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Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor.

ABSTRACT: Expression of genes belonging to the pho regulon in Streptomyces coelicolor is positively regulated (as shown by comparing the wild-type and a DeltaphoP mutant) by binding of the response regulator PhoP to 11-nt direct repeats (DRus). These sequences have been found in over 100 genes of Streptomyces coelicolor; 20 of them were cloned and the binding of PhoP(DBD) to most of their promoters has been shown by electrophoretic mobility shift assays. Deletion experiments showed that at least two DRus are required for proper binding of PhoP(DBD). Deletion of 1 nt leaving a 10-nt direct repeat reduced drastically binding of PhoP(DBD). Three different types of operators have been identified. Complex operators (class III) contain up to six DRus, some of them with poor conservation of the 11-nt consensus sequence, which however were protected by PhoP(DBD) in footprinting analyses. A cooperative binding of PhoP(DBD) molecules initiated at conserved core DRus appears to be the mechanism involved in binding of several PhoP(DBD) monomers to those complex operators. The information theory-based model that incorporates the positive or negative contribution to the binding of PhoP(DBD) of adjacent sequences has been used to deduce the structure of PHO boxes and the relevance of each DRu.

SUBMITTER: Sola-Landa A

PROVIDER: S-EPMC2275107 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor.

Sola-Landa Alberto A Rodríguez-García Antonio A Apel Alexander Kristian AK Martín Juan F JF

Nucleic acids research 20080110 4

Expression of genes belonging to the pho regulon in Streptomyces coelicolor is positively regulated (as shown by comparing the wild-type and a DeltaphoP mutant) by binding of the response regulator PhoP to 11-nt direct repeats (DRus). These sequences have been found in over 100 genes of Streptomyces coelicolor; 20 of them were cloned and the binding of PhoP(DBD) to most of their promoters has been shown by electrophoretic mobility shift assays. Deletion experiments showed that at least two DRus ...[more]

PMID: 18187507

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Project description:The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimRSARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding.IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM The expression of this gene is controlled by the Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator PimR, which binds a series of heptameric direct repeats in its promoter region. The structure and importance of such repeats in protein binding, transcriptional activation, and polyene production have been investigated. These findings should provide important clues to understand the regulatory machinery that modulates antibiotic biosynthesis in Streptomyces and open new possibilities for the manipulation of metabolite production. The presence of PimR orthologues encoded by gene clusters for different secondary metabolites and the conservation of their operators suggest that the improvements observed in the activation of pimaricin biosynthesis by Streptomyces natalensis could be extrapolated to the production of different compounds by other species.

Dataset Information

Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor.

Publications

Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor.

Similar Datasets

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