrate, or 104 mM sodium propionate. When citrate was used as carbon source, the medium contained in addition 100 mM MgCl2. When appropriate, the medium was supplemented with 25 µg of kanamycin/ml. C. glutamicum strains carrying plasmid pEKEx2 and derivatives thereof were cultivated in the presence of 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside. For cloning purposes, E. coli DH5{alpha} (Invitrogen Life Technologies, Inc.) was used and grown at 37 °C in LB medium (20). E. coli BL21(DE3) (22) was used for overproduction of the C. glutamicum AcnR protein with expression plasmids based on pET24 or pET28 (Novagen, Madison, WI).


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  		TABLE I
Bacterial strains and plasmids used in this study

 
Determination of Aconitase Activity—For the determination of the aconitase activity, cells from the CGXII main culture (~25 ml) were harvested with ~25 g of crushed ice (precooled to -20 °C) by centrifugation at 4,000 x g for 5 min. The cell pellet was resuspended in 900 µl of Tris-HCl (90 mM; pH 8.0), and the cells were mechanically disrupted by 3x 20 s bead beating with 1 g of zirconia-silica beads (diameter 0.1 mm; Roth, Karlsruhe, Germany) using a Silamat S5 (Vivadent, Ellwangen, Germany). After centrifugation (5 min; 18,320 x g; 4 °C), the supernatant was used immediately for the enzyme assay. Aconitase activity was assayed by following the formation of cis-aconitate from isocitrate (23) at 20 °C. The assay mixture contained 950-995 µl of 90 mM Tris-HCl pH 8.0 containing 20 mM DL-trisodium isocitrate. The reaction was started by the addition of 5-50 µl of cell extract, and cis-aconitate formation was determined by measuring the absorbance increase at 240 nm using a Jasco V560 spectrophotometer. An extinction coefficient for cis-aconitate of 3.6 mM-1 cm-1 at 240 nm was used. One unit of activity corresponds to 1 µmol of isocitrate converted to cis-aconitate per min.

Recombinant DNA Work—The enzymes for recombinant DNA work were obtained from Roche Diagnostics (Mannheim, Germany) or New England Biolabs (Frankfurt, Germany). The oligonucleotides used in this study were obtained from MWG Biotech (Ebersberg, Germany) and are listed in Table II. Routine methods like PCR, restriction, or ligation were carried out according to standard protocols (20). Chromosomal DNA from C. glutamicum was prepared as described (7). Plasmids from E. coli were isolated with the QIAprep spin miniprep kit (Qiagen, Hilden, Germany). E. coli was transformed by the RbCl method (24), C. glutamicum by electroporation (25). DNA sequencing was performed with a Licor 4200 sequencer (Licor Inc., Lincoln, NB). Sequencing reactions were carried out with the Thermo Sequenase Primer Cycle Sequencing Kit (Amersham Biosciences, Freiburg, Germany).


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  		TABLE II
Oligonucleotides used in this study

 
An in-frame acnR deletion mutant of C. glutamicum was constructed via a two-step homologous recombination procedure as described previously (26). The acnR up- and downstream regions (~500 bp each) were amplified using the oligonucleotide pairs tetR-A-for/tetR-B-rev and tetR-C-for/tetR-D-rev, respectively, followed by a crossover PCR with oligonucleotides tetR-A-for and tetR-D-rev. The resulting PCR product of ~1 kb was digested with EcoRI and HindIII and cloned into pK19mobsacB (27) cut with the same enzymes. DNA sequence analysis confirmed that the cloned PCR product did not contain spurious mutations. Transfer of the resulting plasmid pK19mobsacB-{Delta}acnR into C. glutamicum and screening for the first and second recombination event was performed as described previously (26). The genomic structure of the mutant was first checked by PCR analysis of chromosomal DNA with the primer pair tetR-amp-for/tetR-amp-rev (Table II). The PCR products obtained with wild-type DNA (1.6 kb) and {Delta}acnR mutant DNA (1.1 kb) had the expected sizes. Furthermore, the mutant was controlled by Southern blot analysis (26) using EcoRI-digested DNA and a digoxigenin-labeled PCR product covering the acnR gene and the 500-bp up- and downstream DNA as probe. The expected fragment sizes were obtained for wild type (1.8 kb) and the {Delta}acnR mutant (1.3 kb), confirming the successful acnR deletion (data not shown).

