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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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Ratio/intensity (m/a) plot deduced from DNA microarray hybridizations comparing the transcriptome of the zur mutant C. glutamicum JS2502 with that of the wild-type strain C. glutamicum ATCC 13032. Two biological replicates including label swapping were used for DNA microarray hybridizations. Genes showing significantly enhanced expression in C. glutamicum JS2502 are marked by black dots, decreased transcript levels are indicated by triangles, and genes without differential expression pattern are shown by grey diamonds. Genes were regarded as differentially expressed using the following cut-offs: m-value ≥ 1.0, upregulation; m-value ≤- 1.0, downregulation. The cut-offs correspond to relative changes in gene expression of at least two-fold.
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Figure 5: Ratio/intensity (m/a) plot deduced from DNA microarray hybridizations comparing the transcriptome of the zur mutant C. glutamicum JS2502 with that of the wild-type strain C. glutamicum ATCC 13032. Two biological replicates including label swapping were used for DNA microarray hybridizations. Genes showing significantly enhanced expression in C. glutamicum JS2502 are marked by black dots, decreased transcript levels are indicated by triangles, and genes without differential expression pattern are shown by grey diamonds. Genes were regarded as differentially expressed using the following cut-offs: m-value ≥ 1.0, upregulation; m-value ≤- 1.0, downregulation. The cut-offs correspond to relative changes in gene expression of at least two-fold.

Mentions: To identify C. glutamicum genes that are under transcriptional control by Zur, the zur gene was deleted in the chromosome of the wild-type strain C. glutamicum ATCC 13032 by an allelic exchange procedure, resulting in the mutant strain C. glutamicum JS2502. Growth of the zur-deficient mutant C. glutamicum JS2502 in minimal medium CGXII was indistinguishable from the parental wild-type strain (data not shown), indicating that deregulation of the Zur regulon is not detrimental to any basic physiological functions in C. glutamicum. The genome-wide expression profile of C. glutamicum JS2502 was compared with that of C. glutamicum ATCC 13032 by DNA microarray hybridizations. The resulting ratio/intensity (m/a) plot of the normalized data, based on two hybridization experiments with label swapping, is presented in Fig. 5. By applying a ratio cut-off of ± 1, which is equivalent to relative expression changes of at least two-fold, 23 genes exhibited higher transcript levels in the zur mutant when compared to the wild-type strain, whereas three genes showed lower transcript levels in C. glutamicum JS2502.


The Zur regulon of Corynebacterium glutamicum ATCC 13032.

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

Ratio/intensity (m/a) plot deduced from DNA microarray hybridizations comparing the transcriptome of the zur mutant C. glutamicum JS2502 with that of the wild-type strain C. glutamicum ATCC 13032. Two biological replicates including label swapping were used for DNA microarray hybridizations. Genes showing significantly enhanced expression in C. glutamicum JS2502 are marked by black dots, decreased transcript levels are indicated by triangles, and genes without differential expression pattern are shown by grey diamonds. Genes were regarded as differentially expressed using the following cut-offs: m-value ≥ 1.0, upregulation; m-value ≤- 1.0, downregulation. The cut-offs correspond to relative changes in gene expression of at least two-fold.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823685&req=5

Figure 5: Ratio/intensity (m/a) plot deduced from DNA microarray hybridizations comparing the transcriptome of the zur mutant C. glutamicum JS2502 with that of the wild-type strain C. glutamicum ATCC 13032. Two biological replicates including label swapping were used for DNA microarray hybridizations. Genes showing significantly enhanced expression in C. glutamicum JS2502 are marked by black dots, decreased transcript levels are indicated by triangles, and genes without differential expression pattern are shown by grey diamonds. Genes were regarded as differentially expressed using the following cut-offs: m-value ≥ 1.0, upregulation; m-value ≤- 1.0, downregulation. The cut-offs correspond to relative changes in gene expression of at least two-fold.
Mentions: To identify C. glutamicum genes that are under transcriptional control by Zur, the zur gene was deleted in the chromosome of the wild-type strain C. glutamicum ATCC 13032 by an allelic exchange procedure, resulting in the mutant strain C. glutamicum JS2502. Growth of the zur-deficient mutant C. glutamicum JS2502 in minimal medium CGXII was indistinguishable from the parental wild-type strain (data not shown), indicating that deregulation of the Zur regulon is not detrimental to any basic physiological functions in C. glutamicum. The genome-wide expression profile of C. glutamicum JS2502 was compared with that of C. glutamicum ATCC 13032 by DNA microarray hybridizations. The resulting ratio/intensity (m/a) plot of the normalized data, based on two hybridization experiments with label swapping, is presented in Fig. 5. By applying a ratio cut-off of ± 1, which is equivalent to relative expression changes of at least two-fold, 23 genes exhibited higher transcript levels in the zur mutant when compared to the wild-type strain, whereas three genes showed lower transcript levels in C. glutamicum JS2502.

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