The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum
© Brune et al; licensee BioMed Central Ltd. 2006
Received: 09 December 2005
Accepted: 09 February 2006
Published: 09 February 2006
The knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks. Since iron is essential for a variety of cellular processes but also poses problems in biological systems due to its high toxicity, bacteria have evolved complex transcriptional regulatory networks to achieve an effective iron homeostasis. Here, we apply a combination of transcriptomics, bioinformatics, in vitro assays, and comparative genomics to decipher the regulatory network of the iron-dependent transcriptional regulator DtxR of Corynebacterium glutamicum.
A deletion of the dtxR gene of C. glutamicum ATCC 13032 led to the mutant strain C. glutamicum IB2103 that was able to grow in minimal medium only under low-iron conditions. By performing genome-wide DNA microarray hybridizations, differentially expressed genes involved in iron metabolism of C. glutamicum were detected in the dtxR mutant. Bioinformatics analysis of the genome sequence identified a common 19-bp motif within the upstream region of 31 genes, whose differential expression in C. glutamicum IB2103 was verified by real-time reverse transcription PCR. Binding of a His-tagged DtxR protein to oligonucleotides containing the 19-bp motifs was demonstrated in vitro by DNA band shift assays. At least 64 genes encoding a variety of physiological functions in iron transport and utilization, in central carbohydrate metabolism and in transcriptional regulation are controlled directly by the DtxR protein. A comparison with the bioinformatically predicted networks of C. efficiens, C. diphtheriae and C. jeikeium identified evolutionary conserved elements of the DtxR network.
This work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by DtxR. The DtxR protein has a major role in controlling the expression of genes involved in iron metabolism and exerts a dual regulatory function as repressor of genes participating in iron uptake and utilization and as activator of genes responsible for iron storage and DNA protection. The data suggest that the DtxR protein acts as global regulator by controlling the expression of other regulatory proteins that might take care of an iron-dependent regulation of a broader transcriptional network of C. glutamicum genes.
One of the major challenges in post-genomic research is to decipher and reconstruct the complete connectivity of transcriptional regulatory networks encoded by a bacterial genome sequence [1, 2]. The key components in regulation of bacterial gene expression are DNA-binding transcription factors that are able to sense changing environmental conditions and to modulate the expression of relevant target genes. An important prerequisite for understanding the regulation of gene expression in a bacterial cell in its entirety is the identification of the repertoire of regulatory proteins encoded by a genome sequence along with the complete set of genes that are under transcriptional control by each of the identified regulators [3, 4]. The transcriptional network of the Gram-negative bacterium Escherichia coli is currently the best-understood regulatory system of a single cell. It includes 314 DNA-binding transcription factors and is characterized by a hierarchical and modular architecture that is composed of eight modules with distinct physiological functions. Even for this well-studied model organism, only a small fraction of the transcriptional regulatory interactions are currently known [3, 5].
The long-term objective of our post-genomic work is to characterize the complete repertoire of regulatory proteins of the Gram-positive bacterium Corynebacterium glutamicum along with the complete set of their target genes and corresponding DNA binding sites within the known genome sequence [6, 7]. Since we also want to understand the evolution of the transcriptional regulatory network of C. glutamicum, we took advantage of the published genome sequences of Corynebacterium efficiens , Corynebacterium diphtheriae  and Corynebacterium jeikeium  to perform comparative genomic analyses. C. glutamicum and C. efficiens are widely used in biotechnological fermentation processes, whereas C. diphtheriae and C. jeikeium represent the most hazardous human pathogens among the corynebacteria. Therefore, the four species represent attractive targets to elucidate and compare not only the complete collection of DNA-binding transcriptional regulators but also the connectivity of regulatory interactions encoded by each genome sequence.
In a recent study, we have determined the complete sets of DNA-binding transcriptional regulators of the four corynebacteria and have performed a comparative content analysis of these genomes . A collection of 127 DNA-binding transcriptional regulators was identified in the genome sequence of C. glutamicum, whereas 103 regulators were identified in C. efficiens, 63 in C. diphtheriae and 55 in C. jeikeium. The common set of transcriptional regulators encoded by the four corynebacterial genomes consists of only 28 proteins. Considering functional assignments deduced from computational predictions the common DNA-binding transcriptional regulators were grouped into five modules with distinct physiological functions . The functional module "macroelement and metal homeostasis" includes, for instance, the transcriptional regulator McbR, directing the global regulation of almost all aspects of sulphur metabolism, a FurB homolog, most likely involved in regulation of zinc metabolism , and a transcriptional regulator that is homologous to the diphtheria toxin repressor DtxR of C. diphtheriae . The C. glutamicum DtxR protein shares 71% identical amino acid residues with the orthologous counterpart of C. diphtheriae.
