Abstract
Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM. This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. PimM, truncated forms of the protein without the PAS domain (PimM(ΔPAS)), and forms containing just the DNA-binding domain (DBD) (PimM(DBD)) were overexpressed in Escherichia coli as GST-fused proteins. GST-PimM binds directly to eight promoters of the pimaricin cluster, as demonstrated by electrophoretic mobility shift assays. Assays with truncated forms of the protein revealed that the PAS domain does not mediate specificity or the distinct recognition of target genes, which rely on the DBD domain, but significantly reduces binding affinity up to 500-fold. Transcription start points were identified by 5'-rapid amplification of cDNA ends, and the binding regions of PimM(DBD) were investigated by DNase I protection studies. In all cases, binding took place covering the -35 hexamer box of each promoter, suggesting an interaction of PimM and RNA polymerase to cause transcription activation. Information content analysis of the 16 sequences protected in target promoters was used to deduce the structure of the PimM-binding site. This site displays dyad symmetry, spans 14 nucleotides, and adjusts to the consensus TVGGGAWWTCCCBA. Experimental validation of this binding site was performed by using synthetic DNA duplexes. Binding of PimM to the promoter region of one of the polyketide synthase genes from the Streptomyces nodosus amphotericin cluster containing the consensus binding site was also observed, thus proving the applicability of the findings reported here to other antifungal polyketides.
Highlights
Streptomycetes are filamentous soil bacteria that have a complex life cycle that involves differentiation and sporulation
We report the direct binding of PimM to upstream sequences of eight promoters of the pimaricin gene cluster in Streptomyces natalensis and provide evidence that binding specificity relies on the DNA-binding domain, whereas the PAS domain significantly reduces the affinity of binding to target promoters
Organization of pim Cluster Transcriptional Units—Organization of the pimaricin gene cluster and transcription of the genes were previously largely deduced by analyzing gene chromosomal arrangement and by the different expression profiles of certain genes in reverse transcription-polymerase chain reaction (RT-PCR) experiments [10, 13]; a more accurate identification of operons was needed in order to define an overall picture of the transcriptional arrangement of the pim genes
Summary
DIRECT BINDING OF THE REGULATOR PimM TO EIGHT PROMOTERS OF PIMARICIN GENES AND IDENTIFICATION OF BINDING BOXES*□S. Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. Gene expression analyses by reverse transcription-polymerase chain reaction (RT-PCR) of the pimaricin gene cluster revealed the targets for the PimM regulatory protein According to these analyses, the genes responsible for initiation (pimS0) and the first cycles of polyketide chain extension (pimS1), were among the major targets for regulation, other pim genes were differentially affected, accounting for the lack of pimaricin production [13]. We report the direct binding of PimM to upstream sequences of eight promoters of the pimaricin gene cluster in Streptomyces natalensis and provide evidence that binding specificity relies on the DNA-binding domain, whereas the PAS domain significantly reduces the affinity of binding to target promoters. This study constitutes the first molecular characterization of the mode of action of a polyene macrolide regulator and makes PimM the first pathway-specific regulator of antibiotic biosynthesis, not belonging to the SARP family, whose binding site has been determined
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