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Molecular insights into the metal selectivity of the copper(I)-sensing repressor CsoR from Bacillus subtilis.

ABSTRACT: Bacillus subtilis CsoR (Bsu CsoR) is a copper-sensing transcriptional repressor that regulates the expression of the copZA operon encoding a copper chaperone and a Cu efflux P-type ATPase, respectively. Bsu CsoR is a homologue of Mycobacterium tuberculosis CsoR (Mtb CsoR), representative of a large Cu(I)-sensing regulatory protein family. We show here that Bsu CsoR binds approximately 1 mol equiv of Cu(I) per monomer in vitro with an affinity >or=10(21) M(-1). X-ray absorption spectroscopy shows Cu(I) adopts a trigonal S(2)N coordination like Mtb CsoR. Both apo and Cu(I)-bound Bsu CsoR are stable tetramers in the low micromolar monomer concentration range by sedimentation velocity and equilibrium ultracentrifugation. Apo-Bsu CsoR binds to a pseudopalindromic 30 bp copZA operator-promoter DNA with a stoichiometry of two tetramers per DNA and stepwise affinities of K(1)(apo) = 3.1(+/-0.8) x 10(7) M(-1) and K(2)(apo) = 8.3 (+/-2.2) x 10(7) M(-1) (0.4 M NaCl, 25 degrees C, pH 6.5). Cu(I) Bsu CsoR binds to the same DNA with greatly reduced affinities, K(1)(Cu) = 2.9(+/-0.4) x 10(6) M(-1) and K(2)(Cu)

SUBMITTER: Ma Z

PROVIDER: S-EPMC2728441 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Molecular insights into the metal selectivity of the copper(I)-sensing repressor CsoR from Bacillus subtilis.

Ma Zhen Z Cowart Darin M DM Scott Robert A RA Giedroc David P DP

Biochemistry 20090401 15

Bacillus subtilis CsoR (Bsu CsoR) is a copper-sensing transcriptional repressor that regulates the expression of the copZA operon encoding a copper chaperone and a Cu efflux P-type ATPase, respectively. Bsu CsoR is a homologue of Mycobacterium tuberculosis CsoR (Mtb CsoR), representative of a large Cu(I)-sensing regulatory protein family. We show here that Bsu CsoR binds approximately 1 mol equiv of Cu(I) per monomer in vitro with an affinity >or=10(21) M(-1). X-ray absorption spectroscopy shows ...[more]

PMID: 19249860

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Project description:The copper-sensitive operon repressor (CsoR) family, which is the main Cu(I)-sensing family, is widely distributed and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥3 g/liter copper ions). Here, we identified CsoR in hyper-copper-resistant Acidithiobacillus caldus (CsoRAc), an organism used in the bioleaching process of copper ores. CsoRAc possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoRAc monomer in vitro combined with atomic absorption spectroscopy analysis. CuI-CsoRAc and apo-CsoRAc share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (KD = 2.26 × 10-18 M and 0.53 × 10-15 M) and Cu(I) binding affinity of apo-CsoRAc were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoRAc did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants had the same order of magnitude of Cu(I) binding affinity as apo-CsoRAc. Moreover, apo-CsoRAc could bind to the DNA fragment P08430 in vitro, although with low affinity. Finally, a working model was developed to illustrate putative copper resistance mechanisms in A. caldus. IMPORTANCE Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus. Here, we characterized a major Cu(I)-sensing family protein, CsoRAc, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator bathocuproine disulfonate. In particular, CsoR family proteins were identified only in A. caldus, rather than A. ferrooxidans and A. thiooxidans, which are both used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

Dataset Information

Molecular insights into the metal selectivity of the copper(I)-sensing repressor CsoR from Bacillus subtilis.

Publications

Molecular insights into the metal selectivity of the copper(I)-sensing repressor CsoR from Bacillus subtilis.

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