Manganese is an important trace element required as an enzyme cofactor and for protection against oxidative stress. In this study, we characterized the DtxR-type transcriptional regulator MntR (cg0741) of Corynebacterium glutamicum ATCC 13032 as a manganese-dependent repressor of the predicted ZIP family metal transporter Cg1623. Comparative transcriptome analysis of a ΔmntR strain and the wild type led to the identification of cg1623 as potential target gene of MntR which was about 50-fold upregulated when cells were grown in glucose minimal medium. Using electrophoretic mobility shift assays, a conserved 18 bp inverted repeat (TGTTCAATGCGTTGAACA) was identified as binding motif of MntR in the cg1623 promoter and confirmed by mutational analysis. Promoter fusion of Pcg1623 to eyfp confirmed that the MntR-dependent repression is only abolished in the absence of manganese. However, neither deletion of mntR nor cg1623 resulted in a significant growth phenotype in comparison to the wild type—strongly suggesting the presence of further manganese uptake and efflux systems in C. glutamicum. The control of cg1623 by the DtxR-type regulator MntR represents the first example of a predicted ZIP family protein that is regulated in a manganese-dependent manner in bacteria.

INTRODUCTION

The DtxR/MntR family of metalloregulators represents a central class of transcriptional regulators being involved in the control of metal ion homeostasis in a wide range of Gram-positive and Gram-negative bacteria (Hantke 2001; Andrews, Robinson and Rodriguez-Quinones 2003; Guedon and Helmann 2003). One of the earliest reports is about the iron-dependent expression of the diphtheria toxin in Corynebacterium diphtheria, mediated by DtxR (Pappenheimer and Johnson 1936; Tao et al.1994). However, besides virulence genes, DtxR controls the expression of a variety of different genes involved in iron uptake, siderophore synthesis or iron storage in this species (Boyd, Oza and Murphy 1990; Schmitt and Holmes 1991). The active form of DtxR is a homodimer, with each monomer consisting of two domains connected by a flexible tether. The N-terminal domain contains the helix-turn-helix motif, responsible for DNA binding as well as two binding sites for Fe2+ ions (Spiering et al.2003; D'Aquino et al.2005). The C-terminal domain shares structural similarity with eukaryotic SH3 domains.

MntR of Bacillus subtilis was the first manganese-responsive DtxR-type regulator that has been characterized. It was shown to repress the two manganese uptake systems mntH (proton-coupled NRAMP transporter) and mntABCD (ABC transporter) under conditions of sufficient manganese supply (Que and Helmann 2000). Furthermore, it also activates the mntABCD operon when manganese is limited to increase manganese uptake. A B. subtilis mntR deletion mutant shows a significantly increased sensitivity towards manganese compared to the wild type (Que and Helmann 2000). MntR of C. diphtheriae (DIP0619) represses a five-gene operon in a manganese-dependent manner that contains, besides its own gene, a potential ABC metal ion transporter (mntABCD) (Fig. 1A) (Schmitt 2002). Deletion of this transporter had no effect on growth even under Mn2+ limiting conditions (Schmitt 2002), which is not unexpected as many bacteria possess more than one manganese uptake system (Que and Helmann 2000; Andrews et al.2013).

Figure 1.

Phylogenetic conservation of cg0741 (mntR) among different Corynebacteria and growth of an mntR deletion mutant. (A) Comparison of the organization of the genomic locus of mntR in C. glutamicum and related species. mntR and homologous genes are highlighted in red. hp, hypothetical protein; mp, membrane protein of unknown function; dnaE2, error-prone DNA polymerase; Sir2, Sir2-type NAD-dependent protein deacetylase; cumB, cytidine and deoxycytidylate deaminase. Data were taken from MicrobesOnline (Alm et al.2005). (B) Transcriptional organization of mntR. According to Pfeifer-Sancar et al. (2013) two different transcripts are formed. (C) Cultivation of ATCC 13032 and the mntR deletion mutant on standard CGXII minimal medium with 2% glucose (w v−1), without manganese (-Mn) and in the presence of manganese excess (10xMn). Presented are the average and standard deviation of three biological replicates.

Figure 1.

Phylogenetic conservation of cg0741 (mntR) among different Corynebacteria and growth of an mntR deletion mutant. (A) Comparison of the organization of the genomic locus of mntR in C. glutamicum and related species. mntR and homologous genes are highlighted in red. hp, hypothetical protein; mp, membrane protein of unknown function; dnaE2, error-prone DNA polymerase; Sir2, Sir2-type NAD-dependent protein deacetylase; cumB, cytidine and deoxycytidylate deaminase. Data were taken from MicrobesOnline (Alm et al.2005). (B) Transcriptional organization of mntR. According to Pfeifer-Sancar et al. (2013) two different transcripts are formed. (C) Cultivation of ATCC 13032 and the mntR deletion mutant on standard CGXII minimal medium with 2% glucose (w v−1), without manganese (-Mn) and in the presence of manganese excess (10xMn). Presented are the average and standard deviation of three biological replicates.

The genome of the non-pathogenic Gram-positive soil bacterium Corynebacterium glutamicum encodes three DtxR-type regulators. One of them, DtxR (cg2103), has been characterized as the master regulator of iron homeostasis controlling the transcription of more than 60 target genes in an iron-dependent manner (Brune et al.2006; Wennerhold and Bott 2006). Although manganese has been shown to be crucial for the function of the superoxide dismutase, (El Shafey et al.2008) nothing is known regarding the control of manganese homeostasis in C. glutamicum to date. One prime candidate is the DtxR-type regulator encoded by cg0741, which shares 52% amino acid sequence identity with MntR of C. diphtheriae.

