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The two-component signal transduction system CopRS of Corynebacterium glutamicum is required for adaptation to copper-excess stress.

Schelder S, Zaade D, Litsanov B, Bott M, Brocker M - PLoS ONE (2011)

Bottom Line: Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified.Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289.Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress.

View Article: PubMed Central - PubMed

Affiliation: Institut für Bio-und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, Jülich, Germany.

ABSTRACT
Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu(2+) was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress.

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Mutational analysis of the putative CopR binding site within the cg3286-copR intergenic region.To determine the importance of the predicted binding motif (sequence in the grey boxes) for CopR binding, fragments with different mutations were tested in EMSAs with phosphorylated CopR. According to the results of the shifts, the fragments were classified into two categories: +, mutated fragment shifted like the wild type fragment; -, the fragment was not shifted.
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pone-0022143-g005: Mutational analysis of the putative CopR binding site within the cg3286-copR intergenic region.To determine the importance of the predicted binding motif (sequence in the grey boxes) for CopR binding, fragments with different mutations were tested in EMSAs with phosphorylated CopR. According to the results of the shifts, the fragments were classified into two categories: +, mutated fragment shifted like the wild type fragment; -, the fragment was not shifted.

Mentions: Analysis of the identified DNA region with the motif alignment and search tool MAST (http://meme.sdsc.edu/meme4_4_0/cgi-bin/mast.cgi) revealed the presence of a perfect direct repeat separated by a two base pair linker (TGAAGATTTgaTGAAGATTT). The relevance of this direct repeat for CopR binding was tested by EMSAs with DNA fragments in which two to four nucleotides of the motif or of the surrounding sequence were exchanged (Fig. 5). Mutations in the motifs' flanking sequence or in the linker did not affect CopR binding, whereas mutations of three base pairs within the binding motif led to a complete inhibition of CopR binding. These results confirmed that the predicted DNA motif is relevant for CopR binding.


The two-component signal transduction system CopRS of Corynebacterium glutamicum is required for adaptation to copper-excess stress.

Schelder S, Zaade D, Litsanov B, Bott M, Brocker M - PLoS ONE (2011)

Mutational analysis of the putative CopR binding site within the cg3286-copR intergenic region.To determine the importance of the predicted binding motif (sequence in the grey boxes) for CopR binding, fragments with different mutations were tested in EMSAs with phosphorylated CopR. According to the results of the shifts, the fragments were classified into two categories: +, mutated fragment shifted like the wild type fragment; -, the fragment was not shifted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140484&req=5

pone-0022143-g005: Mutational analysis of the putative CopR binding site within the cg3286-copR intergenic region.To determine the importance of the predicted binding motif (sequence in the grey boxes) for CopR binding, fragments with different mutations were tested in EMSAs with phosphorylated CopR. According to the results of the shifts, the fragments were classified into two categories: +, mutated fragment shifted like the wild type fragment; -, the fragment was not shifted.
Mentions: Analysis of the identified DNA region with the motif alignment and search tool MAST (http://meme.sdsc.edu/meme4_4_0/cgi-bin/mast.cgi) revealed the presence of a perfect direct repeat separated by a two base pair linker (TGAAGATTTgaTGAAGATTT). The relevance of this direct repeat for CopR binding was tested by EMSAs with DNA fragments in which two to four nucleotides of the motif or of the surrounding sequence were exchanged (Fig. 5). Mutations in the motifs' flanking sequence or in the linker did not affect CopR binding, whereas mutations of three base pairs within the binding motif led to a complete inhibition of CopR binding. These results confirmed that the predicted DNA motif is relevant for CopR binding.

Bottom Line: Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified.Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289.Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress.

View Article: PubMed Central - PubMed

Affiliation: Institut für Bio-und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, Jülich, Germany.

ABSTRACT
Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu(2+) was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress.

Show MeSH
Related in: MedlinePlus