For the purification of AcnR with a C-terminal StrepTag-II (28), the acnR coding region was amplified using the Expand High Fidelity polymerase mix (Roche Diagnostics) and oligonucleotides that introduced an NdeI restriction site overlapping the start codon (acnR-FP-for) and an XhoI restriction site preceding the stop codon (acnR-FP-24rev). The purified PCR product was cloned into the expression vector pET24b-Streptag cut with NdeI and XhoI. The AcnR protein encoded by the pET24 derivative (AcnR-C) contains ten additional amino acid residues (LEWSHPQFEK) at the C terminus. For the purification of AcnR with an N-terminal StrepTag-II, the acnR coding region was amplified with the primers acnR-FP-for and acnR-FP-28rev, and the PCR product was cloned into pET28a-Streptag (29) cut with NdeI and XhoI. The AcnR protein encoded by the pET28 derivative (AcnR-N) contains 14 additional amino acid residues (MASWSHPQFEKGAH) at the N terminus. DNA sequence analysis confirmed that the AcnR coding sequence of the resulting plasmids pET24b-acnR-C and pET28aacnR-N did not contain spurious mutations. For the overproduction of the streptagged AcnR derivatives, the plasmids were transferred into E. coli BL21(DE3).

In order to construct plasmids pEKEx2-acnR, pEKEx2-acnR60, pEKEx2-acnR89, and pEKEx2-acnR-C-Strep, the acnR fragments were amplified with oligonucleotide acnR-for as forward primer and acnR-rev, acnR-rev-60, acnR-rev-89, or acnR-strep-rev, respectively, as reverse primers. After digestion with BamHI and EcoRI, the purified PCR products were cloned into the vector pEKEx2 cut with the same enzymes. All PCR-derived parts of the resulting plasmids were checked by DNA sequence analysis in order to exclude unwanted mutations.

Preparation of Total RNA—Cultures of the wild type and the {Delta}acnR mutant were grown in CGXII minimal medium containing 4% (w/v) glucose. In the exponential growth phase at an OD600 of 5-6, 25 ml of the cultures were used for the preparation of total RNA as described previously (30, 31). Isolated RNA samples were analyzed for quantity and quality by UV spectrophotometry and denaturing formaldehyde agarose gel electrophoresis (20), respectively, and stored at -70 °C until use.

DNA Microarray Analyses—The generation of whole genome DNA microarrays (32), synthesis of fluorescent-labeled cDNA from total RNA, microarray hybridization, washing, and data analysis were performed as described previously (33-35). Genes that exhibited significantly changed mRNA levels (p < 0.05 in a Student's t test) by at least a factor of three were determined in four series of DNA microarray experiments: (i) 10 comparisons of the wild type and the {Delta}acnR mutant cultivated in regular CGXII minimal medium with 4% (w/v) glucose (contains 36 µM iron before autoclaving and ~8 µM iron after autoclaving); (ii) four comparisons of the wild type and the {Delta}acnR mutant cultivated in CGXII-glucose medium with an elevated iron concentration (addition of 100 or 500 µM FeSO4 after autoclaving); (iii) three comparisons of the wild type grown with ~8 µM iron and with 508 µM iron; (iv) three comparisons of the {Delta}acnR mutant grown with ~8 µM iron and with 508 µM iron. The 34 genes that were differentially expressed in one or more of these series of experiments were subjected to a hierarchical cluster analysis (36) as described previously (33, 35).

Primer Extension Analysis—Non-radioactive primer extension analysis was performed using the IRD800-labeled oligonucleotides acn-PE1* and acn-PE3* (Table II) and 15 µg of total RNA as described previously (29). The length of the primer extension products was determined by running the four lanes of a DNA sequencing reaction set up with the same oligonucleotide as used for primer extension alongside the primer extension products on the denaturing polyacrylamide gel. A PCR product obtained with the primer pair acn-PEK-fw/acn-PEK-rev was used as template for the DNA sequencing reaction.

Overproduction and Purification of Streptagged AcnR—E. coli BL21(DE3) carrying pET24b-acnR-C was cultivated in 100 ml of LB medium containing 50 µg/ml kanamycin in a 500-ml Erlenmeyer flask at 30 °C until the OD600 reached a value between 0.3 and 0.5. Synthesis of the AcnR-C was induced by addition of isopropyl-1-thio-{beta}-D-galactopyranoside to a final concentration of 1 mM, and the culture was incubated for another 3 h. Cells (final OD600 ~1) were washed once and resuspended in 10 ml of buffer W (100 mM Tris-HCl, pH 8.0, 1 mM EDTA). After addition of 1 mM diisopropylfluorophosphate and 1 mM phenylmethylsulfonyl fluoride, the cell suspension was passed three times through a French pressure cell (SLM Aminco, Spectronic Instruments, Rochester) at 207 MPa. Intact cells and cell debris were removed by centrifugation (20 min, 5,000 x g, 4 °C). The cell-free extract was subjected to ultracentrifugation (1 h, 150,000 x g, 4 °C) and the supernatant applied to a StrepTactin Sepharose column with a bed volume of 1 ml (IBA, Göttingen, Germany). The column was washed with 15 ml of buffer W and streptagged AcnR was eluted with 10 x 1 ml buffer W containing 15 mM desthiobiotin (Sigma-Aldrich). Fractions containing AcnR-C were pooled, and the elution buffer was exchanged against 40 mM Tris-HCl buffer, pH 7.5, containing 10% (v/v) glycerol by gel filtration with Sephadex G-25 (PD-10 column, Amersham Biosciences). Protein concentrations were determined with the BCA protein assay kit (Pierce) using bovine serum albumin as standard. The purity of the protein preparation was assessed by SDS-polyacrylamide gel electrophoresis (37) and subsequent protein detection with Gel Code blue stain reagent (Pierce). Using this protocol, about 1 mg of AcnR-C was purified to apparent homogeneity. The same procedure as described above was used for the isolation of AcnR with an N-terminal StrepTag-II using E. coli BL21(DE3)/pET28a-acnR-N.

