Zur is a zinc-dependent transcriptional repressor of zinc uptake systems in bacteria. In the present study, we examined the role of Corynebacterium glutamicum Zur in the zinc-inducible expression of two genes: one encoding a cation diffusion facilitator (zrf) and the other a metal-translocating P-type ATPase (zra). Both genes were shown to be involved in zinc resistance. Disruption of the zur gene encoding Zur resulted in constitutive expression of zrf and zra mRNAs. An electrophoretic mobility shift assay revealed that the Zur protein binds to the zrf and zra promoters, for which the in vivo activities were up-regulated in response to excess zinc. Interestingly, the in vitro DNA binding activity of Zur was inhibited by zinc, in contrast to its zinc-dependent binding to the promoter region of a zinc-repressible ABC transporter gene znuB2. A 21-bp motif found in the Zur binding site overlaps the putative –35 region of both the zrf and zra promoters. This new motif is a 10-1-10 direct repeat sequence distinct from the 10-1-10 inverted repeat sequence of a previously identified Zur box for zinc-dependent binding. Nevertheless, their 10-bp elements share some sequence similarities. Overexpression of zur in the zur deletion mutant background, as well as deletion of zur in the zrf and zra double deletion mutant background, resulted in decreased resistance to zinc. These results suggest that the direct negative control of both zinc uptake and export systems by Zur is central to C. glutamicum zinc homeostasis and is effected in distinct ways.
Zinc (Zn) has a crucial role in various enzymatic reactions and in protein structures. At the same time, Zn, similar to other essential transition metals, is toxic to living cells when present in excess. In bacteria, the intricate control of uptake, sequestration/storage and export systems for Zn maintains the homeostatic balance of this metal in a cell in response to changing environmental conditions . Several types of metal-sensing transcriptional regulators for metal homeostasis have been identified [2-5]. Among them, Zur, belonging to the iron (Fe) uptake regulator Fur family, acts as a repressor of genes related to Zn availability; the Zur-dependent expression is derepressed under Zn deficiency . This regulatory system is conserved in many bacteria. A high-affinity ABC transporter for Zn uptake is regulated by Zur in Escherichia coli [7, 8], Bacillus subtilis [9, 10], Mycobacterium tuberculosis  and Streptomyces coelicolor [12, 13], amongst others. Zur also directly regulates other genes. For example, Zn-containing ribosomal proteins are replaced with their non-Zn-containing paralogues whose Zur-dependent expression is derepressed under Zn limiting conditions [11-16]. These ribosomal proteins, abundant in a cell, play a role in the storage and mobilization of Zn.
Corynebacterium glutamicum is a high-GC Gram-positive bacterium, which is widely used for the industrial production of amino acids, and is expected to be a versatile workhorse for biofuels and biochemicals [17-23]. In addition, this nonpathogenic soil bacterium should be a model organism for the suborder Corynebacterineae, including medically important pathogens such as Corynebacterium diphtheriae and M. tuberculosis [24-26]. The regulation of homeostasis of Zn that is required for various types of enzymes is of interest for establishing fundamental characteristics of the cellular metabolism in C. glutamicum, and these findings are also valuable from the biotechnological point of view. The Zur regulon in C. glutamicum was recently investigated by transcriptome analysis . Five transcription units, including two sets of ABC transporters, are directly regulated by Zur. Zn-dependent binding of Zur to their promoter regions in vitro has been confirmed. It has been also reported that, in C. diphtheriae, the expression of three genes putatively involved in Zn uptake is repressed by Zur under Zn-replete conditions .
In the present study, we describe the role of Zur in expression of two Zn-inducible transporter genes: cgR_1359 (designated here zrf) and cgR_0148 (designated here as zra), both of which are involved in Zn resistance in C. glutamicum R. zrf encodes a member of the cation diffusion facilitator (CDF) family, whereas zra encodes a putative metal-translocating P-type ATPase. Disruption of zur results in constitutive expression of the zrf and zra mRNAs. We show that the Zur protein binds to the promoter regions of zrf and zra in vitro, and also that the binding is inhibited by Zn. Interestingly, the newly-found Zur binding consensus sequence is a 10-1-10 direct repeat, in contrast to the 10-1-10 inverted repeat of the previously identified Zn-dependent binding sites in the promoter regions of Zn-repressible genes. These results indicate that C. glutamicum Zur directly represses both the Zn-repressible and Zn-inducible genes in different ways.