Here, we characterized Cg0741 (in the following designated as MntR) and we were able to show that this DtxR-type transcriptional regulator functions as a manganese-dependent repressor of a predicted ZIP metal transport system in C. glutamicum.

MATERIALS AND METHODS

Bacterial strains, plasmids and growth media

The bacterial strains and plasmids used in this study are listed in Table 1. The C. glutamicum type strain ATCC 13032 was used as wild type. Growth experiments were performed at 30°C and 1200 rpm in a BioLector system (m2p-labs, Baesweiler, Germany) in 48-well FlowerPlates containing 750 μL CGXII minimal medium (Keilhauer et al.1993) supplemented with 3,4-dihydroxybenzoate (30 mg L−1) and 2% (w v−1) glucose as carbon source. If appropriate, 25 μg mL−1 kanamycin or 10 μg mL−1 chloramphenicol were added. The standard concentrations for metals in CGXII are as follows: 36 μM FeSO4, 59 μM MnSO4 and 3.48 μM ZnSO4. For growth experiment with metal starvation conditions, the relevant metal salt was omitted from the trace element solution. For growth experiments with metal-excess conditions, 10 times the standard concentration of the relevant metal was used (e.g. 360 μM for FeSO4). All cloning was performed in Escherichia coli DH5α cultivated at 37°C in lysogeny broth (LB) (Sambrook and Russell 2001) with 50 μg mL−1 kanamycin or 34 μg mL−1 chloramphenicol.

Table 1.

Strains and plasmids used in this study.

Strain or plasmid Relevant characteristics Source or reference 
E. coli   
DH5α F Φ80dlacΔ(lacZ)M15 Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (rK, mK+) deoR thi-1 phoA supE44 λgyrA96 relA1; strain used for cloning procedures Hanahan (1983
BL21(DE3) F- ompT hsdSB (rB-, mB-) gal dcm (DE3); host for protein production Studier and Moffatt (1986
C. glutamicum   
ATCC13032 Biotin-auxotrophic wild type Kinoshita et al. (1957
ATCC13032 ΔmntR ATCC13032 with an in-frame deletion of cg0741 This work 
ATCC13032 Δcg1623 ATCC13032 with an in-frame deletion of cg1623 This work 
Plasmids   
pK19mobsacB KanR; plasmid for allelic exchange in C. glutamicum; (pK18 oriVE.c., sacB, lacZα) Schäfer et al. (1994
pK19mobsacBmntR KanR; pK19mobsacB derivative containing a PCR product covering the up- and downstream regions of mntR (cg0741) This work 
pK19mobsacB-Δcg1623 KanR; pK19mobsacB derivative containing a PCR product covering the up- and downstream regions of cg1623 This work 
pET24b KanR; vector for overexpression of genes in E. coli, with optional C-terminal hexahistidine affinity tag (pBR322 oriVE.c. PT7 lacINovagen 
pET24b-mntR-Strep KanR; pET24b derivative for overproduction of MntR (Cg0741) with a C-terminal STREP-tag This work 
pJC1-venus-term KanR; pJC1 derivative carrying the venus coding sequence and additional terminators Baumgart et al. (2013
pJC1-Pcg1623-eYFP KanR; pJC1-venus-term derivative carrying the promoter of cg1623 fused to eyfp for promoter activity studies This work 
pEC-XC99E CmR; C. glutamicum/E. coli shuttle vector for regulated gene expression using the Ptac promoter Kirchner and Tauch ( 2003
pEC-mntR CmR; pEC-XC99E-derivative for expression of mntR under control of the Ptac promoter This work 
pAN6 KanR; C. glutamicum/E. coli shuttle vector for regulated gene expression using the Ptac promoter Frunzke et al. (2008
pAN6-cg1623 KanR; pAN6-derivative for expression of cg1623 under control of the Ptac promoter This work 
Strain or plasmid Relevant characteristics Source or reference 
E. coli   
DH5α F Φ80dlacΔ(lacZ)M15 Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (rK, mK+) deoR thi-1 phoA supE44 λgyrA96 relA1; strain used for cloning procedures Hanahan (1983
BL21(DE3) F- ompT hsdSB (rB-, mB-) gal dcm (DE3); host for protein production Studier and Moffatt (1986
C. glutamicum   
ATCC13032 Biotin-auxotrophic wild type Kinoshita et al. (1957
ATCC13032 ΔmntR ATCC13032 with an in-frame deletion of cg0741 This work 
ATCC13032 Δcg1623 ATCC13032 with an in-frame deletion of cg1623 This work 
Plasmids   
pK19mobsacB KanR; plasmid for allelic exchange in C. glutamicum; (pK18 oriVE.c., sacB, lacZα) Schäfer et al. (1994
pK19mobsacBmntR KanR; pK19mobsacB derivative containing a PCR product covering the up- and downstream regions of mntR (cg0741) This work 
pK19mobsacB-Δcg1623 KanR; pK19mobsacB derivative containing a PCR product covering the up- and downstream regions of cg1623 This work 
pET24b KanR; vector for overexpression of genes in E. coli, with optional C-terminal hexahistidine affinity tag (pBR322 oriVE.c. PT7 lacINovagen 
pET24b-mntR-Strep KanR; pET24b derivative for overproduction of MntR (Cg0741) with a C-terminal STREP-tag This work 
pJC1-venus-term KanR; pJC1 derivative carrying the venus coding sequence and additional terminators Baumgart et al. (2013
pJC1-Pcg1623-eYFP KanR; pJC1-venus-term derivative carrying the promoter of cg1623 fused to eyfp for promoter activity studies This work 
pEC-XC99E CmR; C. glutamicum/E. coli shuttle vector for regulated gene expression using the Ptac promoter Kirchner and Tauch ( 2003
pEC-mntR CmR; pEC-XC99E-derivative for expression of mntR under control of the Ptac promoter This work 
pAN6 KanR; C. glutamicum/E. coli shuttle vector for regulated gene expression using the Ptac promoter Frunzke et al. (2008
pAN6-cg1623 KanR; pAN6-derivative for expression of cg1623 under control of the Ptac promoter This work 