Size Exclusion Chromatography—The size of purified AcnR-C and AcnR-N was estimated by size exclusion chromatography using a HiLoad 26/60 Superdex 200 prep grade column (Amersham Biosciences) integrated into an Äkta Explorer system (Amersham Biosciences). The column was equilibrated with 20 mM HEPES-buffer pH 8.0 containing 300 mM NaCl and 1 mM dithiothreitol. After application of 0.5-1 mg of purified protein, elution was performed at 4 °C with a flow rate of 2 ml/min. The column was calibrated with a premixed protein molecular mass marker (MWGF-200, Sigma).

DNase I Footprinting Assays—The binding site of purified AcnR protein at the acn promoter was analyzed by DNase I footprinting as described previously (29). PCR products covering the acn promotor region from position -346 to +52 or from position -268 to +241 relative to the proposed TTG start codon were obtained with the primer pairs acn-PEK-for/acn-PE1* (labeled non-template strand) and acn-FP1*/acn-PEK-rev (labeled template strand) respectively. The primers with an asterisk were IRD800-labeled at the 5'-end. The DNA-AcnR mixtures were treated with DNase I, and the reaction products were separated by denaturing PAGE. The regions, which were protected from DNase I digestion, were localized by running besides the footprinting reactions the four lanes of a DNA sequencing reaction set up with the oligonucleotide acn-PE1* or acn-FP1* and a PCR product obtained with the primer pair acn-PEK-for/acn-PEK-rev as template.

Gel Shift Assays—Purified AcnR-C (500 nM) was mixed with ~50 nM DNA fragments (80-300 bp) covering the acn promoter region in a total volume of 20 µl. The buffer contained 10 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 5% (v/v) glycerol, 0.5 mM dithiothreitol, 0.005% (v/v) Triton X-100, 50 mM NaCl, 5 mM MgCl2, and 2.5 mM CaCl2. After incubation for 20 min at room temperature, the samples were separated by agarose gel electrophoresis with Tris acetate/EDTA buffer (20) at 4 °C and 75 V. The gel was subsequently stained with ethidium bromide and photographed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Influence of the Carbon Source on Aconitase Activity of C. glutamicum—Previous studies revealed that in C. glutamicum the carbon flux through the tricarboxylic acid cycle varies with the carbon source (12). In cells growing on acetate, the tricarboxylic acid cycle flux was about 0.4 µmol min-1 mg protein-1 and thus 4-fold higher than in cells growing on glucose. Remarkably, the activities of citrate synthase (0.5-0.8 units (mg of protein)-1; Ref. 7) and isocitrate dehydrogenase (0.9-1.1 units (mg of protein)-1; Ref. 8) as measured in cell extracts were reported to be similar in glucose- and acetate-grown cells and are sufficient to allow the maximally measured carbon fluxes. We therefore became interested to determine whether the activity of aconitase does or does not vary with different carbon sources and measured its activity in extracts of cells cultivated on glucose, citrate, acetate, or propionate. As shown in Table III, the aconitase activity of glucose-grown cells was 0.2 units/mg of protein, whereas the activity of citrate-, acetate-, and propionate-grown cells was 0.5, 0.8, and 0.5 units/mg protein, respectively, i.e. up to 4-fold higher. Thus, aconitase activity is regulated and might represent a rate-limiting step in the tricarboxylic acid cycle flux. The increased activity on acetate correlates with the increased tricarboxylic acid cycle flux on this carbon source (12), which might also occur with citrate. Propionate is metabolized in C. glutamicum via the methylcitrate cycle (38), in the course of which methylcitrate is converted to methylisocitrate. In E. coli, which also degrades propionate via the methylcitrate cycle (39), this conversion requires two enzymes, i.e. methylcitrate dehydratase (PrpD) and aconitase (AcnB) (40). This could explain the increased aconitase activity in C. glutamicum during growth on propionate.


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  		TABLE III
Influence of the carbon source on the aconitase activity of C. glutamicum wild type and the {Delta}acnR mutant strain

C. glutamicum wild type and {Delta}acnR mutant were grown in CGXII minimal medium containing either 222 mM glucose, 50 mM sodium citrate (+ 100 mM MgCl2), 244 mM sodium acetate, or 104 mM sodium propionate as carbon source. In the exponential growth phase at an OD600 of about 5, cells were harvested, washed, and disrupted by bead beating. After centrifugation at 15,000 x g, the supernatant (cell extract) was immediately used for the aconitase assay. Aconitase activity was determined with DL-trisodium isocitrate as substrate by measuring the formation of cis-aconitate at 240 nm.