The effects of the deletion of zrf, zra and zur on Zn resistance
Although it was recently reported that Zur directly represses the expression of two sets of putative ABC transporters for Zn uptake in C. glutamicum , the physiological role of this regulatory system has remained unclear. We hypothesized that the up-regulation of the Zn uptake systems by zur disruption results in decreased resistance to Zn. However, growth of a zur (cgR_2151) deficient mutant strain under various concentrations of Zn was comparable to that of the wild-type strain (data not shown). This could be the result of a Zn export system.
CDF and P-type ATPase family members act as Zn export systems in bacteria . On the genome of C. glutamicum ATCC 13032 [29, 30] and C. glutamicum R , there are multiple genes belonging to the CDF and the P-type ATPase families. Possible diversity in the function and regulation of these genes is largely unknown, although a CDF gene of C. glutamicum ATCC 13032 has been implicated in cobalt (Co) resistance . In the present study, the involvement of a CDF gene (zrf) and a P-type ATPase gene (zra) of C. glutamicum R in Zn resistance was examined. Transposon-insertion mutants deficient in each of zrf and zra (the zrf::Tn and zra::Tn strains, respectively) were cultured in minimal BTM medium (containing 3.5 μm ZnSO4) or the same medium supplemented with 100 μm ZnSO4, and their growth was compared with that of the wild-type strain. Disruption of each of these genes had no effect on growth under low Zn conditions (Fig. 1A). Under high Zn conditions, growth of the zra::Tn strain was slower and its final cell density was lower than that of the wild-type strain (Fig. 1B). By contrast, disruption of zrf had no effect on Zn resistance. However, growth of a zra zrf double in-frame deletion mutant (ΔzraΔzrf) was inhibited more severely than that of a zra single in-frame deletion mutant (Δzra) under high Zn conditions (Fig. 1C) despite their growth being comparable under low Zn conditions (data not shown). A plasmid carrying each of the zra and zrf genes under the control of a constitutive promoter was introduced into the ΔzraΔzrf strain, and growth of the resultant strains was compared with that of the double deletion mutant strain transformed with a vector plasmid without the target gene. Introduction of each of these genes restored growth of the ΔzraΔzrf strain under high Zn conditions (Fig. 1D) but had minimal effect on growth under low Zn conditions (data not shown). These results indicate that each of zra and zrf confers Zn resistance in C. glutamicum; zra is more effective than zrf.
To examine the effects of inactivation of Zur, a transcriptional repressor of putative Zn uptake systems, on the mutants deficient in the Zn export systems, a zra zur double deletion mutant (ΔzraΔzur) and a zra zrf zur triple deletion mutant (ΔzraΔzrfΔzur) were constructed. The ΔzraΔzrfΔzur strain did not grow well in minimal BTM medium. When cultured in nutrient-rich A medium supplemented with 100 μm ZnSO4, its growth was significantly slower than that of the parent double deletion mutant (Fig. 1E). The effects of zur deletion in the ΔzraΔzrf strain on growth were small when cultured without the Zn supplementation (data not shown). On the other hand, growth of the ΔzraΔzur strain was slightly better than that of the Δzra strain when cultured in nutrient-rich medium supplemented with 1 mm ZnSO4 (Fig. 1F). Their growth was comparable without Zn supplementation (data not shown). These results suggest that the effects of the up-regulation of Zn uptake systems by zur disruption on Zn resistance are counteracted by the presence of both the Zn export systems encoded by zrf and zra.