Recombinant DNA work and construction of deletion mutants

Routine methods such as PCR, DNA restriction and ligation were performed using standard protocols (Hanahan 1983; van der Rest et al.1999; Sambrook and Russell 2001). The oligonucleotides used in this study were obtained from Eurofins MWG Operon (Ebersberg, Germany) and are listed in Table S1 (Supporting Information). DNA sequencing was performed by Eurofins MWG Operon (Ebersberg, Germany). The ΔmntR and Δcg1623 mutants of C. glutamicum were constructed via a two-step homologous recombination protocol as described previously (Niebisch and Bott 2001). For further details regarding plasmid and mutant construction, see the supplemental methods in Supplementary Information.

DNA microarrays

Comparative transcriptome analysis was performed as described previously (Vogt et al.2014). Briefly, C. glutamicum wild-type and ΔmntR cells were grown in 5 mL brain–heart infusion (BHI, Difco) for about 6 h at 30°C. A second precultivation was performed overnight in CGXII minimal medium containing 2% (w v−1) glucose as carbon source. The main cultures were inoculated to an OD600 of 0.5 in CGXII minimal medium with 2% (w v−1) glucose. At an OD600 of 5, the cells were harvested by centrifugation (4120 x g, 10 min and 4°C). The cell pellet was subsequently frozen in liquid nitrogen and stored at −70°C. The preparation of total RNA was performed using the RNeasy Kit from Qiagen (Hilden, Germany). Synthesis of fluorescently labeled cDNA was carried out using SuperScript III reverse transcriptase (Life Technologies, Darmstadt, Germany). Purified cDNA samples of the wild-type and the ΔmntR strain were pooled and the prepared two-color samples were hybridized at 65°C while rotating for 17 h using Agilent's Gene Expression Hybridization Kit, hybridization oven and hybridization chamber. After hybridization, the arrays were washed using Agilent's Wash Buffer Kit according to the manufacturer's instructions. Fluorescence of hybridized DNA microarrays was determined at 532 nm (Cy3) and 635 nm (Cy5) at 5 μm resolution with a GenePix 4000B laser scanner and GenePix Pro 7.0 software (Molecular Devices, Sunnyvale, CA, USA). Fluorescence images were saved to raw data files in TIFF format (GenePix Pro 7.0). Quantitative TIFF image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 7.0). For background correction of spot intensities, ratio calculation and ratio normalization, GPR-files were processed using the BioConductor R-packages limma and marray (http://www.bioconductor.org).

Overproduction and purification of MntR

E. coli BL21(DE3) carrying the expression plasmid pET24b-mntR-strep was grown in LB medium at 37°C and 120 rpm. MntR overproduction of MntR with a C-terminal Strep-tag was induced by addition of 250 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) followed by cultivation at 20°C for 6–8 h before the cells were harvested by centrifugation. StrepTactin affinity chromatography was performed as described previously (Niebisch et al.2006). The protein was frozen in 20 μL aliquots and stored at −20°C. For determination of the molecular weight, gel filtration was performed using a Superdex™ 200 10/300 GL column (GE Healthcare, Munich, Germany) at a flow rate of 0.5 mL min−1 in gel filtration buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl and 1 mM DTT) containing either 1 mM MnCl2 or 1 mM EDTA.

Electrophoretic mobility shift assays

Electrophoretic mobility shift assays (EMSAs) were performed as described previously (Wennerhold and Bott 2006). Briefly, purified MntR was incubated with DNA fragments (30–500 bp, final concentration 0.027–1 μM) in binding buffer (50 mM Tris-HCl pH 7.5, 40 mM KCl, 5 mM MgCl2) and metal ions, as indicated in the figure legends, in a total volume of 20 μL. Electrophoresis was performed using 10–15% native polyacrylamide gels at room temperature and 150 or 180 V for 45–60 min (depending on the size of the DNA fragments) and the gels were subsequently stained with SYBR green.

Promoter fusion studies

In order to analyze the regulation of the cg1623 promoter by MntR in vivo, a DNA fragment covering the cg1623 promoter region was fused to the eyfp-coding sequence (pJC1-Pcg1623-eyfp). Wild-type and ΔmntR cells were transformed with the resulting plasmid. Using a BioLector system (m2p labs), production of biomass was measured as the backscattered light (620 nm) and the eYFP fluorescence was measured at an excitation of 485 nm and an emission of 520 nm. The specific fluorescence for the cells is defined as eYFP fluorescence per scattered light intensity (given in a.u.). Trace elements were added as indicated in the figure legends. For the complementation experiment, the two strains were additionally transformed with plasmid pEC-mntR or the empty plasmid pEC-XC99E as control. The strains were cultivated as described above, without or with 100 μM IPTG to induce mntR transcription.