 
Identification of a Transcriptional Repressor Gene Downstream of the Aconitase Gene—Since previous DNA microarray experiments had shown that the mRNA level of acn is 4-fold higher in acetate-grown cells (41), we assumed that the acn gene is transcriptionally regulated. As in bacteria genes encoding transcriptional regulators are often located in the vicinity of their target genes, we inspected the C. glutamicum genome region encoding aconitase. In contrast to e.g. E. coli, C. glutamicum contains only a single aconitase gene (42) encoding a protein of 943 amino acid residues with a calculated molecular mass of 102,168 Da (43). It shows 58.8% sequence identity to AcnA of E. coli (44) (45) but less than 20% identity to AcnB of E. coli (46) and thus belongs to the bacterial AcnA group of aconitases, which are homologous to the iron-responsive proteins (IRPs) of eukaryotes (47).

As shown in Fig. 1, immediately downstream of acn, a gene encoding a putative transcriptional regulator of 188 amino acid residues (21,184 Da) was identified, which we named acnR. Interestingly, the same gene organization was also found in C. efficiens, Corynebacterium diphtheriae, Mycobacterium tuberculosis, M. bovis, M. marinum, and Rhodococcus strain IG124 (Fig. 1). In M. leprae, the acnR gene is disrupted by frameshifts and stop codons, as found for many other genes of this organism (48). An amino acid sequence alignment of the AcnR homologs (Fig. 2) revealed that the primary sequences are highly conserved, particularly in the N-terminal part from position 16 to 62, where nearly 75% of the amino acids are identical. In silico analysis revealed that this conserved N-terminal part of the AcnR proteins belongs to the TetR family of bacterial regulatory proteins (PFAM family PF00440; (49). Members of this family contain a multihelical DNA binding domain at their N-terminal end and a highly divergent C terminus, which is involved in the binding of an inducer molecule (50).



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  		FIG. 1.
Genomic locus of acn from C. glutamicum and other species of the suborder Corynebacterineae. In all cases except Mycobacterium leprae, a gene encoding a transcriptional repressor was identified downstream of acn. This gene (shown in dark gray) was named acnR. In the case of Rhodococcus sp., only the 3'-terminal part of the acn gene was available. Other genes were numbered and annotated as follows: 1, invasin; 2, 3, 4, and 8, hypothetical proteins; 5, hypothetical cytosolic protein; 6, putative GMP synthase-glutamine amidotransferase domain; 7, ABC transporter, ATP-binding protein; 9, putative NAD+-dependent dehydrogenase. Data were taken from the National Center for Biotechnology Information (NCBI) and the bioinformatics software ERGO (Integrated Genomics).

 



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  		FIG. 2.
Sequence alignment of AcnR proteins from different Corynebacterineae species. Amino acids identical in all sequences are shaded in black, other conserved amino acids in gray. The N-terminal region of the AcnR proteins (amino acids 16-62) shows clear similarity to the N-terminal part of the TetR family of bacterial regulatory proteins (TetR-N; PFAM00440). The consensus sequence of TetR-N and the amino acids identical to the consensus in the AcnR proteins are shown above the alignment.

 
Operon Structure of the acn-acnR Genes—The transcriptional organization of the acn-acnR locus was analyzed by RT-PCR (see Fig. 3 and "Experimental Procedures"). Total RNA isolated from C. glutamicum wild type was transcribed into cDNA using three different primers (RT-1-rev, RT-2-rev, and RT-3-rev) in the reverse transcriptase reactions A, B, and C. The resulting products were then used for the PCR tests no. 1-5 (Fig. 3A). As shown in Fig. 3B, evidence was obtained that acn and acnR are co-transcribed, since a cDNA created with a primer annealing at the 3'-end of acnR (RT reaction A) allowed not only the amplification of an acnR fragment (no. 1), but also of an acn fragment (no. 2). As an internal control in the RT-PCR assays we used dnaE, which encodes a subunit of DNA polymerase. Besides the control reaction no. 3, where no acnR product could be obtained with a cDNA covering only acn, five additional control reactions (no. 6-10) were performed which were identical to reactions no. 1-5, respectively, except that reverse transcriptase was omitted from the initial reactions A-C. The fact that no PCR products were obtained in these reactions confirmed that the RNA was not contaminated with chromosomal DNA.