The diphtheria toxin repressor DtxR has been shown to be a global transcriptional regulator of iron metabolism in C. diphtheriae . Iron is essential for a variety of cellular processes in virtually all organisms, since it plays important roles as enzyme cofactor or as integral part of cytochromes in oxidation-reduction and energy-generating systems . On the other hand, iron poses problems in biological systems since the oxidized form is poorly soluble and the reduced form is highly toxic. Therefore, bacteria have evolved various mechanisms to counter these problems and to achieve an effective iron homeostasis. In addition, expression of the iron homeostatic machinery is subject to iron-dependent transcriptional regulation, for instance of genes encoding high-affinity iron uptake systems, intracellular iron storage proteins, redox-stress resistance systems, and iron-containing proteins to control overall iron consumption of the cell . In C. diphtheriae, almost 20 binding sites of DtxR have been identified either by targeted genetic experiments or by a global repressor titration assay [14, 15]. Major physiological functions that are controlled by DtxR in response to the iron level include the expression of the diphtheria toxin , the synthesis and export of siderophores , the siderophore-dependent uptake of iron , and the synthesis of specific systems for the utilization of iron from heme, hemin and hemoglobin [17, 18].
In the present study, we have characterized the regulon of the transcriptional regulator DtxR of C. glutamicum by a genome-wide approach using DNA microarray technology. This global strategy along with bioinformatics predictions permitted the identification of DtxR binding sites in the C. glutamicum genome sequence along with a set of target genes that are under direct transcriptional control by DtxR. Binding of purified His-tagged DtxR protein to the upstream region of the respective genes was verified in vitro by DNA band shift assays. The resulting data were used for a comparative analysis of the deduced DtxR regulons in the four sequenced corynebacterial species.
Phenotypic characterization of the dtxR mutant strain C. glutamicum IB2103
Genome-wide transcriptional profiling of the dtxR mutant C. glutamicum IB2103 in comparison to the wild-type strain ATCC 13032 by DNA microarray hybridization
To identify genes that are differentially expressed in the transcriptome of C. glutamicum IB2103, the global gene expression pattern of the dtxR mutant was compared with that of the wild-type strain by DNA microarray hybridization. Since C. glutamicum IB2103 was unable to grow in CGXII minimal medium (Figure 1), both strains were cultivated in shaking flasks to mid-exponential growth phase by using low-iron CGXII medium. The cultures revealed a very similar growth behavior that was characterized by almost identical doubling times of the dtxR mutant (4.8 ± 0.41 h) and the wild-type (4.4 ± 0.18 h), indicating that differences in growth of both strains were apparently minimized under the selected cultivation conditions. At the time when the C. glutamicum strains were grown to an optical density of four (OD600 nm), 10 mg/l FeSO4 was added to the growth medium, and the cultures were cultivated for further 15 min before harvesting cells and preparing total RNA. Addition of iron to the wild-type culture should result in a "switch-off" of expression of those genes that are part of the DtxR regulon, whereas the respective genes should be further on expressed in the dtxR mutant C. glutamicum IB2103. Therefore, genes revealing an enhanced expression in C. glutamicum IB2103 when compared to the wild-type are candidates that might belong to the transcriptional regulatory network of DtxR.
By applying a ratio cut-off of ± 1, corresponding to relative expression changes equal or greater than twofold, a total of 257 genes revealed a differential expression in C. glutamicum IB2103 (see Additional file 1). This number includes 131 genes with significantly increased expression in C. glutamicum IB2103 when compared to the wild-type strain (m-value equal or greater than +1.0) and 126 genes with decreased expression in the dtxR mutant (m-value equal or smaller than -1.0). Several genes showing derepression of transcription in C. glutamicum IB2103 when compared to the wild-type strain encode iron-containing proteins and proteins that are apparently involved in iron transport and metabolism (Figure 2). Consequently, the upstream regions of the respective genes were ideally suited to search for the presence of a common DtxR binding motif.