RESULTS AND DISCUSSION

Genomic and transcriptional organization of mntR

In the genome of C. glutamicum ATCC 13032, mntR is organized in an operon with two predicted membrane proteins of unknown function (Fig. 1A). Recent RNAseq data revealed that two separate leaderless transcripts are formed, one containing all three genes and one encoding just mntR (Pfeifer-Sancar et al.2013) (Fig. 1B). The homolog of MntR in C. diphtheriae (DIP1969) is the terminal gene of an operon including, besides mntR, four genes (mntABCD) encoding an ABC transporter responsible for manganese uptake (Schmitt 2002) (Fig. 1A). This operon is repressed by DIP1969 in the presence of manganese (Schmitt 2002). However, a homolog of this ABC transporter is missing in C. glutamicum and Corynebacterium efficiens and consequently the genomic organization is different here.

Construction and growth of a ΔmntR strain

To gain insight into the function and possible target genes of MntR, an in-frame deletion mutant was constructed and analyzed. Growth rate and final OD600 of the wild-type and the deletion mutant were identical when grown in standard CGXII minimal medium (Fig. 1C). The morphology was analyzed by microscopy, but revealed no differences between the strains (data not shown). Even cultivation in the presence of excess manganese, zinc or iron (10 x standard concentrations) or under metal limitation disclosed no significant growth phenotype of the mutant (data for Mn2+ in Fig. 1C, data for Zn2+ and Fe2+ not shown).

Transcriptome analysis of the mntR mutant

In order to elucidate the transcriptional changes caused by the deletion of mntR, DNA-microarrays were performed of cells grown in CGXII minimal medium with glucose as carbon source. In total, 11 genes showed an altered mRNA level of ≥ 2-fold (Table S2, Supporting Information). The mRNA level of mntR was 28-fold reduced, confirming the successful deletion of the corresponding gene. The transcription of the other genes of the mntR operon, cg0739 and cg0740, was unchanged in comparison to the wild-type reference (ratio 1.12 and 0.89, respectively). Remarkably, the gene cg1623, annotated as a zinc transporter of the ZIP family, exhibited an about 50-fold increased mRNA level in the ΔmntR strain. Among the other regulated genes were several, but not all, members of the arg-operons (cg1580-85 and cg1586-1580) responsible for arginine biosynthesis, a glutamine 2-oxoglutarate aminotransferase, a putative allophanate hydrolase and the operon cg3226-27, encoding a lactate permease and a lactate hydrolase. The latter is an operon which very often shows an altered mRNA level in DNA microarray experiments and was therefore not treated as putative target (≥2-fold regulated in about 40% of all microarray experiments in our in-house database).

Promoter fusion studies with cg1623

To study the influence of MntR on the expression of cg1623 in vivo, we fused the cg1623 promoter to eyfp and monitored the fluorescence output in the wild-type and the ΔmntR strain in CGXII medium with different trace element substitutions (Fig. 2A). The growth of the two strains did not differ significantly under the tested conditions (data not shown). When grown in standard CGXII minimal medium, the specific fluorescence of the mntR deletion mutant carrying the promoter fusion plasmid was about 20-fold higher compared to the wild type harboring the same plasmid, which indicates that MntR functions as a repressor of cg1623 transcription. Remarkably, the specific fluorescence is almost identical in the two strains when the trace elements (Zn2+, Cu2+, Mn2+ and Ni2+) were omitted from the medium. In the following, we tested the impact of these four trace elements separately (Fig. 2A, last four media). Here, only manganese starvation resulted in a comparable increase of fluorescence in the wild type. This indicates that the MntR-dependent regulation of cg1623 is responsive to manganese and suggests Cg1623 as a novel transport system involved in manganese uptake in C. glutamicum.

Figure 2.

cg1623 promoter fusion studies with C. glutamicum ATCC 13032 wild type (wt) and ΔmntR in CGXII glucose minimal medium. All strains used in these experiments carry the plasmid pJC1-Pcg1623-eYFP containing the promoter fusion of cg1623 to eyfp. (A) Specific fluorescence of wild type and ΔmntR in CGXII glucose minimal medium lacking one or several metal ions. The media composition is given below the x-axis. TE stands for a combination of the four trace element salts ZnSO4, CuSO4, NiCl2 and MnSO4. (B) Complementation of the fluorescence with plasmid-encoded MntR. C. glutamicum wild type and ΔmntR carrying the reporter plasmid were additionally transformed with a plasmid encoding MntR under control of a leaky, IPTG inducible promoter (or the empty plasmid pEC-XC99E as control). The first preculture was grown in BHI medium and the second preculture in CGXII minimal medium with glucose, both with either kanamycin (A) or kanamycin and chloramphenicol (B). Presented is the specific fluorescence in the stationary growth phase after 24 h of cultivation (average and standard deviation of three biological replicates, the specific fluorescence of the ΔmntR strain in standard CGXII-medium was set to 1).

Figure 2.

cg1623 promoter fusion studies with C. glutamicum ATCC 13032 wild type (wt) and ΔmntR in CGXII glucose minimal medium. All strains used in these experiments carry the plasmid pJC1-Pcg1623-eYFP containing the promoter fusion of cg1623 to eyfp. (A) Specific fluorescence of wild type and ΔmntR in CGXII glucose minimal medium lacking one or several metal ions. The media composition is given below the x-axis. TE stands for a combination of the four trace element salts ZnSO4, CuSO4, NiCl2 and MnSO4. (B) Complementation of the fluorescence with plasmid-encoded MntR. C. glutamicum wild type and ΔmntR carrying the reporter plasmid were additionally transformed with a plasmid encoding MntR under control of a leaky, IPTG inducible promoter (or the empty plasmid pEC-XC99E as control). The first preculture was grown in BHI medium and the second preculture in CGXII minimal medium with glucose, both with either kanamycin (A) or kanamycin and chloramphenicol (B). Presented is the specific fluorescence in the stationary growth phase after 24 h of cultivation (average and standard deviation of three biological replicates, the specific fluorescence of the ΔmntR strain in standard CGXII-medium was set to 1).