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  		FIG. 3.
Transciptional organization of the acn-acnR locus in C. glutamicum analyzed by RT-PCR. A, scheme showing the acn-acnR intergenic region containing two putative stem loop structures. Below the RT-PCR, reactions used to determine co-transcription of acn and acnR are shown schematically. RNA from C. glutamicum wild type was transcribed into cDNA with three different primers in the reverse transcriptase reactions A-C. Afterward these cDNAs were used as templates for the PCR reactions labeled 1-5. B, results from the RT-PCR analyses described above. The lower DNA fragment visible in lanes 1-5 represents dnaE, and RT-PCR of dnaE served as positive control in all reactions. The upper bands correspond to the products of the PCR reactions 1-5 indicated in A. Reactions 6-10 represent controls confirming the absence of DNA in the RNA preparation. The reactions were identical to the PCR reactions as shown in lanes 1-5 except that reverse transcriptase was omitted in reactions (A-C).

 
Aconitase Activity and Growth of a {Delta}acnR Mutant—Since AcnR was found to be a member of the TetR family, we suggested that it functions as transcriptional repressor of the neighboring aconitase gene. In order to test this hypothesis, an in-frame deletion mutant of C. glutamicum was constructed (13032 {Delta}acnR) in which codons 7-177 of acnR were replaced by a 21-bp sequence tag. The genomic structure of the mutant was confirmed by PCR and Southern blot analysis (51; see "Experimental Procedures"). In a first set of experiments, the influence of the acnR deletion on the aconitase activity was determined. Cells were cultivated in CGXII minimal medium with different carbon sources and used in the early exponential growth phase (OD600 ~6) for the determination of enzyme activity. As shown in Table III, the aconitase acitivity in glucose-grown cells was about 1 unit/mg protein and thus 5.3-fold higher than in the wild type. This result clearly supported the proposed function of AcnR as repressor of acn expression. In {Delta}acnR mutant cells grown on citrate, acetate, or propionate, the aconitase activity was 3.4-fold, 1.9-fold, and 5.0-fold higher than in wild-type cells grown with the same carbon sources and 1.7-fold, 1.5-fold, and 2.3-fold higher than in {Delta}acnR cells grown on glucose. This indicates that the increased aconitase activity of wild-type cells grown on acetate is partially because of a lower repression by AcnR and that an additional transcriptional regulator besides AcnR or another regulatory mechanism for aconitase might exist.

In the cultivations for the determination of aconitase activity it was observed that in most cases the {Delta}acnR mutant grew somewhat faster than the wild type on glucose and acetate minimal medium, sometimes also in citrate minimal medium. In glucose medium, for example, the growth rate of the {Delta}acnR mutant was 0.40 ± 0.02 h-1 and that of the wild type 0.36 ± 0.01 h-1 (values from seven replicates). This behavior was also evident when the cells were allowed to grow to stationary phase (Fig. 4) and might be caused by an increased tricarboxylic acid cycle activity in the mutant.



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  		FIG. 4.
Growth of C. glutamicum wild type (circles) and the {Delta}acnR mutant (triangles) on glucose or acetate minimal medium. The strains were cultivated at 30 °C and 120 rpm in CGXII minimal medium containing either 222 mM glucose (open symbols) or 244 mM sodium acetate (filled symbols) as carbon source.

 
Transcriptome Analyses—To determine the effect of the acnR deletion on global gene expression and in particular on acn expression, the transcriptomes of the {Delta}acnR mutant and the wild type were analyzed by DNA microarray analyses. RNA was isolated from cells growing exponentially in CGXII minimal medium with 4% (w/v) glucose (OD600 ~6). In ten comparisons with RNA from five independent cultivations (experiments A01-A10, Fig. 5) the acn mRNA level was always higher in the {Delta}acnR mutant than in the wild type (4.2-fold ±2.3), confirming that the increased aconitase activity of the mutant is caused by increased acn expression. Besides acn, several other genes showed mRNA levels that were on average ≥3-fold increased in the {Delta}acnR mutant, most of which are presumably involved in the uptake of heme and iron siderophores (Fig. 5). This indicates that the {Delta}acnR mutant faces iron limitation, a situation which might be due to the increased production of aconitase, a [4Fe-4S] protein. Indeed, after supplementation of the medium with 100 µM or 500 µM FeSO4 these genes showed no longer an increased mRNA level in the {Delta}acnR mutant, whereas the acn mRNA ratio was still raised (5.8-fold ±0.6) in the mutant (experiments B01-B04, Fig. 5). These data indicate that acn is the only gene whose mRNA level is significantly increased in the {Delta}acnR mutant of C. glutamicum.



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  		FIG. 5.
Hierarchical cluster analysis of gene expression changes observed in four series of DNA microarray experiments. The cluster includes those genes, whose average mRNA level was changed ≥3-fold or ≤3-fold in at least one of four series of microarray experiments (totally 20). The following series of experiments were performed: A01-A10, {Delta}acnR versus wild type cultivated in CGXII-glucose medium with ~8 µM iron; B01-B04, {Delta}acnR versus wild type cultivated in CGXII-glucose medium with 108 µM iron (B01, B02) or 508 µM iron (B03, B04); C01-C03, wild type with 508 µM iron versus wild type with 8 µM iron; D01-D03, {Delta}acnR with 508 µM iron versus {Delta}acnR mutant with 8 µM iron. The scale bar indicates the color coding of the relative RNA ratios.

