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The Two-component Signal Transduction System CopRS of Corynebacterium glutamicum is Required for Adaptation to Copper-excess Stress

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 26 genes that showed a ≥3-fold increased mRNA level, including cg3280-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase and a copper-transport ATPase. 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 and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3280 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.

ORGANISM(S): Corynebacterium glutamicum ATCC 13032

PROVIDER: GSE27510 | GEO | 2011/09/05

SECONDARY ACCESSION(S): PRJNA138577

REPOSITORIES: GEO

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Project description:Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes including frozen dough baking. The cell viability and fermentation activity after freeze-thaw were dramatically decreased due to freeze-thaw injury. Because freeze-thaw injury involves complex phenomena, the mechanisms of it are not fully understood. We attempted to analyze the mechanisms of freeze-thaw injury by indirect gene expression analysis during post-thaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that a high frequency of the genes involved in the homeostasis of metal ions were up-regulated depending on the freezing period. The phenotype of the deletion mutants of the up-regulated genes extracted by indirect gene expression analysis was assessed. The deletion strains of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. Supplementation with copper ions during post-thaw incubation increased intracellular superoxide dismutase activity. Inverse correlated with intracellular superoxide dismutase activity, intracellular levels of reactive oxygen species were decreased. Moreover, cell viability increased by supplementation with copper ions under specific assessment conditions. This study suggested that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury. Total RNA was extracted from the stress-treated yeast cells by using a hot phenol method. Poly(A)+ RNA was enriched from total RNA by using an Oligotex dT30 (Super) mRNA purification kit (Takara Bio, Ohtsu, Japan). cDNA synthesis, cRNA synthesis, and labeling were performed according to the Affymetrix user’s manual (Affymetrix, Santa Clara, USA). Biotinyated cRNA was fragmented and then used as a probe.Affimetrix Yeast Genome 2.0 arrays (Affymetrix) were used as DNA microarrays. All experiments were done in triplicate independently.

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The Two-component Signal Transduction System CopRS of Corynebacterium glutamicum is Required for Adaptation to Copper-excess Stress

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