Zn-inducible expression of zrf and zra under the control of Zur
To examine the expression of the zrf and zra genes in response to Zn, C. glutamicum R wild-type cells were cultured in minimal BTM medium for 4 h, and the exponentially-growing cells were then supplemented with 100 μm ZnSO4. The time course of changes in gene expression after supplementation with the suboptimal concentration of ZnSO4 was analyzed by quantitative RT-PCR. zrf mRNA that was barely detected before supplementation with ZnSO4 increased markedly within 15 min of supplementation (Fig. 2A). The level of zra mRNA increased 2.5-fold upon 60 min of exposure to excess Zn (Fig. 2B). Disruption of zur resulted in increased levels of zrf and zra mRNAs under low Zn conditions (data not shown). This is consistent with results of recent transcriptome analysis of C. glutamicum ATCC 13032 showing that the expression of cg1447 (which corresponds to zrf in C. glutamicum R) is up-regulated in a zur mutant . The zra orthologue is absent on the genome of C. glutamicum ATCC 13032. This might be a result of indirect effects acting through the derepression of high-affinity Zn uptake systems under the control of Zur. To determine the contribution of two sets of the Zur-dependent, Zn-repressible ABC transporter genes, znuA1B1C1 (cgR_2535-cgR_2537-cgR_2536) and znuA2B2C2 (cgR_0041-cgR_0042-cgR_0043), to the effects of zur deletion, a zur znuB1 znuB2 triple deletion mutant (ΔzurΔznuB1ΔznuB2) was constructed. However, the high expression level of zrf mRNA was observed irrespective of Zn concentrations in the ΔzurΔznuB1ΔznuB2 strain, as well as in the Δzur strain (data not shown), indicating that the effects of zur deletion on zrf expression are not dependent on the up-regulation of these Zn uptake system genes.
When a plasmid carrying the zur gene under the control of a constitutive promoter was introduced into the Δzur strain, the up-regulation of the zrf and zra genes in response to exposure to excess Zn was restored, as expected (Fig. 2C,D). As a control, the Δzur strain transformed with a vector plasmid without the target gene was used, and the constitutive expression of zrf and zra was confirmed. We found that the zur-complemented strain showed more severe growth defect compared to the Δzur control strain when cultured in minimal BTM medium supplemented with 100 μm ZnSO4 (Fig. 2F). Smaller inhibitory effects of the zur overexpression on growth were observed without Zn supplementation. It should be noted that the expression of zrf (Fig. 2C) and zra (Fig. 2D) in addition to the znu genes (znuA2 expression representatively shown in Fig. 2E) was significantly repressed in the zur-complemented strain compared to the Δzur control strain. Taken together, it is possible that Zur directly represses the expression of not only the Zn-repressible znu genes, but also the Zn-inducible zrf and zra genes.
Zn-inducible activity of the zrf and zra promoter
In an attempt to determine the mechanism responsible for the Zn-inducible expression of zrf and zra, their promoter regions were located. On the chromosome of C. glutamicum R, zrf is oriented in the same direction as its upstream and downstream hypothetical genes (Fig. 3). The intergenic region between cgR_1358 and zrf is 62 bp in length, whereas that between zrf and cgR_1360 is 15 bp in length. When C. glutamicum wild-type cells were exposed to excess Zn as described above, the level of cgR_1358 mRNA did not change, and that of cgR_1360 mRNA increased two-fold in response to excess Zn (data not shown). These results are in contrast to the marked responsiveness of zrf mRNA to Zn (Fig. 2A). Therefore, it is likely that the zrf gene is transcribed under the control of its own promoter as a monocistronic mRNA.
zra is oriented in the same direction as the upstream hypothetical cgR_0147 gene and the downstream transposase-related cgR_0149 gene (Fig. 3). The cgR_0147-zra and zra-cgR_0149 intergenic regions are 123 and 288 bp in length, respectively. Expression of the upstream cgR_0147 gene showed no discernible response to excess Zn, unlike the zra gene (data not shown), suggesting that zra is transcribed under the control of its own promoter.
To confirm the promoter activity, a 400-bp region upstream of zrf and a 600-bp region upstream of zra were fused to a lacZ reporter gene, and the respective gene fusions were integrated into the chromosome of the C. glutamicum R wild-type strain. The resulting strains were cultured in minimal BTM medium supplemented with ZnSO4 at various concentrations, and β-galactosidase activity in their logarithmic growth and stationary phases was measured (Fig. 4). Some inhibition of cell growth was observed in the presence of over 100 μm ZnSO4 (Fig. 4C). In the logarithmic growth phase, activity of the zrf promoter was barely detected at 3.5 μm ZnSO4, although the activity increased as the concentration of ZnSO4 increased to 50 μm (Fig. 4A). When cells were cultured in the presence of over 25 μm ZnSO4, the zrf promoter activity increased two- to four-fold in the stationary phase relative to that in the logarithmic growth phase. The activity remained strictly repressed at lower concentrations of ZnSO4.
