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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 Cu2+ 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.
Highlights
Due to its ability to change between the oxidised Cu2+ and reduced Cu+ state, copper has become a versatile cofactor for enzymes involved in electron transport or redox reactions such as cytochrome c oxidases or monooxygenases [1]
This assumption is supported by the fact that several of these genes code for proteins that are obviously linked to copper homeostasis, such as a putative copper-transporting ATPase (CopB, Cg3281; Accession UniProtKB Q8NLI0) and a secreted multicopper oxidase (CopO, Cg3287; Accession UniProtKB Q8NLH5)
To enable the cell to react to copper-induced stress, several copper ion sensors were evolved like CueR of E. coli, CopY of E. hirae and CsoR of M. tuberculosis [2,4,12,24]
Summary
Due to its ability to change between the oxidised Cu2+ and reduced Cu+ state, copper has become a versatile cofactor for enzymes involved in electron transport or redox reactions such as cytochrome c oxidases or monooxygenases [1]. In E. hirae the cop operon is mainly responsible for copper homeostasis It consists of four genes coding for a transcriptional repressor (CopY), a copper chaperon (CopZ) and two copper Ptype ATPases (CopA and CopB). In the presence of elevated copper concentrations CopZ donates Cu+ to CopY resulting in a derepression of the cop operon and subsequently in copper export by CopB [2]. In the presence of elevated copper concentrations, CueR activates the transcription of copA and cueO encoding a P-type ATPase and an oxygen-dependent multicopper oxidase, respectively [8,9]. The two-component system CusRS was found to play a role in copper homeostasis under anoxic conditions It represents a prototypical two-component system [10] where the membrane-bound sensor kinase CusS monitors the periplasmic copper concentration and autophosphorylates a histidine residue at elevated copper concentrations. The translation products CusCBA (a proton-cation antiporter) and CusF (a copper chaperone) contribute to copper tolerance under copper stress conditions
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