Computational identification of potential DtxR binding sites in the genome sequence of C. glutamicum ATCC 13032
Identification of DtxR binding sites in the genome sequence of C. glutamicum
Predicted DtxR binding motif2
Distance to translational start codon3
Differential gene expression in C. glutamicum IB2103 measured by
DNA microarray (ratio)4
Real-time RT-PCR (relative expression)5
Verification of differential expression of genes with predicted DtxR binding sites by real-time RT-PCR experiments
Differential expression of the 40 genes identified by bioinformatics screening of the C. glutamicum genome sequence for the presence of DtxR binding sites was examined further by real-time RT-PCR assays using the same experimental setup as applied for DNA microarray hybridizations. The resulting data are summarized in Table 1. The real-time RT-PCR assays demonstrated that the expression of 31 genes was enhanced up to 2000-fold in the dtxR mutant C. glutamicum IB2103 when compared with the wild-type strain. Accordingly, the results can be divided into three categories: (i) Differential expression in C. glutamicum IB2103 of the 24 genes initially identified by the DNA microarray experiment was confirmed by the RT-PCR approach. (ii) Among the genes additionally predicted by HMM searches seven showed indeed a differential expression in the dtxR mutant strain C. glutamicum IB2103. Expression of four genes was enhanced only up to 5-fold, and a further examination of the DNA microarray data indicated that at least two of the respective ratios were close to the experimental cut-off (data not shown). (iii) In the case of nine potential motifs, the prediction by the HMM profile was most likely false-positive, since the corresponding genes showed no significant changes of the expression level in C. glutamicum IB2103, neither by DNA microarray hybridization nor by real-time RT-PCR measurements. Consequently, the combination of DNA microarray hybridization, bioinformatics screening approaches and real-time RT-PCR assays led to a set of genes that are differentially expressed in the dtxR mutant C. glutamicum IB2103 and that are characterized by a putative DtxR binding site within the upstream region.
Binding of purified DtxR protein to the identified sequence motifs as analyzed by DNA band shift assays
To demonstrate binding of the DtxR regulator to the predicted 19-bp motifs by DNA band shift assays, a modified DtxR protein was constructed and subsequently purified. For this purpose, the coding region of the dtxR gene was fused with a 3' extension encoding six histidine residues and amplified by PCR. The resulting DNA fragment was cloned in E. coli into the expression vector pEC-XK99E  resulting in plasmid pIB4001 that is suitable for heterologous overexpression of the modified DtxR protein upon IPTG induction. The His-tagged DtxR protein was purified to homogeneity from an induced E. coli culture by means of Ni-TED columns and positively identified by MALDI-TOF mass spectrometry and peptide mass fingerprinting (data not shown). Moreover, plasmid pIB4001 was transferred into the dtxR mutant C. glutamicum IB2103 by electroporation to prove that the His-tagged DtxR protein is functional in vivo. This control experiment demonstrated that the growth deficiency of the dtxR mutant C. glutamicum IB2103 in CGXII minimal medium can be complemented by plasmid pIB4001 but not by the empty cloning vector pEC-XK99E (data not shown).
Subsequent DNA band shift assays were performed with 42 pmol of purified DtxR protein (Figure 3D). The presence of purified DtxR protein caused a band shift of almost all tested 40 mer DNA fragments, with exception of a 40 mer region located in front of the cg1120 gene that encodes the ArsR-type regulatory protein RipA . The reason for this observation is currently unknown since binding of the DtxR protein to the upstream region of cg1120 was described earlier . Nevertheless, the DNA band shift assays confirmed in vitro the binding of the DtxR regulator to specific DNA fragments containing the common 19-bp motif that was identified in the upstream region of differentially expressed genes.
Localization of DtxR binding sites in mapped promoter regions of differentially expressed genes
Bioinformatics characterization and functional dissection of the DtxR regulon of C. glutamicum
The genes belonging to the DtxR regulon of C. glutamicum can be divided into seven functional categories (Figure 5) according to the predicted physiological roles of the deduced proteins . The functional classification yielded a prominent group of 26 genes that encode iron transport systems or siderophore interacting proteins. Accordingly, this functional category includes almost half of the genes that are under direct transcriptional control by DtxR. Additionally, the genes cg2445 (hmuO), cg2782 (ftn) and cg3327 (dps) were identified as part of the DtxR regulon of C. glutamicum. The respective proteins specify the heme oxygenase HmuO involved in the release of iron from the heme moiety , a putative ferritin most likely involved in iron storage  and a Dps homolog that can act either in iron storage or as DNA-protecting anti-redox agent . Expression of the ftn and dps genes was decreased in the dtxR mutant C. glutamicum IB2103 as was the expression of the sdh gene cluster that encodes the components of the iron-containing succinate dehydrogenase complex of C. glutamicum . The genes belonging to other categories of the DtxR regulon encode proteins without a precise functional prediction, secreted proteins of unknown function or even hypothetical proteins (Figure 5), with the exception of the cgl gene cluster that represents the main DNA restriction and modification system of C. glutamicum ATCC 13032  and the cysI-fpr2 gene region involved in assimilatory sulphate reduction .
