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Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR).

ABSTRACT: A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoR(Sl)) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and Cu(I)-CsoR(Sl) to be a tetramer assembly, and a 1.7-? resolution crystal structure of apo-CsoR(Sl) reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), which highlighted that next to the csoR gene itself, the regulon consists of two Cu(I) efflux systems involving a CopZ-like copper metallochaperone protein and a CopA P(1)-type ATPase. Although deletion of csoR has only minor effects on S. lividans development when grown under high copper concentrations, mutations of the Cu(I) ligands decrease tolerance to copper as a result of the Cu(I)-CsoR mutants failing to disengage from the DNA targets, thus inhibiting the derepression of the regulon. RNA-seq experiments carried out on samples incubated with exogenous copper and a ?csoR strain showed that the set of genes responding to copper stress is much wider than anticipated and largely extends beyond genes targeted by CsoR. This suggests more control levels are operating and directing other regulons in copper homeostasis beside the CsoR regulon.

SUBMITTER: Dwarakanath S

PROVIDER: S-EPMC3366776 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR).

Dwarakanath Srivatsa S Chaplin Amanda K AK Hough Michael A MA Rigali Sébastien S Vijgenboom Erik E Worrall Jonathan A R JAR

The Journal of biological chemistry 20120326 21

A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoR(Sl)) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and Cu(I)-CsoR(Sl) to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoR(Sl) reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), whic ...[more]

PMID: 22451651

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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

Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR).

Publications

Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR).

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