Under iron starvation conditions (second medium), the growth of both strains is strongly decreased. Therefore, the increased fluorescence of both strains is probably due to the higher contribution of the autofluorescence of the cells to the total fluorescence in these samples.

To further confirm the specificity of the regulation of cg1623 by MntR, we performed a complementation experiment with plasmid encoded MntR under control of the IPTG-inducible Ptac promoter (Fig. 2B). Under standard manganese conditions, the basal expression of mntR by the leaky Ptac promoter is already sufficient to suppress transcription of cg1623 in the ΔmntR strain. We also tested the complementation with induced MntR (100 μM IPTG), but these strains showed a growth defect and the specific fluorescence was not further reduced (data not shown).

MntR is a dimer, independent of the presence of Mn2+

For in vitro studies, MntR was heterologously expressed in E. coli BL21 (DE3) and purified as a C-terminal strep-tag fusion (Fig. S1A, Supporting Information). In line with the report of Lieser et al. for B. subtilis MntR, size exclusion chromatography revealed that C. glutamicum MntR forms a dimer in the presence or absence (+EDTA) of Mn2+ (Fig. S1B, Supporting Information) (Lieser et al.2003). Upon addition of EDTA the peak shifts to slightly higher molecular weight, possibly because the absence of manganese leads to a conformational change. For B. subtilis, it was described that in the absence of metal ions, the two DNA binding domains are spread farther apart than in the metal bound state (DeWitt et al.2007).

Identification of MntR target genes

The microarrays and promoter fusion studies suggested cg1623 to be a direct target gene of MntR. Therefore, a DNA fragment covering the promoter region of cg1623 was tested for complex formation with MntR in EMSAs. An obvious shift was observed for this DNA fragment, whereas the promoters of cg0739 and mntR itself were not bound by MntR, in vitro (Fig. S2, Supporting Information). The promoter regions of further putative targets identified in the transcriptome analysis were also tested, but no considerable interaction with MntR was observed in EMSA studies (Fig. S3, Supporting Information). Only the promoter of cg1580 showed a slight shift with the highest protein concentration in vitro. But the genes of this operon (cg1580-cg1585) were regulated in different directions (ratios cg1580-cg1506: 2.0, 1.0, 0.5, 0.2, 0.15, 0.15) and a putative binding motif could not be identified in this region. A potential regulation of this operon by MntR was therefore regarded as unlikely to be physiologically relevant and not further elucidated.

A nearly perfect 18 bp inverted repeat with high sequence identity to the MntR binding motif of C. diphtheriae was identified in the promoter region of cg1623, centering 24 bp upstream of the transcriptional and translational start site (leaderless transcript, personal communication Jörn Kalinowski) (Fig. 3A). A 30 bp fragment containing this motif was indeed bound by MntR with high affinity in the presence of manganese (Fig. 3B). In the following, the high specificity of MntR for its palindromic binding site was confirmed by a mutational analysis revealing that the outer six base pairs of the binding motif are most important for complex formation (Fig. 4). A further MntR motif in the promoter of cg0343 was identified by a genome-wide in silico search and was also bound by MntR in EMSAs but with slightly lower affinity (Fig. 3B). Cg0343 encodes a MarR-type transcriptional regulator of unknown function which is not conserved among Corynebacteria and Mycobacteria. The mRNA level of cg0343 was not significantly altered in the comparative transcriptome analysis (average ratio of three experiments: 0.84, P-value: 0.090). A possible reason for this could be that cg0343 is regulated by further regulators or other regulatory mechanisms which counteract the effect of mntR deletion under the tested conditions. Therefore, the relevance of MntR for cg0343 regulation remains to be elucidated.

Figure 3.

Manganese-dependent binding of MntR to its target genes. (A) Localization of the MntR binding motif in the cg1623 promoter (box) and comparison with the MntR binding site of C. diphtheriae (above the box), −10 and −35 region as indicated in bold letters. The transcript is leaderless; +1 therefore indicates the transcriptional and translational start. (B) EMSAs of purified MntR binding its target promoters. A 30 bp oligonucleotide pair located in the promoter region of cg1918 was used as negative control. (C) Manganese dependence of MntR binding. Oligonucleotides (30 bp, 1 μM) were incubated with MntR, MnSO4 and EDTA in the given concentrations, analyzed using 15% native polyacrylamide gels and stained with SybrGreen I.

Figure 3.

Manganese-dependent binding of MntR to its target genes. (A) Localization of the MntR binding motif in the cg1623 promoter (box) and comparison with the MntR binding site of C. diphtheriae (above the box), −10 and −35 region as indicated in bold letters. The transcript is leaderless; +1 therefore indicates the transcriptional and translational start. (B) EMSAs of purified MntR binding its target promoters. A 30 bp oligonucleotide pair located in the promoter region of cg1918 was used as negative control. (C) Manganese dependence of MntR binding. Oligonucleotides (30 bp, 1 μM) were incubated with MntR, MnSO4 and EDTA in the given concentrations, analyzed using 15% native polyacrylamide gels and stained with SybrGreen I.

Figure 4.

Mutational analysis and verification of the MntR binding site. The predicted binding site is printed in bold letters. Three nucleotides were exchanged in each oligonucleotide as indicated (M1–M8). + indicates that the mutated fragment was bound with the same affinity as the unaltered wild-type fragment (positive control); (+) indicates that the mutated fragment was shifted, but with lower affinity; (−) indicates that the mutated fragment was not shifted or with much lower affinity. Oligonucleotides (30 bp, 1 μM) were incubated with MntR in the given concentrations and analyzed using 15% native polyacrylamide gels. A 30 bp oligonucleotide pair located in the promoter region of cg1918 was used as negative control.