Among the set of regulatory genes that are under direct control by DtxR, three DNA-binding transcriptional regulators were identified. This set includes the cg1120 (ripA) gene that was recently shown to be involved in iron metabolism of C. glutamicum  as well as the cg0527 and cg3082 genes encoding members of the ArsR family of regulatory proteins that typically exert metal-dependent transcriptional regulations in bacterial cells . These data clearly indicate that the DtxR protein not only regulates the expression of genes playing a direct role in iron metabolism of C. glutamicum but also that of genes encoding transcriptional regulators, which can direct the expression of further target genes at another level of regulatory hierarchy.
Genome-wide identification of putative DtxR binding sites in corynebacterial genome sequences and comparative content analysis of the deduced DtxR regulons
Predicted DtxR binding sites in corynebacterial genome sequences
Predicted DtxR binding site
iron ABC transport system
diphtheria toxin precursor
succinate dehydrogenase complex
ArsR-family regulatory protein
preprotein translocase subunit SecY
iron ABC transport system
siderophore biosynthesis protein
iron ABC transport system
preprotein translocase subunit SecA
AraC-family regulatory protein
iron ABC transport system
peptide transport protein
DNA binding protein
ubiquinol-cytochrome C reductase
non-ribosomal peptide synthase
succinate dehydrogenase complex
ArsR-family regulatory protein
iron ABC transport system
iron ABC transport system
cation transporting P-type ATPase
DNA binding protein
iron ABC transport system
ABC transport system
collagen binding protein
iron ABC transport system
Fe-S cluster assembly system
iron utilization protein
ABC transport system
solute binding protein
iron ABC transport system
siderophore synthesis system
iron ABC transport system
HTH_3-family regulatory protein
iron ABC transport system
To identify either species-specific or common genes that are under transcriptional control by DtxR in corynebacteria, functional predictions of the coding regions located downstream of the determined DtxR binding sites were compared between the four sequenced species. Orthologous proteins were identified within the complete collection of potentially DtxR-regulated genes by using the BLASTP algorithm  to detect amino acid sequence similarities and by performing synteny analyses of the respective genomic context. The resulting data were visualized as Venn diagrams (Figure 6B). First of all, it is apparent that the four corynebacteria share no common gene that is under direct transcriptional control by the DtxR regulator. This unexpected result might be due to the exceptional collection of species-specific genes that are involved in iron metabolism of C. jeikeium . The set of orthologous genes that are most likely under transcriptional regulation by DtxR in three corynebacterial species includes the orthologs of the cg0466 gene cluster (Figure 5) that encode components of an iron ABC transport system in C. glutamicum, C. diphtheriae and C. jeikeium and the orthologs of the cg0926 gene cluster that are apparently involved in iron uptake in C. glutamicum, C. efficiens and C. jeikeium. Orthologs of the sdh gene cluster and the ftn gene, whose expression decreased in the dtxR mutant C. glutamicum IB2103, are apparently under direct transcriptional control by DtxR in C. efficiens and C. diphtheriae, indicating that the DtxR regulator might be involved in the activation of gene expression also in these species. Regulatory genes orthologous with cg0527 of C. glutamicum are present in the genomes of C. efficiens and C. diphtheriae  and all are presumably regulated by DtxR, suggesting similar hierarchical topologies of the respective DtxR regulons. However, most of the identified genes that are under transcriptional control by DtxR are species-specific, although one has to keep in mind that some non-orthologous genes might share similar physiological functions. This comparative content analysis led to the conclusion that the DtxR regulons of corynebacteria are quite different in their genetic composition, although they rely on an orthologous regulatory protein and similar 19-bp motifs for regulator binding. Nevertheless, some interesting regulatory features deduced from the experimental characterization of the DtxR regulon of C. glutamicum are apparently realized also in the topology of DtxR regulons of other corynebacteria.
In the present study, the genetic network of the transcriptional regulator DtxR was examined in C. glutamicum, and the topology of the DtxR regulon was deduced from DNA microarray hybridizations, bioinformatics approaches, in vivo studies of differential gene expression, and DNA band shift assays. For these purposes, a defined deletion was constructed within the coding region of the dtxR gene, resulting in the mutant strain C. glutamicum IB2103 that was unable to grow in CGXII minimal medium under high-iron conditions. However, when the dtxR mutant C. glutamicum IB2103 was cultivated in low-iron CGXII medium, it revealed the same growth behavior as the wild-type control. These experiments showed that the dtxR gene is dispensable in C. glutamicum under specific culture conditions, as it was previously demonstrated for the orthologous regulatory genes dtxR, dmdR1 and ideR of C. diphtheriae, Streptomyces coelicolor and Mycobacterium smegmatis, respectively [33–35]. On the other hand, the ideR gene of Mycobacterium tuberculosis can be inactivated only in the presence of a second functional copy of the gene or when a second-site suppressor mutation alleviates the lethal effects of ideR inactivation, making ideR an essential gene in this bacterial species .