The zra promoter activity was also enhanced by Zn, although its responsiveness to Zn was not as prominent as that of the zrf promoter (Fig. 4B). The weak responsiveness of zra to Zn under these conditions is consistent with observations for its mRNA expression level (Fig. 2B).
In vitro binding of Zur to the promoter regions of the zrf and zra genes
To examine DNA binding activity in vitro, the Zur protein was expressed in E. coli and purified. An electrophoretic mobility shift assay (EMSA) was carried out with the Zur protein and the 400-bp promoter region of zrf used in the above lacZ reporter assay (Fig. 5). The Zur protein reduced the electrophoretic mobility of this probe; the amount of Zur–DNA complex increased as the concentration of the Zur protein increased (Fig. 5, lanes 1–6). By contrast, the mobility of the promoter region of the copB operon, which is putatively involved in the copper (Cu) transport system, was unaffected by the presence of the Zur protein (Fig. 5, lanes 7–12). These results indicate that Zur binds to the zrf promoter region in a sequence-specific manner.
The effects of ZnSO4 on the DNA binding of Zur were examined by an EMSA (Fig. 6). The binding of Zur to the zrf promoter region was inhibited depending on the concentration of ZnSO4, from 50 to 200 μm. When the 200-bp zra promoter region was used as a DNA probe, a DNA–protein complex was also detected, although more abundant Zur protein was required than in the case of the zrf promoter region. The binding of Zur to the zra promoter region was significantly inhibited by ZnSO4. By contrast, the binding of the Zur protein to the 265-bp promoter region of a Zn-repressible gene, znuB2, was enhanced by the addition of ZnSO4, which is consistent with the results of a previous study .
The effects of other metals on the DNA binding of Zur in the EMSA were examined (Fig. 7). When either CuSO4 or MnSO4 was added at a concentration of 100 or 200 μm to the mixture of the Zur protein and the zrf promoter region, no significant effect on DNA binding activity was observed. On the other hand, both CoCl2 and FeSO4 showed negative effects on the Zur binding activity comparable to that of ZnSO4. NiCl2 also reduced the binding activity, although to a smaller extent than that of ZnSO4. Similar effects on biding of Zur to the zra promoter region were observed when these metals were used at 100 μm.
When the promoter regions of the zrf and zra genes were compared, a 21-bp consensus sequence consisting of a 10-bp direct repeat separated by 1 bp was found (Figs 3 and 8B). Therefore, we hypothesized that the consensus sequence (Zur box) is involved in transcriptional regulation by Zur. Interestingly, this motif is different from the 10-1-10 inverted repeat previously identified in the promoter regions for Zur-dependent expression . The 10-bp element in the newly-found Zur box is similar to the right-half of the previously identified Zur box (i.e. the corresponding sequences present in the promoter regions of the Zn uptake system genes shown in Fig. 8B). The transcription start sites of zrf and zra were determined by RACE and found to coincide with the adenine of their respective translation start sites (Fig. 3). Leaderless transcripts have been frequently found in C. glutamicum . The consensus sequences of the –10 and –35 regions of C. glutamicum SigA-dependent promoter  are found in the 5′-upstream region of the zrf and zra genes (Fig. 3). The Zur box overlaps the putative –35 region of both genes.
