The Two-Component Signal Transduction System CopRS of Corynebacterium glutamicum Is Required for Adaptation to Copper-Excess Stress

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

CopRS and CsoR: two regulatory systems involved in copper homeostasis of Corynebacterium glutamicum\textit{Corynebacterium glutamicum}

Many organisms, from bacteria to humans, require copper for life, as it is part of many enzymes. On the other hand, copper causes problems due to its toxicity when present in high amounts. Consequently, a tight control of copper usage is a cellular necessity. The interest in understanding bacterial copper homeostasis systems has increased in the past years, since it became obvious that it is partially conserved in humans and also responsible for full virulence of pathogenic microorganisms like e.g. $\textit{Mycobacterium tuberculosis}$. In this work the regulation of the copper stress response was investigated in the Gram-positive soil bacterium $\textit{Corynebacterium glutamicum}$, a non-pathogenic relative of $\textit{M. tuberculosis}$. The following results were obtained: 1. Via genome-wide analysis of the transcriptional response of $\textit{C. glutamicum}$ to excess copper (20 μM) the copper stimulon was determined. It includes heme biosynthesis and putative copper resistance genes, and the regulatory genes $\textit{copRS}$ (cg3285-cg3284) and $\textit{csoR}$ (cg0463). 2. The $\textit{copRS}$ genes encode a classical two-component system, which is active only in the presence of excess copper. Under these conditions, the response regulator CopR binds to a single operator sequence within the genome and activates transcription of two divergently located gene regions ($\textit{cg3286-copO-cg3288-tlpA}$ and $\textit{copR-copS-cg3282-copB}$), encoding copper resistance proteins. Evidence was provided that two histidine residues (H41, H56) within the ‘periplasmic’ loop of the sensor kinase CopS are important for copper sensing or signal transfer. 3. The transcriptional regulator CsoR belongs to the family of cytoplasmic CsoR-type repressors. Besides the CopRS target genes cg3282 and $\textit{copB}$ encoding a ‘periplasmic’ copper-binding protein and a putative copper-exporting P-type ATPase, respectively, the copper chaperone genes cg3402 and cg3411 and the $\textit{ctpV-csoR}$ operon, encoding the copper export P-type ATPase CtpV, are direct target genes of CsoR. It was demonstrated that CsoR represses their transcription in the absence of excess copper by binding to a conserved DNA sequence motif within the promoter regions of the target genes. 4. Mutational analyses of the CopRS and CsoR target genes revealed that $\textit{C. glutamicum}$ possesses two main pathways for detoxification of excess copper. CopRS confers ‘periplasmic’ copper resistance via the multicopper oxidase CopO, whereas CsoR controls the cytoplasmic copper resistance via the copper export P-type ATPase CtpV