As observed with the defined dtxR mutant strain C. glutamicum IB2103, growth of the dtxR transposon mutant C7(β)18.5 of C. diphtheriae was dependent on the amount of iron that was added to the culture medium . The C7(β)18.5 mutant of C. diphtheriae exhibited an extended lag phase and slower exponential growth in low-iron medium when compared with the parental C7(β) wild-type strain. Moreover, the C7(β)18.5 mutant grew only poorly in high-iron medium when compared with a control medium containing no additional iron sources, and it was more-easily killed by exposure to high-iron conditions and H2O2 than was the parental strain . Since the DtxR protein apparently regulates the expression of iron uptake systems, it might also be involved in protecting the cell from damage that is caused by high intracellular iron concentrations. Transcriptional deregulation of iron homeostasis favors the Fenton reaction, leading to the production of hydroxyl radicals that can damage all biological macromolecules . Therefore, transcriptional regulation of iron metabolism by the DtxR protein in C. glutamicum is not only necessary for economic reasons but also to avoid inevitable iron toxicity. In E. coli, iron homeostasis and oxidative stress response are closely interconnected and strictly controlled by the transcriptional regulator Fur . Inactivation of the fur gene enhanced the sensitivity of the cell to redox stress, a physiological effect that can be reversed by iron chelation, by inhibiting ferric iron transport or by enhancing the iron storage capacity of the cell .
The dtxR mutant C. glutamicum IB2103 was further on used to compare the genome-wide expression pattern with that of the wild-type strain by DNA microarray hybridizations. In principle, inactivation of a gene encoding a repressor results in constitutive expression of those genes that are directly regulated by the respective protein. Therefore, comparative DNA microarray hybridizations are typically carried out in such a way that the relevant genes of the wild-type control are repressed and those of the mutant strain are de-repressed . Since the dtxR mutant C. glutamicum IB2103 grew only in low-iron medium containing a trace amout of iron, the genes of the DtxR regulon are apparently de-repressed in both the mutant strain and the wild-type control. To detect differential gene expression in C. glutamicum IB2103, we applied an alternative experimental setup by the subsequent addition of an appropriate amount of iron to low-iron growth medium. Indeed, the DtxR repressor switched off the expression of its target genes in the wild-type strain, whereas the target genes were further on expressed in the dtxR mutant. This physiological effect was detectable on a global scale by DNA microarray experiments and enabled the screening for differentially expressed genes that were later on assigned to the DtxR regulon of C. glutamicum. In addition to genes belonging to the DtxR regulon, the DNA microarray hybridization identified numerous coding regions whose expression was positively or negatively influenced in response to the iron stimulus. These genes are considered to be subject to indirect regulation by iron and are apparently not part of the DtxR regulon of C. glutamicum since they lack DtxR binding sites in their upstream regions. Investigating the physiological role of these genes in the cellular response to the subsequent addition of iron might eventually allow a better understanding of the molecular mechanisms used by C. glutamicum to counter high-iron conditions.
Furthermore, the combination of bioinformatics predictions and DNA band shift assays resulted in the identification of DtxR binding sites within the upstream region of 31 genes and gene clusters, suggesting that at least 64 genes are under direct transcriptional control by DtxR in C. glutamicum. For a closer examination of the identified DtxR binding sites, transcriptional start sites of four differentially expressed genes were determined and the corresponding promoters were mapped. Three DtxR binding sites were found to overlap the deduced promoter regions, suggesting that DtxR acts as transcriptional repressor by blocking RNA polymerase binding and thus preventing expression of the respective genes . Similar results were reported upon promoter mapping and localization of DtxR binding sites in C. diphtheriae  and of IdeR binding sites in M. smegmatis and M. tuberculosis [41, 42]. On the other hand, the DtxR binding sites of cg2782, cg3327 and cg0445 were located upstream of the deduced -35 promoter region. Since the respective genes showed a decreased expression in C. glutamicum IB2103 during DNA microarray hybridization, this location is more consistent with an activating function of the corresponding transcriptional regulator . In general, transcriptional regulators activate the expression of genes in such a way that they facilitate interaction of the RNA polymerase with the respective promoters. However, the large distance of the DtxR binding site to the -35 promoter region of the cg0445 gene cluster that encodes the iron-containing succinate dehydrogenase complex of C. glutamicum suggests an alternative activation mechanism. In this context it is noteworthy that the cg0445 gene cluster is also under direct transcriptional control by the RipA repressor , a member of the AraC-family of DNA-binding transcriptional regulators . Some members of the AraC protein family fulfill their regulatory function by using two DNA binding sites and a DNA looping mechanism to control gene expression . The DtxR binding site in front of cg0445 is located directly adjacent to the RipA binding site A, and DtxR binding might therefore interfere with RipA binding to exert its regulatory function. Since expression of the ripA gene is also controlled by DtxR, the corresponding network topology of the DtxR regulon represents a coherent feed-forward loop of type 4, in which the direct connection to the target gene is activating and the indirect connections are both repressing . In such a way environmental input signals can be integrated into the transcriptional regulatory network at different levels of regulatory hierarchy to control expression of the succinate dehydrogenase complex.
