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The Zur regulon of Corynebacterium glutamicum ATCC 13032.

Schröder J, Jochmann N, Rodionov DA, Tauch A - BMC Genomics (2010)

Bottom Line: The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794).Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression.Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie, Universität Bielefeld, D-33615 Bielefeld, Germany.

ABSTRACT

Background: Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators.

Results: The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays.

Conclusion: Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.

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Comparative analysis of Zur proteins from actinobacteria. (A), Multiple amino acid sequence alignment of actinobacterial Zur proteins, including FurB from M. tuberculosis H37Rv. The winged-helix DNA binding domain is highlighted in grey. Three zinc binding sites (Zn 1 to Zn 3) deduced from the crystal structure of the mycobacterial FurB protein [23] are specifically coloured. Zn 1 (yellow): Asp-71, Cys-85, His-91, and His-93; Zn 2 (red): Cys-96, Cys-99, Cys-136, and Cys-139; Zn 3 (blue): His-90, His-92, Glu-111, and His-128 (according to the C. glutamicum protein positions). (B), Maximum likelihood phylogenetic tree of Zur protein orthologues from actinobacteria. The source of the abbreviated Zur-like proteins is indicated by the respective GenBank identifiers.
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Figure 1: Comparative analysis of Zur proteins from actinobacteria. (A), Multiple amino acid sequence alignment of actinobacterial Zur proteins, including FurB from M. tuberculosis H37Rv. The winged-helix DNA binding domain is highlighted in grey. Three zinc binding sites (Zn 1 to Zn 3) deduced from the crystal structure of the mycobacterial FurB protein [23] are specifically coloured. Zn 1 (yellow): Asp-71, Cys-85, His-91, and His-93; Zn 2 (red): Cys-96, Cys-99, Cys-136, and Cys-139; Zn 3 (blue): His-90, His-92, Glu-111, and His-128 (according to the C. glutamicum protein positions). (B), Maximum likelihood phylogenetic tree of Zur protein orthologues from actinobacteria. The source of the abbreviated Zur-like proteins is indicated by the respective GenBank identifiers.

Mentions: The Cg2502 (Zur) protein of C. glutamicum ATCC 13032 has a predicted size of 144 amino acids, a theoretical molecular mass of 15.7 kDa and belongs to the small core set of 24 transcription regulators that were detected in all hitherto sequenced corynebacterial genomes [5,19]. Protein domain predictions performed with the SUPERFAMILY [20] and the Conserved Domain Database tools [21] showed that the Zur protein contains an amino-terminal helix-turn-helix motif of the winged-helix type and is a member of the Zur (zinc uptake regulator) subgroup of the Fur (ferric uptake regulator) family of metalloregulatory proteins [14]. According to BLASTP data [22], the C. glutamicum Zur protein revealed high amino acid sequence similarities to orthologous proteins encoded in other sequenced corynebacterial genomes, ranging from 56% to 80% identical amino acids (Fig. 1A). Furthermore, Zur orthologues in other actinobacteria are well conserved and corroborated by the phylogenetic tree for these proteins (Fig. 1B). The Zur orthologue in Mycobacterium tuberculosis H37Rv (57% identity with Cg2502) is the zinc metalloregulator FurB, whose crystal structure has been elucidated recently [23]. The multiple alignment of Zur proteins from actinobacteria demonstrates the conservation of all amino acid residues forming three distinct zinc binding sites in the FurB protein (Fig. 1A). The zinc binding site 1 is surrounded by conserved aspartate, cysteine and histidine residues, whereas the zinc binding site 2 is represented by a cluster of four cysteines. The putative zinc binding site 3 is build by three histidines and one glutamate, but the exact biological function of this protein site remains to be determined [23]. These structural protein data strongly suggested that the Cg2502 (Zur) protein of C. glutamicum is a zinc-binding protein and involved in the transcriptional regulation of zinc metabolism in this species.