In addition to the Zur box (Zur box-1) in the intergenic region of cgR_1358-zrf, another Zur box-like sequence (Zur box-2) was found in the upstream cgR_1358 coding region (Fig. 8A,B). To examine the binding of Zur to these sites, four DNA probes were used in an EMSA (Fig. 8A). The P1 probe containing both Zur box-1 and Zur box-2 corresponds to the 400-bp zrf promoter region described above. Deletion of Zur box-1 and its flanking downstream region from the P1 probe resulted in the 342-bp P2 probe. Deletion of Zur box-2 and its flanking upstream region from the P2 probe resulted in the 192-bp P3 probe. The P4 probe corresponds to the 342-bp DNA region immediately downstream of Zur box-1. When the P2 probe containing only Zur box-2 was incubated with the Zur protein, a minor DNA–protein complex was observed, although its binding affinity appeared to be much lower than that of the Zur–P1 complex (Fig. 8C). Furthermore, no DNA–protein complex was observed when the P3 and P4 probes, both of which do not contain either Zur box-1 or Zur box-2, were used. It was also confirmed that Zur bound to another DNA probe containing only Zur box-1, as well as to the P1 probe containing both Zur box-1 and Zur box-2 (Fig. S1). These results indicate that Zur primarily binds to the Zur box-1 in the zrf promoter region. DNase I footprinting analysis showed that Zur protected a region overlapping Zur box-1 (Fig. 9).
The findings of the present study indicate that C. glutamicum Zur directly represses two Zn-inducible genes, zrf and zra, which encode a CDF and a metal-translocating P-type ATPase, respectively. The two genes appear to be responsible for the export of excess Zn out of cells. The purified Zur protein binds to the promoter regions of zrf and zra. Zn supplementation inhibits the binding of Zur to these promoter regions in vitro, and enhances the promoter activities in vivo in a dose-dependent manner. We observed that disruption of zur stimulates the expression of zrf and zra, and consequently eliminates the Zn-dependent induction of these genes. These results indicate that C. glutamicum Zur acts as a transcriptional repressor of these genes, and the repression is relieved in the presence of excess Zn. It is possible that zur disruption may have indirect effects through the up-regulation of the Zn uptake system genes whose expression is directly repressed by Zur . However, this does not appears to be the case in the present study because inactivation of two sets of the Zn-repressible ABC transporters in the zur mutant background had no effect on zrf expression. Furthermore, overexpression of zur resulted in decreased resistance to Zn, probably as a result of the down-regulation of the Zn export system genes. These results imply that Zur negatively regulates the Zn export systems independently of the Zn uptake systems.
In both Gram-negative and Gram-positive bacteria, Zur acts as a transcriptional repressor of multiple Zn-repressible genes . High-affinity Zn uptake systems are representative targets of Zur, and this highly conserved regulatory system plays a pivotal role in replenishment of Zn in a bacterial cell under Zn deficiency. So far, Zur proteins have been reported to bind to promoter regions of its target genes in the presence of Zn, a finding that is consistent with the derepression of these genes under Zn deficiency. Indeed, our in vitro DNA binding assay confirmed that Zn acts as a positive effector of C. glutamicum Zur binding to the promoter region of the Zn-repressible znuB2 gene encoding a component of an ABC transporter, as reported previously . Interestingly, this is in contrast to our new finding showing that Zn acts as a negative effector of this Zur protein binding to the promoter regions of two Zn-inducible genes: zrf and zra. Zur was shown to bind to a site containing a 21-bp consensus sequence between their promoter regions. The Zur binding site overlaps the putative –35 region of these target genes, suggesting that the binding of Zur prevents RNA polymerase from interacting with these promoters. It is interesting to note that the newly-identified Zur binding motif is a 10-1-10 direct repeat, which is different from the inverted repeat previously identified in Zur binding sites of C. glutamicum  and other bacteria, such as E. coli , B. subtilis [9, 10], M. tuberculosis , S. coelicolor [12, 13], and Yersinia pestis , amongst others. The 10-bp elements of the newly-identified motif present in the C. glutamicum Zn-inducible gene promoters and the previously identified motif (10-1-10 inverted repeat) present in the Zn-repressible gene promoters share an 8-bp similar sequence. These findings suggest that the Zur protein, with or without the regulatory Zn, binds to its respective different set of target DNA sites with a different motif in C. glutamicum. The crystal structure of M. tuberculosis Zur, which has 51% amino acid sequence identity to C. glutamicum Zur, has been reported previously . The amino acid residues, corresponding to the ligands for three Zn binding sites, are conserved in C. glutamicum Zur . The molecular details of the two distinct Zn-responsive binding modes of C. glutamicum Zur remain an interesting subject for future studies. Direct transcriptional regulation of both Zn uptake and export systems has been reported for Xanthomonas campestris Zur, however, which acts as an activator of the Zn export system gene , in contrast to the negative regulation in C. glutamicum. X. campestris Zur binds to both the Zn-repressible and Zn-inducible gene promoters in the presence of Zn in vitro. Its binding motif for the latter promoter (59-bp GC-rich sequence) is different from that for the former (an ~ 30-bp AT-rich sequence), and has no apparent sequence similarity to the newly-identified Zur box in C. glutamicum. It is noteworthy that the Fe-sensing transcriptional regulator Fur, structurally related to Zur, generally acts as a repressor of Fe-repressible genes in the presence of Fe in bacteria , although Helicobacter pylori apo-Fur (with no Fe bound) uniquely acts as a repressor of Fe-inducible genes in addition to the typical repressor function of Fe-bound Fur for Fe-repressible genes . It is unlikely that the H. pylori apo-Fur binding motif shares any common features with the C. glutamicum apo-Zur binding motif. The findings of the present study shed some light on a new functional aspect of the Fur family of transcriptional regulators.
