Dataset Information

Cu(I)-mediated allosteric switching in a copper-sensing operon repressor (CsoR).

ABSTRACT: The copper-sensing operon repressor (CsoR) is representative of a major Cu(I)-sensing family of bacterial metalloregulatory proteins that has evolved to prevent cytoplasmic copper toxicity. It is unknown how Cu(I) binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes. A phylogenetic analysis of 227 DUF156 protein members, including biochemically or structurally characterized CsoR/RcnR repressors, reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)-bound Gt CsoR reveals that Cu(I) binding induces a kink in the ?2-helix between two conserved copper-ligating residues and folds an N-terminal tail (residues 12-19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo-state with these dynamics quenched upon Cu(I) binding. Small angle x-ray scattering experiments on an N-terminally truncated Gt CsoR (?2-10) reveal that the Cu(I)-bound tetramer is hydrodynamically more compact than is the apo-state. The implications of these findings for the allosteric mechanisms of other CsoR/RcnR repressors are discussed.

SUBMITTER: Chang FM

PROVIDER: S-EPMC4081955 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Cu(I)-mediated allosteric switching in a copper-sensing operon repressor (CsoR).

Chang Feng-Ming James FM Coyne H Jerome HJ Cubillas Ciro C Vinuesa Pablo P Fang Xianyang X Ma Zhen Z Ma Dejian D Helmann John D JD García-de los Santos Alejandro A Wang Yun-Xing YX Dann Charles E CE Giedroc David P DP

The Journal of biological chemistry 20140515 27

The copper-sensing operon repressor (CsoR) is representative of a major Cu(I)-sensing family of bacterial metalloregulatory proteins that has evolved to prevent cytoplasmic copper toxicity. It is unknown how Cu(I) binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes. A phylogenetic analysis of 227 DUF156 protein members, including biochemically or structurally characterized CsoR/RcnR repressors, reveals that Geobacillus thermodenitrificans (Gt) CsoR charac ...[more]

PMID: 24831014

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Project description:Purpose: CsoR, a copper responsive negative regulator of Mycobacterium tuberculosis has been shown to respond to copper and bind to its own regulon in in vitro experiments. Here we examine the role of CsoR in vivo by examining the impact of deletion of csoR on the host transcriptome. Methods: csoR was knocked out of M. tuberculosis H37Rv using the specialized transduction method developed by Bardarov et al (2002), followed by removal of the hygromycin marker with plasmid pYUB870. Both wild type and confirmed csoR knockout were grown in copper free Sauton's media to late log phase before harvesting for transcriptomic studies. RNA was extracted using a modified Trizol method prior to DNAse treatment and rRNA depletion. Sequencing was done on an Illumina HiSeq 2000, filtered for quality using the FASTX-Toolkit, and mapped using Bowtie, and counts per locus generated with BedTools in the Galaxy platform. Differential expression analysis was carried out in edgeR with significantly differentially expressed genes being identified as those with ≥|2| fold differential expression between ΔcsoR and wild type strains, and an FDR < 0.05. Results: We found that 223 genes were differentially expressed between the ΔcsoR and wild type strains, 52 induced and 71 repressed. Differential expression of 10 of these genes, 6 induced and 4 repressed, was confirmed by qRT-PCR using SYBR green methodology. The only copper responsive genes identified were those within the csoR operon: csoR, Rv0968, ctpV, Rv0970, suggesting that csoR may only regulate its own operon in response to copper. Genes in the RicR regulon, another copper responsive transcriptional regulator, were not significantly differentially expressed, but some of these copper inducible genes were slightly down regulated suggesting that copper levels may be lower in the mutant strain as compared to wild type. Notably, 44 of the 48 members of the dosR regulon, responsive to hypoxic and NO stress, were induced in the mutant vs wild type suggesting that csoR is necessary for maintaining homeostasis, likely through copper regulation, within the cell. Conclusions: We have shown that CsoR is likely only directly regulating its own operon in response to copper, however it is required to maintain homeostasis, preventing a hypoxia-type stress response in the absense of copper.

Dataset Information

Cu(I)-mediated allosteric switching in a copper-sensing operon repressor (CsoR).

Publications

Cu(I)-mediated allosteric switching in a copper-sensing operon repressor (CsoR).

Similar Datasets

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