 
Further microarray experiments, in which the influence of different iron concentrations (508 µM versus 8 µM) on global gene expression was analyzed separately for the wild type (experiments C01-C03, Fig. 5) and the {Delta}acnR mutant (experiments D01-D03, Fig. 5), revealed that the acn mRNA level was increased under iron excess, both in the wild type (3.0-fold ±1.0) and in the {Delta}acnR mutant (4.8-fold ±0.7). Thus, AcnR is not directly involved in iron regulation. Besides acn, several other genes encoding iron-containing proteins (52) showed increased mRNA levels under iron excess, indicating that the synthesis of these iron-containing proteins is to some extent controlled by the availability of iron in C. glutamicum.

Primer Extension Analysis of the acn Promoter—In order to confirm the results of the DNA microarray experiments with an alternative method and to locate the acn promoter, primer extension was performed using total RNA samples of wild type and {Delta}acnR mutant, reverse transcriptase and two different primers, acn-PE1* (Fig. 6, A and C) and acn-PE3* (Fig. 6B). With both primers, the major transcriptional start point of acn was localized 110 bp upstream of the proposed start codon. In addition, a weaker transcriptional start point was observed 113 bp upstream of the TTG start codon. The intensity of the primer extension products was stronger in the {Delta}acnR mutant than in the wild type, in accordance with the DNA microarray data (Fig. 6B). This was true not only for RNA isolated from glucose-grown cells, but also for RNA isolated from citrate- and acetate-grown cells (Fig. 6C), which qualitatively agrees with the aconitase activity measurements (Table III).



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  		FIG. 6.
Determination of the transcriptional start sites of the C. glutamicum acn gene and expression analysis. 12.5 µg (A and B) or 15 µg (C) of total RNA isolated from cells of C. glutamicum wild type and strain {Delta}acnR cultivated in CGXII minimal medium with different carbon sources was used for primer extension analysis with the oligonucleotides acn-PE1* (A and C) and acn-PE3* (B). The transcriptional start sites are indicated by asterisks. The sequencing reactions were generated with a PCR product covering the corresponding DNA region as template and the same IRD-800-labeled oligonucleotide as in the primer extension reaction.

 
Purification of AcnR and Determination of Its Native Mass—The increased level of acn mRNA and of aconitase activity in the {Delta}acnR mutant indicated that AcnR represses acn transcription. In order to identify the operator region, the AcnR protein containing either an N-terminal or a C-terminal StrepTag-II was overproduced in E. coli and purified by StrepTactin affinity chromatography to apparent homogeneity as described under "Experimental Procedures." The apparent mass on an SDS gel corresponded to the calculated mass of 22.5 kDa. Similar results were obtained for AcnR-N (data not shown). The native molecular mass of the purified AcnR proteins was determined by size exclusion chromatography. AcnR-C (1 mg) or AcnR-N (0.5 mg) were chromatographed on a Superdex 200 column (Amersham Biosciences) calibrated with five proteins. As shown in Fig. 7, both AcnR proteins were found to have a molecular mass of 44 kDa, indicating a homodimeric structure, which was also found for other members of the TetR family, e.g. TetR (53), CamR (50), QacR (54), or EthR (55). Complementation studies with the streptagged AcnR showed that this tag did not interfere with the repressing activity of AcnR (Table IV).



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  		FIG. 7.
Determination of the native molecular mass of AcnR by size exclusion chromatography. Purified AcnR protein containing either a C-terminal or an N-terminal StrepTag-II was separated on a HiLoad 26/60 Superdex 200 prep grade column using 20 mM HEPES buffer (pH 8.0) containing 300 mM NaCl, and 1 mM dithiothreitol for elution. For calibration, a premixed protein molecular mass marker containing the following proteins was used: cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa), {beta}-amylase (200 kDa). V0 was determined with blue dextran (2,000 kDa).

 


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  		TABLE IV
Aconitase activity in different C. glutamicum strains

All strains were cultivated under the same conditions in CGXII medium with 222 mM glucose. For strains harboring pEKEx2 or derivatives the medium contained in addition 50 µg/ml kanamycin and 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside.

 
Identification of the AcnR Binding Site within the acn Promoter Region—The binding site of AcnR at the acn promoter was determined with DNase I footprint assays. As shown in Fig. 8, the protected region on the template strand extended from position -11 to -28 relative to the first transcription start site (Fig. 8A), whereas on the non-template strand, protection was observed only in the region -18 to -20. Inspection of the upstream regions of the acn genes in C. diphtheriae, C. efficiens, and four Mycobacterium species revealed that they all contained sequence motifs similar to the one protected by AcnR in C. glutamicum. According to the alignment shown in Fig. 9, a putative AcnR consensus sequence could be derived, which contains an imperfect inverted repeat separated by four nucleotides: CAGNACaagcGTACTG. A binding motif of this type and this size is typical for TetR-type transcriptional regulators like TetR or CamR, although larger motifs have also been found, e.g. for QacR (56) or EthR (55).