In addition to the cg0445 gene cluster, the dps (cg3327) and ftn (cg2782) genes showed a decreased expression in the dtxR mutant C. glutamicum IB2103. Dps-like proteins may function either as anti-redox agents or iron storage proteins, whereas ferritins act primarily in iron storage . These data indicated that the DtxR protein can exert a regulatory role as positive modulator of expression of genes that are involved in iron storage and DNA protection. This mechanism of transcriptional regulation is reasonable since the iron storage and protection genes are not expressed under low-iron conditions, whereas both, the iron storage capacity of the cell and the protection of DNA, increase under high-iron conditions by activating the respective genes. In M. tuberculosis, expression of the iron storage genes bfrA and bfrB, encoding a bacterioferritin and a ferritin-like protein, is induced also by iron and IdeR [36, 42]. Binding of IdeR to the regulatory region of bfrA indicated that the gene is transcribed from a promoter that is activated by iron and binding of IdeR, since induction of bfrA gene expression was detected in the wild-type but not in an ideR mutant strain. The transcriptional control of iron storage in E. coli was also shown to be positively regulated by iron but no direct interaction of Fur with the regulatory region of the bfrA and ftn genes has been observed [45, 46].
The comparative content analysis of DtxR binding sites in four corynebacterial genome sequences provided further insights into the topology of the DtxR regulon of C. glutamicum. The deduced consensus sequences of DtxR binding sites are not only very similar among the corynebacterial species but also resemble IdeR and DmdR1 binding sites that were detected in M. tuberculosis and S. coelicolor, respectively [42, 47]. Consequently, DtxR-like regulatory proteins utilize conserved recognition signals in different actinobacteria, as is the situation with DNA binding sites of the Fur repressor in gamma-proteobacteria . On the other hand, the comparative content analysis revealed that most of the DtxR-regulated genes are species-specific in the four sequenced corynebacteria, indicating that a wide variety of genetic information is used in the individual species to provide an effective iron homeostasis. Varying genetic information can be gained for instance by horizontal gene transfer, as suggested for the C. jeikeium genome that contains large sets of DtxR-regulated iron acquisition genes on mobile genetic elements . As iron metabolism is closely connected to iron toxicity, any additional genetic information regarding iron metabolism has to be integrated into the pre-existing DtxR regulon to avoid detrimental consequences for the bacterial cell. Only in case of similar DNA binding sites being present in the regulatory region of the respective genes, it is generally ensured that the genes fall under direct regulation by the DtxR protein and that the bacterium can benefit from the acquired genetic information. The close functional relationship between DNA binding sites of at least DtxR and IdeR is indeed apparent from the observation that the regulatory proteins can interact with operators of the cognate regulons in either corynebacteria or mycobacteria .
In summary, the presented experimental work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by the diphtheria toxin repressor homolog DtxR and of the response of this bacterium to changing environmental iron levels. The results demonstrate that DtxR acts as a dual transcriptional regulator with a major role in controlling the expression of genes involved in iron metabolism. The DtxR protein exerts its dual regulatory function as repressor of genes participating in iron uptake and iron utilization and as activator of genes responsible for iron storage and DNA protection. Moreover, the data suggest that the DtxR protein acts as global regulator by controlling the expression of other regulatory proteins that might take care of an iron-dependent regulation of a broader transcriptional network of C. glutamicum genes.
Bacterial strains and growth conditions
Wild-type strain C. glutamicum ATCC 13032 (American Type Culture Collection, Manassas, VA) was used to characterize the DtxR regulon. C. glutamicum strains were routinely grown at 30°C in Luria-Bertani medium . Growth of C. glutamicum strains in CGXII minimal medium  containing 30 mg/l protocatechuic acid was monitored in time intervals of 1 h with the nephelometer Nephelostar Galaxy (BMG Laboratories, Offenburg, Germany). Standard CGXII minimal medium (10 mg/l FeSO4) and low-iron CGXII medium (0 mg/l FeSO4) were used for growth assays . Growth of shaking flask cultures was monitored by measuring the optical density at 600 nm with an Eppendorf Bio Photometer. E. coli DH5αMCR was used for standard cloning procedures and grown at 37°C in Luria-Bertani medium  supplemented with 2 g/l glucose. Selection for the presence of plasmids was performed with kanamycin (50 μg/ml for E. coli and 25 μg/ml for C. glutamicum). Isopropyl beta-D-thiogalactoside (IPTG) was used to induce dtxR expression by the P trc promoter of pEC-XK99E .