In the present study, we show that disruption of zra and zrf resulted in cells with decreased resistance to Zn. In addition, deletion of zur in the genetic background of the double deletion mutant of zra and zrf resulted in a further decrease in resistance to Zn. On the other hand, overexpression of zur in the zur deletion mutant background also impaired Zn resistance. These findings imply a complicated interplay between the Zn uptake and Zn export systems under the control of Zur. It is interesting to note that, in C. glutamicum, Zur plays a central role in the cell response to both a deficiency and excess of Zn by directly regulating the transcription of the relevant genes in distinct ways. This is in contrast to the involvement of other types of transcriptional regulators in the induction of the Zn export systems under excess Zn in other bacteria. In E. coli, ZntR, a MerR-type transcriptional regulator, acts as a Zn-dependent activator of zntA, a gene that encodes a Zn-exporting ATPase . In Synechocystis sp. PCC 6803, ZiaR, belonging to the ArsR family, acts as a repressor of a Zn-exporting ATPase gene, ziaA . In Staphylococcus aureus plasmid pI258, a P-type ATPase gene, cadA, which is involved in resistance to Zn, is located immediately downstream of cadC encoding an ArsR-type transcriptional repressor of cadA [42, 43]; in the chromosome of this bacterial species, zntR (czrA), encoding another ArsR-type regulator, is adjacent to its target CDF gene, zntA (czrB), which is also involved in Zn resistance [44-46]. In mycobacteria, a gene encoding an ArsR-type transcriptional regulator is linked to the zur gene on the chromosome, and these two regulators are suggested to be involved in Zn homeostasis [11, 47].
In the present study, we observed that in vitro DNA binding of Zur to the zrf and zra promoter regions is inhibited by Fe, nickel and Co, as well as by Zn, but not by Cu and manganese. How the in vivo expression of these genes is affected by multiple transition metals remains unclear. It is noteworthy that C. glutamicum ATCC 13032 cgl1281 (cg1447), corresponding to zrf, is involved in the Co resistance of the microorganism and plays a role in alkali-tolerance . With respect to Fe-dependent global gene expression in C. glutamicum, the involvement of two different types of transcriptional regulators, DtxR and RipA, has been reported [48-50]. Further studies are required to determine the possible interplay among these metal-responsive transcriptional regulators in C. glutamicum.
Materials and methods
Bacterial strains and culture conditions
C. glutamicum R (JCM 18229)  was used as the wild-type strain. Mutant strains deficient in each of zrf (cgR_1359) and zra (cgR_0148) were obtained from a mutant library constructed by transposon-mediated mutagenesis .
For genetic manipulations, E. coli and C. glutamicum strains were grown as described previously .
