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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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Fluorescence microscopy of E. coli DH5α MCR and C. glutamicum ATCC 13032. The cells are carrying either the empty pEPR1 vector, pEPR1 containing the znr upstream region, or pEPR1 containing the znr-zur intergenic region. Images at a 400-fold magnification were taken with transmitted light or UV light at 395 nm to detect GFP fluorescence.
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Figure 3: Fluorescence microscopy of E. coli DH5α MCR and C. glutamicum ATCC 13032. The cells are carrying either the empty pEPR1 vector, pEPR1 containing the znr upstream region, or pEPR1 containing the znr-zur intergenic region. Images at a 400-fold magnification were taken with transmitted light or UV light at 395 nm to detect GFP fluorescence.

Mentions: Operon predictions for C. glutamicum ATCC 13032 suggested that the znr-zur genes are expressed as bicistronic transcript [27]. To provide experimental support for this prediction, the transcription of the znr-zur region was analyzed by marker gene expression using the green fluorescent protein encoded on the promoter-probe vector pEPR1 [28]. Both the znr upstream region and the znr-zur intergenic region were tested for promoter activity in E. coli DH5α MCR and C. glutamicum ATCC 13032 (Fig. 3). For this purpose, a 141-bp DNA fragment covering the znr upstream region and a 107-bp DNA fragment containing the 40-bp znr-zur intergenic region were amplified by PCR and cloned in front of the promoterless gfp gene present on pEPR1. The expression of gfp was detected by fluorescence microscopy only with a pEPR1 derivative containing the znr upstream region, indicating the presence of a promoter in front of znr and supporting the view that znr and zur are organized as operon. This observation was further strengthened by detecting with RT-PCR a 309-bp cDNA fragment that encompasses the intergenic region on the znr-zur transcript (data not shown). The promoter in front of the znr gene was deduced from RACE-PCR experiments with total RNA purified from C. glutamicum ATCC 13032 cultures, showing that transcription starts at a guanine residue located 45 nucleotides upstream of the GTG start codon of znr (Fig. 2B). Based on the known consensus motif for corynebacterial promoters [29], potential -10 (TAAAAT) and -35 (CTCATA) promoter regions with an 18-bp spacing and a putative up-element [30] were detected (Fig. 2B).


The Zur regulon of Corynebacterium glutamicum ATCC 13032.

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

Fluorescence microscopy of E. coli DH5α MCR and C. glutamicum ATCC 13032. The cells are carrying either the empty pEPR1 vector, pEPR1 containing the znr upstream region, or pEPR1 containing the znr-zur intergenic region. Images at a 400-fold magnification were taken with transmitted light or UV light at 395 nm to detect GFP fluorescence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Fluorescence microscopy of E. coli DH5α MCR and C. glutamicum ATCC 13032. The cells are carrying either the empty pEPR1 vector, pEPR1 containing the znr upstream region, or pEPR1 containing the znr-zur intergenic region. Images at a 400-fold magnification were taken with transmitted light or UV light at 395 nm to detect GFP fluorescence.
Mentions: Operon predictions for C. glutamicum ATCC 13032 suggested that the znr-zur genes are expressed as bicistronic transcript [27]. To provide experimental support for this prediction, the transcription of the znr-zur region was analyzed by marker gene expression using the green fluorescent protein encoded on the promoter-probe vector pEPR1 [28]. Both the znr upstream region and the znr-zur intergenic region were tested for promoter activity in E. coli DH5α MCR and C. glutamicum ATCC 13032 (Fig. 3). For this purpose, a 141-bp DNA fragment covering the znr upstream region and a 107-bp DNA fragment containing the 40-bp znr-zur intergenic region were amplified by PCR and cloned in front of the promoterless gfp gene present on pEPR1. The expression of gfp was detected by fluorescence microscopy only with a pEPR1 derivative containing the znr upstream region, indicating the presence of a promoter in front of znr and supporting the view that znr and zur are organized as operon. This observation was further strengthened by detecting with RT-PCR a 309-bp cDNA fragment that encompasses the intergenic region on the znr-zur transcript (data not shown). The promoter in front of the znr gene was deduced from RACE-PCR experiments with total RNA purified from C. glutamicum ATCC 13032 cultures, showing that transcription starts at a guanine residue located 45 nucleotides upstream of the GTG start codon of znr (Fig. 2B). Based on the known consensus motif for corynebacterial promoters [29], potential -10 (TAAAAT) and -35 (CTCATA) promoter regions with an 18-bp spacing and a putative up-element [30] were detected (Fig. 2B).

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