Figure 4.

Mutational analysis and verification of the MntR binding site. The predicted binding site is printed in bold letters. Three nucleotides were exchanged in each oligonucleotide as indicated (M1–M8). + indicates that the mutated fragment was bound with the same affinity as the unaltered wild-type fragment (positive control); (+) indicates that the mutated fragment was shifted, but with lower affinity; (−) indicates that the mutated fragment was not shifted or with much lower affinity. Oligonucleotides (30 bp, 1 μM) were incubated with MntR in the given concentrations and analyzed using 15% native polyacrylamide gels. A 30 bp oligonucleotide pair located in the promoter region of cg1918 was used as negative control.

MntR binding is dependent on the presence of divalent metal cations

Addition of the chelating agent EDTA led to the dissociation of MntR-DNA complexes in vitro (Fig. 3C), confirming that the binding is strictly dependent on the presence of divalent metal ions. For DtxR-regulators, it is known that despite their high specificity in vivo they appear to have low ion selectivity in vitro (Guedon and Helmann 2003). This seems to be the case also for MntR as 100 μM of Mn2+, Fe2+, Zn2+, Ni2+ or Co2+ strengthened complex formation whereas the addition of Cu2+ inhibited binding (Fig. S4, Supporting Information). Together, in vitro protein–DNA interaction studies and in vivo promoter fusion experiments provided convincing evidence that Mn2+ is the major metal ion triggering MntR activity in the living organism.

The putative manganese transporter cg1623

In this work, we show that MntR, as a manganese-responsive regulator, seems to have a similar function as in related organisms, but mediates response to manganese starvation by derepression of a target gene not homologous to previously described MntR targets. Cg1623 is an uncharacterized membrane protein which is annotated as a member of the ZIP family of metal transporters. It is the only ZIP protein of C. glutamicum with a rather low conservation among the Corynebacteriales (homologous proteins are only present in the genomes of C. efficiens and Corynebacterium aurimucosum). It consists of 263 amino acids with seven (SMART; Letunic et al.2012) or eight (PredictProtein; Rost, Yachdav and Liu 2004) predicted transmembrane helices. A deletion mutant of cg1623 was constructed in this study and tested for its behavior under standard and metal starvation conditions. However, no growth phenotype was observed in standard CGXII medium (growth rates: wt: 0.61 ± 0.04, Δcg1623: 0.59 ± 0.02) and without Mn2+ (growth rates: wt: 0.53 ± 0.03, Δcg1623: 0.53 ± 0.02) or Zn2+ (growth rates: wt: 0.48 ± 0.04, Δcg1623: 0.44 ± 0.03). Furthermore, we tested the influence of cg1623 overexpression in the presence of Mn2+, Zn2+ or Fe2+ excess (Fig. S5, Supporting Information). Basal expression from the pAN6 plasmid with the leaky promoter Ptac has no significant influence on growth compared to an empty plasmid control strain. The induction of cg1623 by 100 μM IPTG leads to a strong growth defect already in standard CGXII medium, which is a rather typical consequence of the overproduction of a membrane protein. Hence, with this experimental setup it is not possible to observe ion specific effects to get further hints regarding the function of cg1623.

Proteins of the ZIP family of metal transporters can be found in a wide range of organisms including bacteria, fungi, plants, insects and mammals (Eide 2005) and are known to translocate, besides zinc, also other metal ions such as Fe2+, Mn2+, Cd2+ and Co2+ across cellular membranes (Guerinot 2000; Eide 2005; Taudte and Grass 2010). The discussion with respect to the driving force is controversial, but there are some hints that transport might be triggered by the proton motive force (Taudte and Grass 2010) or bicarbonate (Gaither and Eide 2000). The best characterized members are the ZIP1-4 zinc transporters of Arabidopsis thaliana (Grotz et al.1998), whereas the E. coli ZupT represents the first prokaryotic ZIP transporter identified and characterized in more detail (Grass et al.2002, 2005). E. coli ZupT has a rather broad substrate spectrum and was shown to transport Zn2+, Fe2+, Co2+, Mn2+ and Cd2+ (Grass et al.2005; Taudte and Grass 2010). An E. coli zupT single mutant has only a very slight phenotype which can be well explained by the broad substrate spectrum and the fact that there are several other uptake systems for zinc, manganese and iron in E. coli. This seems to be also the case in C. glutamicum because we did not observe an obvious phenotype for the single deletion mutant Δcg1623.

Different regulatory mechanisms have been described for ZIP homologs—both on transcriptional and post-transcriptional level. In Saccharomyces cerevisiae, the transcriptional activator Zap1 triggers the transcription of zinc uptake systems under zinc limited conditions (Zhao and Eide 1997). Several ZIP transporters of A. thaliana are also known to be induced under zinc deficiency conditions (Grotz et al.1998). Another level of control in yeast is the inactivation of zinc uptake systems by endocytosis and degradation in the presence of high zinc concentration (Gitan et al.1998). In contrast, E. coli ZupT appears to be constitutively expressed (Grass et al.2005). To our knowledge, cg1623 is the first example of a ZIP family protein that is regulated in a manganese-dependent manner. Whether cg1623 really transports Mn2+ and/or other metal ions remains to be elucidated in further experiments.

