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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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Specific Cat activities of C. glutamicum wild type (wt) and the ΔcopRS mutant.Both strains carrying either the pET2, the pET2-IGR (copR promoter) or the pET2-IGR_inverse (cg3286 promoter) vector. Cat activities were measured after growth in CGXII medium containing either 1.25 µM CuSO4 (black bars), 20 µM additional CuSO4 (dark grey bars) or 20 µM additional NiSO4 (light grey bars). A, wt/pET2; B, ΔcopRS/pET2; C, wt/pET2-IGR (copR promoter); D, ΔcopRS/pET2-IGR; E, wt/pET2-IGR_inverse (cg3286 promoter); F, ΔcopRS/pET2-IGR_inverse. The values represent averages and standard deviations of three biological replicates.
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pone-0022143-g006: Specific Cat activities of C. glutamicum wild type (wt) and the ΔcopRS mutant.Both strains carrying either the pET2, the pET2-IGR (copR promoter) or the pET2-IGR_inverse (cg3286 promoter) vector. Cat activities were measured after growth in CGXII medium containing either 1.25 µM CuSO4 (black bars), 20 µM additional CuSO4 (dark grey bars) or 20 µM additional NiSO4 (light grey bars). A, wt/pET2; B, ΔcopRS/pET2; C, wt/pET2-IGR (copR promoter); D, ΔcopRS/pET2-IGR; E, wt/pET2-IGR_inverse (cg3286 promoter); F, ΔcopRS/pET2-IGR_inverse. The values represent averages and standard deviations of three biological replicates.

Mentions: The data reported above showed that CopR binds to the intergenic region between cg3286 and copR, but it was still unclear if CopR activates expression of only the copR promoter, of only the cg3286 promoter or of both promoters. To answer this question, reporter gene assays were performed. The plasmids pET2-IGR and pET2-IGR_inverse, which contain the cg3286-copR intergenic region in both orientations upstream of a promoterless chloramphenicol acetyltransferase (cat) gene, were transferred into C. glutamicum wild type and the ΔcopRS mutant. The resulting strains and the control strains carrying the vector pET2 were cultivated in CGXII minimal medium with or without the addition of 20 µM CuSO4 or 20 µM NiSO4, respectively. When cultivated in standard CGXII medium with 1.25 µM CuSO4 (Fig. 6), none of the strains exhibited Cat activity. Same results were observed when NiSO4 was added (Fig. 6). Thus, CopR did not activate transcription of the reporter gene under these conditions. When the medium was supplemented with 20 µM CuSO4 (Fig. 6), Cat activity was measurable for the wild type strain harbouring the plasmid pET2-IGR or pET2-IGR_inverse, but not for the ΔcopRS mutant harbouring one of these plasmids. Also the control strains carrying pET2 had no measurable Cat activity. These results showed that both the cg3286 promoter and the copR promoter are activated by CopRS in a strictly copper-dependent manner.


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)

Specific Cat activities of C. glutamicum wild type (wt) and the ΔcopRS mutant.Both strains carrying either the pET2, the pET2-IGR (copR promoter) or the pET2-IGR_inverse (cg3286 promoter) vector. Cat activities were measured after growth in CGXII medium containing either 1.25 µM CuSO4 (black bars), 20 µM additional CuSO4 (dark grey bars) or 20 µM additional NiSO4 (light grey bars). A, wt/pET2; B, ΔcopRS/pET2; C, wt/pET2-IGR (copR promoter); D, ΔcopRS/pET2-IGR; E, wt/pET2-IGR_inverse (cg3286 promoter); F, ΔcopRS/pET2-IGR_inverse. The values represent averages and standard deviations of three biological replicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140484&req=5

pone-0022143-g006: Specific Cat activities of C. glutamicum wild type (wt) and the ΔcopRS mutant.Both strains carrying either the pET2, the pET2-IGR (copR promoter) or the pET2-IGR_inverse (cg3286 promoter) vector. Cat activities were measured after growth in CGXII medium containing either 1.25 µM CuSO4 (black bars), 20 µM additional CuSO4 (dark grey bars) or 20 µM additional NiSO4 (light grey bars). A, wt/pET2; B, ΔcopRS/pET2; C, wt/pET2-IGR (copR promoter); D, ΔcopRS/pET2-IGR; E, wt/pET2-IGR_inverse (cg3286 promoter); F, ΔcopRS/pET2-IGR_inverse. The values represent averages and standard deviations of three biological replicates.
Mentions: The data reported above showed that CopR binds to the intergenic region between cg3286 and copR, but it was still unclear if CopR activates expression of only the copR promoter, of only the cg3286 promoter or of both promoters. To answer this question, reporter gene assays were performed. The plasmids pET2-IGR and pET2-IGR_inverse, which contain the cg3286-copR intergenic region in both orientations upstream of a promoterless chloramphenicol acetyltransferase (cat) gene, were transferred into C. glutamicum wild type and the ΔcopRS mutant. The resulting strains and the control strains carrying the vector pET2 were cultivated in CGXII minimal medium with or without the addition of 20 µM CuSO4 or 20 µM NiSO4, respectively. When cultivated in standard CGXII medium with 1.25 µM CuSO4 (Fig. 6), none of the strains exhibited Cat activity. Same results were observed when NiSO4 was added (Fig. 6). Thus, CopR did not activate transcription of the reporter gene under these conditions. When the medium was supplemented with 20 µM CuSO4 (Fig. 6), Cat activity was measurable for the wild type strain harbouring the plasmid pET2-IGR or pET2-IGR_inverse, but not for the ΔcopRS mutant harbouring one of these plasmids. Also the control strains carrying pET2 had no measurable Cat activity. These results showed that both the cg3286 promoter and the copR promoter are activated by CopRS in a strictly copper-dependent manner.

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