DNA isolation, manipulation and transfer
Vector DNA was prepared from E. coli cells by an alkaline lysis technique using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). Chromosomal DNA of C. glutamicum was prepared as described previously . Modification of DNA, analysis by agarose gel electrophoresis and ligation were performed by standard procedures . Transformation of E. coli and C. glutamicum cells was performed by electroporation [52, 53].
PCR techniques and dtxR mutant construction
PCR experiments were carried out with a PTC-100 thermocycler (MJ Research, Watertown, MA), Pwo DNA polymerase (Roche Diagnostics, Mannheim, Germany) and chromosomal C. glutamicum DNA as template. PCR products were purified by using the PCR Purification Spin Kit (Qiagen). Oligonucleotides used for PCR amplification were purchased from Operon Biotechnologies (Cologne, Germany). The gene SOEing method  was applied to construct a pK18mobsacB derivative that is suitable to perform an allelic exchange of the dtxR gene in the chromosome of C. glutamicum ATCC 13032 . The primers used were cg2103del1 (GATCTAGAATTCCCAAGGCGTGAGATGACAG), cg2103del2 (GAGCACGCAGAGGAACAAT), cg2103del3 (ATTGTTCCTCTGCGTGCTCTCAAGCAGATGAGCCTGAT), and cg2103del4 (GATCTAGGATCCTTCTACGCGGACTGCATGT). The DNA fragment was digested with EcoRI and BamHI and cloned into the vector pK18mobsacB. The resulting plasmid pIB2103 carries a modified dtxR gene that is specified by a defined deletion of 338 nucleotides. Gene replacement in the chromosome of C. glutamicum ATCC 13032 resulted in the dtxR mutant strain C. glutamicum IB2103. For genetic complementation of mutant strain C. glutamicum IB2103, the dtxR coding region was amplified by PCR with the primer pair compl1 (GATCTAGAATTCAAAGGAGGACAACCATGAAGGATCTGGTCGATAC) and compl2 (GATCTAGGATCCGTGTGTTAGCCCTCAACC). The PCR product was digested with EcoRI and BamH and cloned into the compatible sites of the IPTG-inducible expression vector pEC-XK99E . The resulting plasmid pIB4000 was transferred into C. glutamicum ATCC 13032 and C. glutamicum IB2103 by electrotransformation.
Total RNA preparation and DNA microarray hybridization
For the preparation of total RNA, C. glutamicum cultures were grown in minimal medium CGXII. Approximately 1 × 109 cells from exponentially growing cultures were harvested by centrifugation with 11,000 g for 15 s and subsequently transferred into liquid nitrogen. RNA isolation, cDNA synthesis, labeling of probes, and DNA microarray hybridization were performed as described previously . The experiments were carried out in duplicate using label swapping. Normalization and evaluation of data was accomplished by the EMMA microarray data analysis software using a m-value cut-off of ± 1, which corresponds to relative expression changes equal or greater than twofold . Since each DNA microarray contains four replicates per gene, a total number of eight spots per gene was available for calculating differential gene expression.
Bioinformatics tools for DtxR binding site prediction
The annotated version of the C. glutamicum ATCC 13032 genome sequence  was used to perform a genome-wide screening for putative DtxR binding sites. The search was accomplished by Hidden Markov model (HMM) analysis using the HMMER software package . A set of predicted DtxR binding sites was aligned by means of the CLUSTAL X program , and the resulting alignment was used to create a HMM by using the HMMBUILD module. The calculated HMM profile along with the HMMSEARCH module was applied to screen the C. glutamicum genome sequence for the presence of DtxR binding sites. The genomic positions of the resulting hits were correlated with coding sequences that revealed differential expression in the dtxR mutant when compared with the wild-type strain by DNA microarray hybridization. The experimentally verified DtxR binding sites of C. glutamicum were aligned with the CLUSTAL X program and used to generate a HMM profile for an iterative screening of the genome sequences of C. efficiens , C. diphtheriae  and C. jeikeium .
Real-time reverse transcription (RT)-PCR
Purified total RNA of C. glutamicum cultures was used for real-time RT-PCR experiments with the LightCycler instrument (Roche Diagnostics) and the Quanti-Tect SYBR Green RT-PCR Kit (Qiagen). Oligonucleotides used to measure relative gene expression were purchased from Operon Biotechnologies. Verification of RT-PCR products was performed by melting curve analysis. Differences in gene expression were determined by comparing the crossing points of two samples measured in duplicate. Crossing points were calculated by the LightCycler software (Roche Diagnostics).
