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.
Highlights
Streptomycetes are well-known for their ability to produce a great variety of secondary metabolites including therapeutic molecules like polyene macrolide antibiotics
PimR was the first of its class to be described, and constitutes the prototype of a new class of regulators. Members of this class include the regulator PteR from S. avermitilis located in the biosynthetic gene cluster for the pentaene filipin [16], the nikkomycin activator in S. ansochromogenes SanG [17], or the polyoxin regulator in S. cacaoi PolR [18] which is directly controlled by PolY [19]
The new strain S. natalensis DpimR2 had growth and morphological characteristics identical to those of S. natalensis wild type when grown on solid or liquid media, suggesting that PimR has no role in bacterial growth or differentiation
Summary
Streptomycetes are well-known for their ability to produce a great variety of secondary metabolites including therapeutic molecules like polyene macrolide antibiotics These constitute a large group of antifungal agents [1,2] whose production, occurs in a growth-phase-dependent manner, at the transition between the rapid growth phase and the stationary growth phase [3]. PimR is a transcriptional activator (its inactivation from the S. natalensis chromosome resulted in complete loss of pimaricin production [4]) with a peculiar architecture It contains an N-terminal SARP (Streptomyces Antibiotic Regulatory Protein) domain [14] with a Cterminal half homologous to guanylate cyclases and LAL regulators (Large ATP-binding regulators of the LuxR family) [15]. Members of this class include the regulator PteR from S. avermitilis located in the biosynthetic gene cluster for the pentaene filipin [16], the nikkomycin activator in S. ansochromogenes SanG [17], or the polyoxin regulator in S. cacaoi PolR [18] which is directly controlled by PolY [19]
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