Control of manganese homeostasis in C. glutamicum

The regulator MntR that was characterized in this study represses transcription of the predicted ZIP family metal transporter cg1623 in the presence of sufficient intracellular concentrations of manganese. In growth experiments, we did not observe any significant phenotype for both the ΔmntR and the Δcg1623 mutants of C. glutamicum, in contrast to what is described for some other organisms (Que and Helmann 2000). This suggests that a different manganese uptake system as well as a manganese efflux system is likely present and regulated by different regulatory system(s) or mechanisms. A good candidate for the efflux system is cg1660, which has a high similarity to MntP (42% identity), a potential manganese efflux pump of E. coli (Waters, Sandoval and Storz 2011). The transcription level for cg1660 did not change upon deletion of mntR (ratio 1.169, P-value: 0.023). For the additional manganese uptake system, there is currently no obvious candidate, as no homologs of MntABCD can be found in the C. glutamicum genome. Interestingly, an MntH homolog was identified in the C. glutamicum R strain (cgR_0158), but not in ATCC 13032. Hence, there are two options: (i) C. glutamicum possesses a currently unknown manganese uptake system or (ii) manganese is taken up as side activity for example by the potential ABC-type zinc transport systems Cg0041–Cg0043 and Cg2911–Cg2913 (Schröder et al.2010). The third DtxR regulator of C. glutamicum, Cg2784, is very likely involved in the regulation of additional manganese homeostasis components, as its ligand binding residues (D12, D104) strongly suggest Mn2+ responsiveness (Guedon and Helmann 2003) (Fig. 5). As an additional point of evidence, the cg2784 gene is located between the two components of the manganese-ribonucleotide reductase NrdEF (Abbouni et al.2009), which could be a hint for the regulation of manganese containing proteins by this regulator. In summary, our study has revealed a first insight into the manganese regulatory network in C. glutamicum, but several further components remain to be elucidated.

Figure 5.

Alignment of the DtxR-type regulators of C. diphtheriae and C. glutamicum. Residues highlighted with a red background are highly conserved. Residues printed in red are partially conserved. Residues marked with a green arrow are involved in metal binding. The secondary structure of DIP1414 is shown above the alignment (Qiu et al.1996). The alignment presentation was prepared using ESPript (Gouet et al.2003).

Figure 5.

Alignment of the DtxR-type regulators of C. diphtheriae and C. glutamicum. Residues highlighted with a red background are highly conserved. Residues printed in red are partially conserved. Residues marked with a green arrow are involved in metal binding. The secondary structure of DIP1414 is shown above the alignment (Qiu et al.1996). The alignment presentation was prepared using ESPript (Gouet et al.2003).

SUPPLEMENTARY DATA

Supplementary data is available at FEMSLE online.

We thank Gerd Seibold and Nathalie Brühl for providing plasmid pK19mobsacB-Δcg1623, Jörn Kalinowski for the information about the cg1623 transcriptional start site as well as Cornelia Gätgens, Laura Beust and Sabrina Fassbender for excellent technical support.

FUNDING

This work was supported by the German Ministry of Education and Research (BMBF, grant 0316017B) and by the Helmholtz Association (Young Investigator grant VH-NG-716).

Conflict of interest statement. None declared.

REFERENCES

Abbouni
B
Oehlmann
W
Stolle
P
et al
Electron paramagnetic resonance (EPR) spectroscopy of the stable-free radical in the native metallo-cofactor of the manganese-ribonucleotide reductase (Mn-RNR) of Corynebacterium glutamicum
Free Radical Res
 
2009
43
943
50
Alm
EJ
Huang
KH
Price
MN
et al
The MicrobesOnline web site for comparative genomics
Genome Res
 
2005
15
1015
22
Andrews
S
Norton
I
Salunkhe
AS
et al
Banci
L
Control of iron metabolism in bacteria
Metallomics and the Cell
 
2013
Vol. 12
Dordrecht, Netherlands
Springer
203
39
Andrews
SC
Robinson
AK
Rodriguez-Quinones
F
Bacterial iron homeostasis
FEMS Microbiol Rev
 
2003
27
215
37
Baumgart
M
Luder
K
Grover
S
et al
IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria
BMC Biol
 
2013
11
122
Boyd
J
Oza
MN
Murphy
JR
Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae
P Natl Acad Sci USA
 
1990
87
5968
72
Brune
I
Werner
H
Hüser
AT
et al
The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum
BMC Genomics
 
2006
7
21
D'Aquino
JA
Tetenbaum-Novatt
J
White
A
et al
Mechanism of metal ion activation of the diphtheria toxin repressor DtxR
P Natl Acad Sci USA
 
2005
102
18408
13
DeWitt
MA
Kliegman
JI
Helmann
JD
et al
The conformations of the manganese transport regulator of Bacillus subtilis in its metal-free state
J Mol Biol
 
2007
365
1257
65
Eide
DJ
Iuchi
S
Kuldell
N
The ZIP family of zinc transporters
Zinc Finger Proteins
 
2005
New York
Springer
261
4
El Shafey
HM
Ghanem
S
Merkamm
M
et al
Corynebacterium glutamicum superoxide dismutase is a manganese-strict non-cambialistic enzyme in vitro
Microbiol Res
 
2008
163
80
6
Frunzke
J
Engels
V
Hasenbein
S
et al
Co-ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2
Mol Microbiol
 
2008
67
305
22
Gaither
LA
Eide
DJ
Functional expression of the human hZIP2 zinc transporter
J Biol Chem
 
2000
275
5560
4
Gitan
RS
Luo
H
Rodgers
J
et al
Zinc-induced inactivation of the yeast ZRT1 zinc transporter occurs through endocytosis and vacuolar degradation
J Biol Chem
 