For analytical purposes, C. glutamicum starter culture was grown aerobically in 10 mL of nutrient-rich A medium  containing 1% glucose at 33 °C in a 100-mL test tube overnight. The cells were inoculated in fresh medium at a dilution of 100-fold or higher. The cells were cultured in 10 mL of nutrient-rich A medium or minimal BTM medium , each containing 1% glucose, at 33 °C in a 100-mL test tube. For quantitative RT-PCR analysis, the cells were cultured in 100 mL of medium in a 500-ml flask. To assess the response to Zn, the medium was supplemented with ZnSO4 at the stated concentrations. Cell growth was monitored by measuring the D610 using a spectrophotometer (DU640; Beckman Coulter, Brea, CA, USA).
Chromosomal and plasmid DNA were prepared from C. glutamicum, and the target DNA regions were amplified by PCR as described previously .
C. glutamicum cells were transformed by electroporation as described previously . E. coli cells were transformed by the CaCl2 procedure .
DNA sequencing was performed with ABI Prism 3100xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). DNA sequence data were analyzed using genetyx (Software Development, Tokyo, Japan).
The upstream and downstream regions of the target gene for deletion were amplified using the sets of primers summarized in Table S1. The resultant amplicons were fused and cloned into pCRA725 , a suicide vector for markerless gene disruption. The resultant plasmids, pCRC310, pCRC311, pCRC312, pCRC319 and pCRC320, were used for in-frame deletion of zrf, zra, zur (cgR_2151), znuB1 (cgR_2537) and znuB2 (cgR_0042), respectively. C. glutamicum was subsequently transformed with the respective plasmid DNA, and screening for deletion mutants was performed as described previously . Deletion of the target genes was confirmed by PCR.
Plasmids for gene expression in C. glutamicum
To obtain plasmids for expression of the target genes, the region for each of the ORFs was amplified by PCR using the C. glutamicum chromosomal DNA as a template and a set of primers with appropriate restriction sites (Table S1). The amplified ORF region was digested with the restriction enzymes, and was inserted into the corresponding site downstream of the lac promoter in an E. coli–Corynebacterium shuttle vector pCRB1 , yielding pCRC314, pCRC315 and pCRC321 for the expression of zrf, zra and zur, respectively.
RT-PCR and RACE
Total RNA was prepared from C. glutamicum cells using an RNeasy minikit and RNAprotect Bacteria reagent (Qiagen, Hilden, Germany), and quantitative RT-PCR analysis was performed using Applied Biosystems 7500 Fast Real-Time PCR System as described previously . The primers used are listed in Table S2. The relative abundance of the target mRNAs was quantified based on the cycle threshold value. To standardize the results, the relative abundance of 16S rRNA was used as the internal standard.
The 5′-end of mRNA was determined by RACE using the primers summarized in Table S3. Using the 5′-Full RACE Core Set (Takara, Osaka, Japan), single-stranded cDNA synthesized from total RNA using the 5′-phosphrylated primer was self-ligated with T4 RNA ligase. The first PCR reaction proceeded using inverted primers, and then the second PCR reaction proceeded using nested inverted primers with an EcoRI site in their 5′-ends. The amplified DNA was digested with EcoRI and inserted into the corresponding site of a plasmid pHSG398 (Takara). More than 12 clones from E. coli transformed with the resulting plasmid were sequenced.
Construction of strains carrying the promoter-lacZ reporter fusion
A DNA fragment containing the target promoter region was amplified by PCR using C. glutamicum chromosomal DNA as a template and a set of primers (Table S1). The amplified DNA was digested with DraI, and was inserted into the corresponding site of pCRA741  to construct the promoter-lacZ fusion. The lacZ fusion was integrated into strain-specific island 7 (SSI7) on the chromosome of C. glutamicum R using markerless gene insertion methods that have been described previously .
C. glutamicum cells were harvested, washed once with Z-buffer , resuspended with the same buffer and treated with toluene. The permeabilized cells were then incubated with O-nitrophenyl-β-d-galactopyranoside, and activity was measured in Miller units, as described previously .