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  		FIG. 8.
DNase I footprinting analysis with AcnR-C and the acn promoter region. 0.95 nM labeled acn template strand (panel A) or 0.75 nM labeled acn non-template strand (panel B) were incubated with increasing concentrations of AcnR-C: lane 0, no protein; lane 1, 5 nM monomeric AcnR; lane 2, 10 nM; lane 3, 25 nM; lane 4, 50 nM; lane 5, 100 nM; lane 6, 250 nM; lane 7, 500 nM (not in panel A); lane 8, 1.0 µM; lane 9, 2.5 µM; lane 10, 5 µM. Regions protected from digestion by DNase I are indicated by a black bar. The DNA sequencing reactions were set up with the IRD-800-labeled oligonucleotides used for generating the labeled PCR fragments for footprinting. Unlabeled PCR fragments, identical in sequence to the footprinting probes, were used as template DNA.

 



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  		FIG. 9.
Sequence of the acn promoter region of C. glutamicum aligned to putative acn promoter regions from other Corynebacterium and Mycobacterium species. Indicated are the start of the acn coding region (start codons bold and underlined), the transcriptional start sites (*) of the C. glutamicum acn gene, putative -10 (underlined and bold) and -35 regions (bold), and the AcnR binding site as determined by DNase I footprints (shaded in gray) for the C. glutamicum acn gene. As shown by the alignment, also the other species possess putative AcnR binding sites in the acn upstream region. The binding site represents an imperfect inverted repeat with the consensus sequence CAGNACnnncGTACTG.

 
In order to confirm the footprint data and the proposed AcnR consensus sequence, gel shift assays were performed. Experiments with a varying AcnR/DNA ratio revealed that a 4-6-fold molar excess of AcnR is sufficient for a shift (data not shown). In the experiment shown in Fig. 10, the shift of nine different DNA fragments was analyzed, in each case with a 10-fold excess of AcnR. The results obtained with fragments 1-5 confirmed that the binding site is located within a 40-bp region, which includes the binding site proposed from the footprints. Fragments containing this region (nos. 1, 2, and 5) were completely shifted, whereas those lacking this region (nos. 3 and 4) were not shifted at all. Fragments 6-9 represent derivatives of fragment 5 with mutations within or outside the proposed binding motif. Exchange of the two inner (fragment 6) or the two outer (fragment 7) bases of the imperfect inverted repeat completely inhibited the shift, as did an exchange of the four bases separating the inverted repeat (fragment 9). In contrast, exchange of four bases outside the proposed binding site (fragment 8) did not prevent the shift. These data provide strong support for the AcnR consensus binding site proposed above.



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  		FIG. 10.
Gel shift experiments with purified AcnR-C. A, DNA fragments used for gel shift analysis (1-9) are indicated by the white numbers in black circles. The gray bar indicates the region required for a shift and the black bar indicates the proposed position of the AcnR binding site. Fragments 6-9 are derivatives of fragment 5 containing mutations that were introduced with the primers 4*-for, 5*-for, 6*-for, and 7*-for. The 24-bp sequence shown beside these fragments includes the proposed AcnR consensus binding site (bold and underlined) and four base pairs up- and downstream. The mutated bases are indicated in bold and double underlined. B, approximately 50 nM DNA fragments nos. 1-9 were incubated either without AcnR-C (lanes labeled with -) or with 500 nM AcnR-C (lanes labeled with +) for 20 min at room temperature. Subsequently, the samples were separated on a 2.5% agarose gel at 4 °C and 75 V in 1x Tris acetate/EDTA buffer, and the gel was stained with ethidium bromide. The lanes labeled M contain a DNA size standard (50-bp ladder, Fermentas, St. Leon-Rot, Germany).