Construction and purification of a His-tagged DtxR protein
A PCR product fusing the coding region of the C. glutamicum dtxR gene with a nucleotide sequence encoding a C-terminal His tag was generated by using the primer pair dtxR-His1 (GATCTAGAATTCAAAGGAGGACAACCATGAAGGATCTGGTCGATA) and dtxR-His2 (GATCTAGGATCCTTAATGGTGATGGTGATGGTGGCCCTCAACCTTTTCTAC). The PCR product was digested with EcoRI and BamHI and cloned into the IPTG-inducible expression vector pEC-XK99E . The resulting plasmid pIB4001 was transferred to E. coli DH5αMCR and to C. glutamicum strains by electroporation. To isolate the His-tagged DtxR protein, E. coli DH5αMCR (pIB4001) was grown for 16 h in Luria-Bertani medium containing 50 μg/ml kanamycin and 0.1 mM IPTG. Approximately 4 × 1010 cells were harvested by centrifugation, resuspended in LEW buffer (50 mM NaH2PO4, 300 mM NaCl, one tablet of Roche Complete Mini protease inhibitors; pH 8.0) and transferred into a RiboLyser tube (Hybaid, Heidelberg, Germany). Cell disruption by means of the RiboLyser instrument was carried out with a speed rate of 6.5 and two time intervals of 30 s. The His-tagged DtxR protein was purified from the protein crude extract with Protino Ni-TED 1000 packed columns (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. After loading of the protein crude extract, the resin column was washed twice with 2 ml LEW buffer. The His-tagged DtxR protein was eluted with 2.5 ml LEW buffer containing 10 mM imidazole and finally stored at -20°C. The resulting eluate was concentrated by using Amicon ultra-4 5000 MWCO centrifugal filter units (Millipore, Schwalbach, Germany) and subsequently analyzed by SDS-PAGE . The protein concentration was determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories, Munich, Germany). To verify the purification of the His-tagged DtxR protein, an aliqout of the eluate was enzymatically digested with modified trypsin (Promega, Mannheim, Germany) and the resulting peptide mass fingerprint was determined by MALDI-TOF mass spectrometry, applying an Ultraflex mass spectrometer (Bruker Daltonics, Bremen, Germany) and the MASCOT software.
DNA band shift assays to characterize binding of the purified DtxR protein
DNA band shift assays were performed with Cy3-labeled 40 mer oligonucleotides (Operon) that were annealed with corresponding complementary oligonucleotides to double-stranded DNA fragments by heating at 94°C for 5 min and annealing on ice for 15 min. During band shift assays, 42 pmol of purified His-tagged DtxR protein were mixed with 0.05 pmol DNA, 200 μM CoCl2, 15% (v/v) glycerol, and DtxR binding buffer (20 mM Na2HPO4, 50 mM NaCl, 5 mM MgCl2, 2 nM DTT, 10% (v/v) glycerol, 100 μg/ml BSA; pH 7.0 ), to get a total volume of 20 μl. The assay was incubated at room temperature for 15 min and then separated with a 2% agarose gel (containing 150 mM CoCl2) prepared in gel buffer (20 mM Na2HPO4; pH 7.0). A voltage of 80 V was supplied for 1.5 h. The agarose gel was scanned with a Typhoon 8600 Variable Mode Imager (Amersham Biosciences Europe, Freiburg, Germany). During displacement experiments, increasing concentrations of non-labeled competitor DNA (0.05 to 1 pmol) were added to the reaction batch.
Identification of transcriptional start sites by the RACE method
For the identification of transcriptional start sites, total RNA was isolated from C. glutamicum wild-type cultures grown in CGXII medium. RACE primers (18 mer oligonucleotides) binding 200 to 300 nucleotides downstream of the annotated translational starts of investigated genes along with 1 μg of total RNA were used for cDNA synthesis. RACE primer sequences will be provided by the authors upon request. The resulting cDNA was modified and amplified by two additional PCRs using the 5'/3' RACE Kit second generation (Roche Diagnostics). PCR products were cloned into the pCR2.1-TOPO vector (Invitrogen, Karlsruhe, Germany) and transferred into competent E. coli TOP10 cells. DNA sequencing of cloned RACE products was performed by IIT Biotech (Bielefeld, Germany).
The authors thank Carola Eck for excellent technical assistance during MALDI-TOF mass spectrometry and Karina Brinkrolf for valuable discussions.
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