Hierarchical Control on Polyene Macrolide Biosynthesis: PimR Modulates Pimaricin Production via the PAS-LuxR Transcriptional Activator PimM

Abstract

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the Streptomyces antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. The PimR SARP domain (PimRSARP) was expressed in Escherichia coli as a glutathione S-transferase (GST)–fused protein. Electrophoretic mobility shift assays showed that GST-PimRSARP binds a single target, the intergenic region between the regulatory genes pimR and pimMs in the pimaricin cluster. The PimRSARP-binding site was investigated by DNaseI protection studies, revealing that it contains three heptameric direct repeats adjusting to the consensus 5′-CGGCAAG-3′. Transcription start points of pimM and pimR promoters were identified by 5′-RACE, revealing that unlike other SARPs, PimRSARP does not interact with the -35 region of its target promoter. Quantitative transcriptional analysis of these regulatory genes on mutants on each of them has allowed the identification of the pimM promoter as the transcriptional target for PimR. Furthermore, the constitutive expression of pimM restored pimaricin production in a pimaricin-deficient strain carrying a deletion mutant of pimR. These results reveal that PimR exerts its positive effect on pimaricin production by controlling pimM expression level, a regulator whose gene product activates transcription from eight different promoters of pimaricin structural genes directly.

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

“…The specific sequences recognized by SARPs generally overlap the Ϫ35 regions of their targets, but sometimes the sequences are far from the transcriptional start point (tsp) of their target genes. Some SARP-binding sites contain three discernible heptamers, such as those upstream of actII-ORF1 and actVI-ORF1 in S. coelicolor (29), claR, cefD, and cefF in S. clavuligerus (216), vlmJ and vlmA-vlmH in Streptomyces viridifaciens (217), dnrD in S. peucetius (218), fdmD in S. griseus (219), pimM in S. natalensis (220), and so on. Other sites contain only two obvious heptamers, such as sanO-sanN in S. ansochromogenes (221), polB and polC in Streptomyces cacaoi subsp.…”

“…Since changes of ADP/ATP concentrations significantly affect the binding activity of PolY in vivo, the ATPase domain may sense endogenous ADP/ATP levels. Other large SARPs studied genetically include PteR (filipin, S. avermitilis) (233) and PimR (pimaricin, S. natalensis) (220,234). It has been pointed out that all the examples of large SARP CSRs are in pathways for antifungal antibiotics, leading to the suggestion that such SARPs might recognize a common signal related to this antifungal activity (220).…”

“…Other large SARPs studied genetically include PteR (filipin, S. avermitilis) (233) and PimR (pimaricin, S. natalensis) (220,234). It has been pointed out that all the examples of large SARP CSRs are in pathways for antifungal antibiotics, leading to the suggestion that such SARPs might recognize a common signal related to this antifungal activity (220). Using our database of 236 antibiotic biosynthetic clusters from streptomycetes other than S. coelicolor, we found 57 with at least one SARP (Table 1).…”

A regulatory gene (ECO‐orf4) required for ECO‐0501 biosynthesis in Amycolatopsis orientalis

“…Pathway‐specific regulatory genes located within the cluster usually regulate relevant antibiotics specifically. Recently, proteins of large ATP‐binding regulators of the LuxR (LAL) family acting as pathway specific regulators motivated the current study, such as PikD , RapH , AveR , GdmRI and GdmRII , PimR . Nevertheless, LAL regulators SCO0877 and SCO7173 were reported to act as pleiotropic modulators .…”

Exploration of the Substrate Promiscuity of Biosynthetic Tailoring Enzymes as a New Source of Structural Diversity for Polyene Macrolide Antifungals

“…Both the biochemistry and the gene regulation of pimaricin production are well known 6. 20, 21 The experimental gene disruption of pimD , encoding a P450 cytochrome responsible for the introduction of an epoxide group in the C4C5 bond (Scheme ) during the last step of pimaricin biosynthesis,22, 23 leads to the accumulation of DEP 12. Owing to its small size in relation to other polyene macrolides, DEP constitutes an excellent candidate in the search for substrate promiscuity in tailoring enzymes as it retains many of the chemical features presumably required for the molecular recognition of their original substrates, whilst avoiding many potential steric hindrance effects.…”