 
Effect of Overexpression of acnR and Derivatives on Aconitase Activity—Since deletion of acnR had caused derepression of acn, overexpression of acnR was supposed to lead to an enhanced acn repression. As shown in Table IV, overexpression of the unmodified acnR gene by the expression plasmid pEKEx-acnR caused an ~2-fold decreased aconitase activity. A comparable decrease was observed upon overexpression of an acnR derivative encoding an AcnR protein with a C-terminal StrepTag-II. Therefore, the tag does not interfere with the repressing activity of AcnR. If two shortened derivatives of acnR were overexpressed, which contained only the N-terminal 60 or 89 amino acid residues of AcnR, there was no effect on aconitase activity. Thus, the N-terminal DNA binding region of AcnR is not sufficient for its repressing activity, but requires the C-terminal effector binding region. Most likely, the C-terminal domain is essential for the dimerization of AcnR, as shown for TetR (53) and QacR (56). When unmodified acnR was overexpressed in the {Delta}acnR mutant, wild-type levels of aconitase activity were obtained (Table IV).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study the first regulator of a tricarboxylic acid cycle gene in C. glutamicum was identified. The acnR gene located downstream of the aconitase gene acn of C. glutamicum was shown to encode a repressor of acn expression. Deletion of acnR led to a 5-fold increased acn mRNA level and to a 5-fold increased aconitase activity. Repression is most likely caused by binding of the dimeric AcnR protein to the acn promoter in the region located 14 to 29 bp upstream of the transcriptional start point, where it interferes with the binding of RNA polymerase. By comparing the experimentally determined AcnR binding site within the C. glutamicum acn promoter (CAGAACGCTTTGTACTG) with the acn upstream regions of other Corynebacterium and Mycobacterium species, a putative consensus sequence CAGNACnnnnGTACTG containing an imperfect inverted repeat was found. Analysis of the C. glutamicum genome sequence revealed that this motif occurs only three times in the entire genome, once within the acn promoter, once within the coding region for the ribosomal protein L31, and once within the coding region of a gene of unknown function. This suggests that acn is the only target gene of AcnR. Further support for this assumption was obtained by comparing the transcriptomes of the {Delta}acnR mutant and the wild type with whole genome DNA microarrays. In these studies, only the acn gene showed reproducibly a ≥3-fold increased mRNA level in the {Delta}acnR mutant. Experiments with different iron concentrations revealed that the acn mRNA level is higher under iron sufficiency than under iron deficiency. A regulation of the aconitase level by the iron availability was previously also reported for M. tuberculosis (57).

Overexpression of the acnR gene by a plasmid-encoded copy under the control of the tac promoter caused a 2-fold decrease of the aconitase activity in glucose-grown wild-type cell extracts. Previous experience with other genes that were cloned into the same vector (pEKEx2) and cultivated under the same growth conditions used for acnR overexpression indicates that at least 10-fold higher AcnR levels should be obtained. Therefore, the question arises why there is only a 2-fold reduction of aconitase activity. Besides other explanations one obvious reason could be that increased acn repression leads to an increase of the concentration of a metabolite, which binds to AcnR, triggers the dissociation of the AcnR-operator complex and thus counteracts the increased repression. Such a mechanism would ensure that aconitase expression is never completely repressed by AcnR, which would be harmful to the cell. In order to identify the proposed metabolite we tested a variety of metabolites of central carbon metabolism with respect to their effects on AcnR binding to the acn promoter using the gel shift assay. However, we could not yet find a metabolite that inhibits binding of AcnR to its operator. Thus, the question of the ligand recognized by AcnR is still open.

Regulation of aconitase gene expression is widespread in bacteria. As mentioned before, E. coli contains two distinct aconitases (46), AcnB, which is the major citric acid cycle enzyme and AcnA, an aerobic stationary phase enzyme induced by iron and redox stress. The acnB gene is activated by CRP and repressed by ArcA, FruR and Fis and acnA expression is activated directly or indirectly by CRP, FruR, Fur, and SoxRS and repressed by ArcA and FNR (58). In B. subtilis, expression of the aconitase gene citB is subject to repression by the LysR-type regulator CcpC and repression can be relieved by citrate (59, 60). Besides CcpC, also CodY and AbrB were reported to be involved in citB expression (61). Similar to E. coli and B. subtilis, we also obtained evidence that acn expression in C. glutamicum might be controlled by more than one regulator. As shown in Table III, the aconitase activity of the {Delta}acnR mutant was 1.5-2.3-fold higher on acetate, citrate, and propionate compared with glucose. Regulators other than AcnR or other regulatory mechanisms might be responsible for this increase. Recently, RamB was identified as a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum (62). The acn gene belongs to the acetate stimulon (41) and two putative RamB binding sites were identified at positions -492 to -480 (+ strand) and -456 to -444 (-strand) upstream of the proposed acn start codon (62). Thus, RamB might represent a second regulator of acn expression besides AcnR. Our future studies will focus on the search for additional regulators of acn expression, for the inducer molecule recognized by AcnR, and for the answer to the question whether the C. glutamicum aconitase also has a regulatory function.


   FOOTNOTES
 
This article is dedicated to Prof. Rudolf K. Thauer on the occasion of his 65th birthday.

* This work was supported by the European Union within the framework of the VALPAN project (QLK 3-2000-00497). 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. Back

{ddagger} To whom correspondence should be addressed: Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany. Tel.: 49-2461-61-5515; Fax: 49-2461-61-2710; E-mail: m.bott{at}fz-juelich.de.

1 The abbreviations used are: acn, C. glutamicum aconitase gene; RT-PCR, reverse transcriptase-PCR; AcnR, TetR-type repressor protein. Back


   ACKNOWLEDGMENTS
 
We thank C. Lange and T. Polen for help with DNA microarrays and data analysis and S. Engels for assistance with DNase I footprint assays.



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