Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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. Four or five biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown with 1.25 µM or with 21.25 µM CuSO4; Exp. 2: WT vs. copRS deletion mutant grown with 21.25 µM CuSO4; For analysis via DNA microarrays, the RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 1.25 uM CuSO4 or 21.25 uM CuSO4.

ORGANISM(S): Corynebacterium glutamicum ATCC 13032

SUBMITTER: Tino Polen

PROVIDER: E-GEOD-27510 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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

Schelder Stephanie S Zaade Daniela D Litsanov Boris B Bott Michael M Brocker Melanie M

PloS one 20110720 7

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 ( ...[more]

PMID: 21799779

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Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum. Three biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown und FeSO4 or heme as iron source; Exp. 2: WT vs. hrrA deletion mutant grown on FeSO4; Exp. 3: WT vs. hrrA mutant grown on heme. For analysis via DNA microarraysose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 2.5 uM FeSO4 or 2.5 uM heme as iron source.

Project description:Pupylation is a posttranslational protein modification in bacteria resembling ubiquitination in eukaryotes. The prokaryotic ubiquitin-like protein Pup is covalently attached to other proteins via an isopeptide bond between its carboxyterminal glutamate residue and a lysine residue in the target. In mycobacteria, pupylation was shown to mark proteins for unfolding by the ATPase Mpa and subsequent degradation by the proteasome. However, the occurrence of pupylation in species without a proteasome like Corynebacterium glutamicum suggests that degradation may not be the only fate of pupylated proteins. The Îpup mutant senses a stronger iron limitation than the wild type. Among the 125 genes showing at least 2-fold changes in transcript levels in the Îpup mutant (p-value â¤ 0.05) were 54% of all genes known to be regulated by the master regulator of iron homeostasis DtxR (Brune et al., 2006; Wennerhold and Bott, 2006). Except for ftn, which is activated by DtxR and showed a 2-fold decreased mRNA level, the other DtxR target genes showed increased mRNA levels in the Îpup strain. These included ripA, encoding a transcriptional regulator of iron proteins, which represses a number of prominent iron-containing proteins under iron limitation, such as aconitase or succinate dehydrogenase (Wennerhold et al., 2005). 79% of the known RipA target genes showed decreased mRNA levels in the Îpup strain. DNA microarray analyses were performed to compare the mRNA levels of the C. glutamicum Îpup mutant and its parent wild type under iron-limited conditions. The two strains precultivated in CGXII medium with 4% (w/v) glucose and 1 ÂµM FeSO4 were inoculated into fresh medium to an OD600 of 1, cultured for 2 h, and harvested on ice by centrifugation (5 min at 4,000 g and 4Â°C). Please note that the GPL16989 array design comprises oligos for 4 different bacterial genomes. In the GPR-files in this study, all IDs/Names (oligonucleotides) which are not from the host C. glutamicum were replaced by the text EMPTY since only C. glutamicum expression was analyzed.

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Dataset Information

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

Publications

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

Similar Datasets

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