Limits...
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.

Show MeSH

Related in: MedlinePlus

Zinc-dependent activity of the cg0042 and cg2911 operon promoters. The promoter activities of the Zur-regulated operons cg0042 and cg2911 was measured in the wild-type strain C. glutamicum ATCC 13032 (WT) and in the zur mutant C. glutamicum JS2502 (zur) under low, high and zinc-chelated (TPEN) conditions. The relative expression of the gfp reporter gene was determined by real-time RT-PCR. The values are means of four measurements. The relative expression was calculated by using a C. glutamicum control carrying the empty expression vector pEPR1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2823685&req=5

Figure 7: Zinc-dependent activity of the cg0042 and cg2911 operon promoters. The promoter activities of the Zur-regulated operons cg0042 and cg2911 was measured in the wild-type strain C. glutamicum ATCC 13032 (WT) and in the zur mutant C. glutamicum JS2502 (zur) under low, high and zinc-chelated (TPEN) conditions. The relative expression of the gfp reporter gene was determined by real-time RT-PCR. The values are means of four measurements. The relative expression was calculated by using a C. glutamicum control carrying the empty expression vector pEPR1.

Mentions: As the putative operons cg0042 and cg2911 are apparently under negative control by the Zur protein in C. glutamicum, we investigated their zinc-dependent expression in vivo by using again the promoterless gfp reporter system. For this purpose, the mapped promoter regions were amplified by PCR and cloned into the promoter-probe vector pEPR1. The resulting plasmids pEPR1_prom_cg0042 and pEPR1_prom_cg2911 (Table 3) were transferred into the C. glutamicum ATCC 13032 wild-type strain and into the zur mutant C. glutamicum JS2502 to detect differential gfp expression by real-time RT-PCR, using high, low and chelated zinc conditions in the growth medium (Fig. 7). C. glutamicum ATCC 13032 carrying the empty cloning vector pEPR1 served as reference for calculating the differential gene expression. In the wild-type strain, the cloned promoters are apparently repressed under high-zinc condition and are derepressed under zinc-depletion, i.e. low-zinc condition and during growth in the presence of the chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). A similar deregulation of gene expression was detected in the zur mutant C. glutamicum JS2502, irrespective of the presence or absence of zinc ions in the growth medium (Fig. 7). These in vivo data suggested that the lack of zinc-dependent regulation of gene expression is caused by the absence of the Zur protein in C. glutamicum JS2502. Furthermore, the data indicated that the Zur protein is sensing zinc ions and that it binds to operator sequences in the presence of zinc, thus acting as a repressor of the cg0042 and cg2911 operons.


The Zur regulon of Corynebacterium glutamicum ATCC 13032.

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

Zinc-dependent activity of the cg0042 and cg2911 operon promoters. The promoter activities of the Zur-regulated operons cg0042 and cg2911 was measured in the wild-type strain C. glutamicum ATCC 13032 (WT) and in the zur mutant C. glutamicum JS2502 (zur) under low, high and zinc-chelated (TPEN) conditions. The relative expression of the gfp reporter gene was determined by real-time RT-PCR. The values are means of four measurements. The relative expression was calculated by using a C. glutamicum control carrying the empty expression vector pEPR1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Zinc-dependent activity of the cg0042 and cg2911 operon promoters. The promoter activities of the Zur-regulated operons cg0042 and cg2911 was measured in the wild-type strain C. glutamicum ATCC 13032 (WT) and in the zur mutant C. glutamicum JS2502 (zur) under low, high and zinc-chelated (TPEN) conditions. The relative expression of the gfp reporter gene was determined by real-time RT-PCR. The values are means of four measurements. The relative expression was calculated by using a C. glutamicum control carrying the empty expression vector pEPR1.
Mentions: As the putative operons cg0042 and cg2911 are apparently under negative control by the Zur protein in C. glutamicum, we investigated their zinc-dependent expression in vivo by using again the promoterless gfp reporter system. For this purpose, the mapped promoter regions were amplified by PCR and cloned into the promoter-probe vector pEPR1. The resulting plasmids pEPR1_prom_cg0042 and pEPR1_prom_cg2911 (Table 3) were transferred into the C. glutamicum ATCC 13032 wild-type strain and into the zur mutant C. glutamicum JS2502 to detect differential gfp expression by real-time RT-PCR, using high, low and chelated zinc conditions in the growth medium (Fig. 7). C. glutamicum ATCC 13032 carrying the empty cloning vector pEPR1 served as reference for calculating the differential gene expression. In the wild-type strain, the cloned promoters are apparently repressed under high-zinc condition and are derepressed under zinc-depletion, i.e. low-zinc condition and during growth in the presence of the chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). A similar deregulation of gene expression was detected in the zur mutant C. glutamicum JS2502, irrespective of the presence or absence of zinc ions in the growth medium (Fig. 7). These in vivo data suggested that the lack of zinc-dependent regulation of gene expression is caused by the absence of the Zur protein in C. glutamicum JS2502. Furthermore, the data indicated that the Zur protein is sensing zinc ions and that it binds to operator sequences in the presence of zinc, thus acting as a repressor of the cg0042 and cg2911 operons.

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