The Zur regulon of Corynebacterium glutamicum ATCC 13032.

Schröder J, Jochmann N, Rodionov DA, Tauch A - BMC Genomics (2010)

Comparative analysis of Zur proteins from actinobacteria. (A), Multiple amino acid sequence alignment of actinobacterial Zur proteins, including FurB from M. tuberculosis H37Rv. The winged-helix DNA binding domain is highlighted in grey. Three zinc binding sites (Zn 1 to Zn 3) deduced from the crystal structure of the mycobacterial FurB protein [23] are specifically coloured. Zn 1 (yellow): Asp-71, Cys-85, His-91, and His-93; Zn 2 (red): Cys-96, Cys-99, Cys-136, and Cys-139; Zn 3 (blue): His-90, His-92, Glu-111, and His-128 (according to the C. glutamicum protein positions). (B), Maximum likelihood phylogenetic tree of Zur protein orthologues from actinobacteria. The source of the abbreviated Zur-like proteins is indicated by the respective GenBank identifiers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2823685&req=5

Figure 1: Comparative analysis of Zur proteins from actinobacteria. (A), Multiple amino acid sequence alignment of actinobacterial Zur proteins, including FurB from M. tuberculosis H37Rv. The winged-helix DNA binding domain is highlighted in grey. Three zinc binding sites (Zn 1 to Zn 3) deduced from the crystal structure of the mycobacterial FurB protein [23] are specifically coloured. Zn 1 (yellow): Asp-71, Cys-85, His-91, and His-93; Zn 2 (red): Cys-96, Cys-99, Cys-136, and Cys-139; Zn 3 (blue): His-90, His-92, Glu-111, and His-128 (according to the C. glutamicum protein positions). (B), Maximum likelihood phylogenetic tree of Zur protein orthologues from actinobacteria. The source of the abbreviated Zur-like proteins is indicated by the respective GenBank identifiers.
Mentions: The Cg2502 (Zur) protein of C. glutamicum ATCC 13032 has a predicted size of 144 amino acids, a theoretical molecular mass of 15.7 kDa and belongs to the small core set of 24 transcription regulators that were detected in all hitherto sequenced corynebacterial genomes [5,19]. Protein domain predictions performed with the SUPERFAMILY [20] and the Conserved Domain Database tools [21] showed that the Zur protein contains an amino-terminal helix-turn-helix motif of the winged-helix type and is a member of the Zur (zinc uptake regulator) subgroup of the Fur (ferric uptake regulator) family of metalloregulatory proteins [14]. According to BLASTP data [22], the C. glutamicum Zur protein revealed high amino acid sequence similarities to orthologous proteins encoded in other sequenced corynebacterial genomes, ranging from 56% to 80% identical amino acids (Fig. 1A). Furthermore, Zur orthologues in other actinobacteria are well conserved and corroborated by the phylogenetic tree for these proteins (Fig. 1B). The Zur orthologue in Mycobacterium tuberculosis H37Rv (57% identity with Cg2502) is the zinc metalloregulator FurB, whose crystal structure has been elucidated recently [23]. The multiple alignment of Zur proteins from actinobacteria demonstrates the conservation of all amino acid residues forming three distinct zinc binding sites in the FurB protein (Fig. 1A). The zinc binding site 1 is surrounded by conserved aspartate, cysteine and histidine residues, whereas the zinc binding site 2 is represented by a cluster of four cysteines. The putative zinc binding site 3 is build by three histidines and one glutamate, but the exact biological function of this protein site remains to be determined [23]. These structural protein data strongly suggested that the Cg2502 (Zur) protein of C. glutamicum is a zinc-binding protein and involved in the transcriptional regulation of zinc metabolism in this species.

Bottom Line: The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794).Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression.Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie, Universität Bielefeld, D-33615 Bielefeld, Germany.

ABSTRACT

Background: Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators.

Results: The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays.

Conclusion: Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.

Show MeSH
Related in: MedlinePlus