Purification of the Zur protein expressed in E. coli
A DNA fragment containing the zur gene was amplified by PCR using a primer pair (Table S1; zur-Ex-F and zur-Ex-R). The amplified DNA was digested with NdeI and EcoRI, and was inserted into the corresponding site of the pET-28a expression vector (Merck KGaA, Darmstadt, Germany). The resulting plasmid, pCRC322, contains the zur gene fused to a His tag sequence at the N-terminus. E. coli BL21(DE3) cells transformed with pCRC322 were grown at 37 °C in 100 mL of LB medium supplemented with kanamycin (50 μg·mL−1). The recombinant gene was expressed in exponentially growing cells (D610 = 0.6) by adding 1 mm isopropyl thio-β-d-galactoside. After 1 h of incubation, the cells were harvested by centrifugation. The His-tagged Zur protein was extracted and purified by affinity column chromatography using an Ni-NTA Fast Start Kit (Qiagen). The His tag was removed from the purified protein by thrombin treatment using a Thrombin Cleavage Capture Kit (Merck KGaA). The Zur protein was loaded onto a gel filtration column (PD-10 column; GE Healthcare UK Ltd, Little Chalfont, UK) equilibrated with buffer containing 20 mm Tris-HCl (pH 7.5), 100 mm NaCl, 10 mm MgCl2 and 1 mm dithiothreitol, and eluted with the same buffer. The resultant Zur protein was used for an EMSA.
An EMSA was performed as described previously  with some modifications. The purified Zur protein at stated concentrations was incubated with 40 ng of a DNA probe in 20 μL of binding buffer containing 10 mm Tris-HCl (pH 7.5), 150 mm KCl, 50 mm NaCl, 5 mm MgCl2, 1.5 mm dithiothreitol and 10% glycerol for 30 min at 25 °C. ZnSO4, CuSO4, NiCl2, CoCl2, MnSO4 and FeSO4 were added at the stated concentrations. The binding reaction mixture was subjected to electrophoresis on a 6% polyacrylamide gel containing 5% glycerol in 0.5 × TB electrophoresis buffer, and the DNA probe was detected with SYBR Green.
DNA probes were prepared by PCR using the primers summarized in Tables S1 and S4; for the 400-bp zrf promoter region P1 probe, zrf-Pr-F1 and zrf-Pr-R1 were used; for the P2 probe, zrf-Pr-F1 and zrf-Pr-R2 were used; for the P3 probe, zrf-Pr-F2 and zrf-Pr-R2 were used; for the P4 probe, zrf-Pr-F3 and zrf-Pr-R3 were used; for the 200-bp zra promoter DNA probe, zra-Pr-F2 and zra-Pr-R1 were used; for the 265-bp znuB2 promoter DNA probe, znuB2-Pr-F and znuB2-Pr-R were used; for the 400-bp promoter region of the copB operon, 0124-Pr-F and 0124-Pr-R were used.
DNase I footprinting analysis
A labelled DNA fragment was prepared by PCR using zrf-Pr-F4 and a 5′-IRD700-labelled primer IR-Rv (Table S4). A plasmid containing the zrf promoter-lacZ fusion described above was used as the template. The obtained DNA fragment contains a region between –189 and +12 bp with respect to the transcription start site of zrf. The purified Zur protein at stated concentrations was incubated with 80 ng of the DNA probe in 20 μL of binding buffer containing 20 mm Tris-HCl (pH 7.5), 50 mm NaCl, 2.5 mm MgCl2, 0.45 mm EDTA, 0.5 mm dithiothreitol, 0.05% Nonidet P-40 and 10% glycerol for 25 min at room temperature. Four microlitres of the binding buffer containing 1–2 mU of DNase I (Takara), 5 mm MgCl2 and 10 mm CaCl2 were then added and incubated for 1 min, followed by the addition of 2 μL of 325 mm EDTA and subsequent heating at 80 °C for 10 min. The samples mixed with IR2 stop solution (Li-Cor, Lincoln, NE, USA) were heated at 95 °C for 3 min, and separated on a 5.5% KB plus gel matrix (Li-Cor) using a Li-Cor 4300 DNA analyzer. The DNA-sequencing reaction mixtures using the same IRD700-labelled primer and a DYEnamic direct cycle sequencing kit with 7-deaza-dGTP (GE Healthcare UK Ltd) were subjected to the same gel.
We thank Crispinus A. Omumasaba (RITE) for critically reading the manuscript. This work was financially supported in part by the New Energy and Industrial Technology Development Organization (NEDO), Japan.