1998
273
28617
24
Gouet
P
Robert
X
Courcelle
E
ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins
Nucleic Acids Res
 
2003
31
3320
3
Grass
G
Wong
MD
Rosen
BP
et al
ZupT is a Zn(II) uptake system in Escherichia coli
J Bacteriol
 
2002
184
864
6
Grass
G
Franke
S
Taudte
N
et al
The metal permease ZupT from Escherichia coli is a transporter with a broad substrate spectrum
J Bacteriol
 
2005
187
1604
11
Grotz
N
Fox
T
Connolly
E
et al
Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency
P Natl Acad Sci USA
 
1998
95
7220
4
Guedon
E
Helmann
JD
Origins of metal ion selectivity in the DtxR/MntR family of metalloregulators
Mol Microbiol
 
2003
48
495
506
Guerinot
ML.
The ZIP family of metal transporters
BBA-Biomembranes
 
2000
1465
190
8
Hanahan
D
Studies on transformation of Escherichia coli with plasmids
J Mol Biol
 
1983
166
557
80
Hantke
K.
Iron and metal regulation in bacteria
Curr Opin Microbiol
 
2001
4
172
7
Keilhauer
C
Eggeling
L
Sahm
H
Isoleucine synthesis in Corynebacterium glutamicum : molecular analysis of the Ilvb-Ilvn-Ilvc operon
J Bacteriol
 
1993
175
5595
603
Kinoshita
S
Udaka
S
Shimono
M
Studies of amino acid fermentation. Part I. Production of L-glutamic acid by various microorganisms
J Gen Appl Microbiol
 
1957
3
193
205
Kirchner
O
Tauch
A
Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum
J Biotechnol
 
2003
104
287
99
Letunic
I
Doerks
T
Bork
P
SMART 7: recent updates to the protein domain annotation resource
Nucleic Acids Res
 
2012
40
D302
5
Lieser
SA
Davis
TC
Helmann
JD
et al
DNA-binding and oligomerization studies of the manganese(II) metalloregulatory protein MntR from Bacillus subtilis
Biochemistry (Moscow)
 
2003
42
12634
42
Niebisch
A
Bott
M
Molecular analysis of the cytochrome bc1-aa3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c1
Arch Microbiol
 
2001
175
282
94
Niebisch
A
Kabus
A
Schultz
C
et al
Corynebacterial protein kinase G controls 2-oxoglutarate dehydrogenase activity via the phosphorylation status of the OdhI protein
J Biol Chem
 
2006
281
12300
7
Pappenheimer
AM
Johnson
SJ
Studies in diphtheria toxin production. I: the effect of iron and copper
Brit J Exp Pathol
 
1936
17
335
41
Pfeifer-Sancar
K
Mentz
A
Rückert
C
et al
Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique
BMC Genomics
 
2013
14
888
Qiu
XY
Pohl
E
Holmes
RK
et al
High-resolution structure of the diphtheria toxin repressor complexed with cobalt and manganese reveals an SH3-like third domain and suggests a possible role of phosphate as co-corepressor
Biochemistry (Moscow)
 
1996
35
12292
302
Que
Q
Helmann
JD
Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins
Mol Microbiol
 
2000
35
1454
68
Rost
B
Yachdav
G
Liu
JF
The PredictProtein server
Nucleic Acids Res
 
2004
32
W321
6
Sambrook
J
Russell
DW
Molecular Cloning: A Laboratory Manual
 
2001
New York, NY
Cold Spring Harbor Laboratory Press
Schäfer
A
Tauch
A
Jäger
W
et al
Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum
Gene
 
1994
145
69
73
Schmitt
MP
Analysis of a DtxR-like metalloregulatory protein, MntR, from Corynebacterium diphtheriae that controls expression of an ABC metal transporter by an Mn2+-dependent mechanism
J Bacteriol
 
2002
184
6882
92
Schmitt
MP
Holmes
RK
Iron-dependent regulation of diphtheria toxin and siderophore expression by the cloned Corynebacterium diphtheriae repressor gene dtxR in C. diphtheriae C7 strains
Infect Immun
 
1991
59
1899
904
Schröder
J
Jochmann
N
Rodionov
DA
et al
The Zur regulon of Corynebacterium glutamicum ATCC 13032
BMC Genomics
 
2010
11
12
Spiering
MM
Ringer
D
Murphy
JR
et al
Metal stoichiometry and functional studies of the diphtheria toxin repressor
P Natl Acad Sci USA
 
2003
100
3808
13
Studier
FW
Moffatt
BA
Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes
J Mol Biol
 
1986
189
113
30
Tao
X
Schiering
N
Zeng
HY
et al
Iron, DtxR, and the regulation of diphtheria-toxin expression
Mol Microbiol
 
1994
14
191
7
Taudte
N
Grass
G
Point mutations change specificity and kinetics of metal uptake by ZupT from Escherichia coli
Biometals
 
2010
23
643
56
van der Rest
ME
Lange
C
Molenaar
D
et al
A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA
Appl Microbiol Biot
 
1999
52
541
5
Vogt
M
Haas
S
Klaffl
S
et al
Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction
Metab Eng
 
2014
22
40
52
Waters
LS
Sandoval
M
Storz
G
The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis
J Bacteriol
 
2011
193
5887
97
Wennerhold
J
Bott
M
The DtxR regulon of Corynebacterium glutamicum
J Bacteriol
 
2006
188
2907
18
Zhao
H
Eide
DJ
Zap1p, a metalloregulatory protein involved in zinc-responsive transcriptional regulation in Saccharomyces cerevisiae
Mol Cell Biol
 
1997
17
5044
52