Characterization and Analysis of the PikD Regulatory Factor in the Pikromycin Biosynthetic Pathway of Streptomyces venezuelae

Abstract

The Streptomyces venezuelae pikD gene from the pikromycin biosynthetic cluster was analyzed, and its deduced product (PikD) was found to have amino acid sequence homology with a small family of bacterial regulatory proteins. Database comparisons revealed two hypothetical domains, including an N-terminal triphosphate-binding domain and a C-terminal helix-turn-helix DNA-binding motif. Analysis of PikD was initiated by deletion of the corresponding gene (pikD) from the chromosome of S. venezuelae, resulting in complete loss of antibiotic production. Complementation by a plasmid carrying pikD restored macrolide biosynthesis, demonstrating that PikD is a positive regulator. Mutations were made in the predicted nucleotide triphosphate-binding domain, confirming the active-site amino acid residues of the Walker A and B motifs. Feeding of macrolide intermediates was carried out to gauge the points of operon control by PikD. Although the pikD mutant strain was unable to convert macrolactones (10-deoxymethynolide and narbonolide) to glycosylated products, macrolide intermediates (YC-17 and narbomycin) were hydroxylated with high efficiency. To study further the control of biosynthesis, presumed promoter regions from pik cluster loci were linked to the xylE reporter and placed in S. venezuelae wild-type and pikD mutant strains. This analysis demonstrated that PikD-mediated transcriptional regulation occurs at promoters controlling expression of pikRII, pikAI, and desI but not those controlling pikRI or pikC.The soil bacteria belonging to the genus Streptomyces have been of great interest due to their well-known capacity to produce a diverse range of antibiotics and other secondary metabolites (4, 24). Production typically occurs according to a growth phase-dependent profile (11,12) and is often accompanied by the development of spore-bearing aerial mycelia (9).The regulatory elements involved in generating antibiotics are of significant interest. An entire family of regulatory genes called SARPs (Streptomyces antibiotic regulatory proteins) has been identified based on sequence and motif homology, as well as by complementation studies (2,8). Sequence analysis links the SARP family together by the presence of OmpR-like DNA-binding domains (25). Members of this family include the positive regulators ActII-ORF4 of the actinorhodin biosynthetic cluster (2), RedD of the undecylprodigiosin gene cluster (26, 36), DnrI of the daunorubicin biosynthetic system (33, 36), and CcaR, which regulates both the cephamycin and clavulanic acid pathways (28, 36). Other genes have been identified that encode transcriptional activators of specific SARPs. These include redZ and dnrN, which activate redD and dnrI, respectively (15, 35).A general network or system of regulatory elements involved in the control of secondary metabolite pathways has not yet emerged. For example, the srmR gene of Streptomyces ambofaciens, which regulates spiramycin production, shows no sequence homology to other regulatory proteins (16). The tylosin biosynthetic pathw...

Roles of rapH and rapG in Positive Regulation of Rapamycin Biosynthesis in Streptomyces hygroscopicus

“…The necessity for ATP binding and hydrolysis was demonstrated by work on PikD, a LAL regulator required for pikromycin biosynthesis. The ability of PikD to act as a transcriptional activator was dependent on the presence of a functional ATP-binding motif (47). RapH fits the profile of the LAL family and has a high sequence identity with a number of Streptomyces polyketide antibiotic biosynthetic cluster regulators of the LAL family, three of which have been identified in S. hygroscopicus strains ( Table 3).…”

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

“…They have often been found as CSRs in Streptomyces spp. Examples include AmphRI and AmphRIII (amphotericin, Streptomyces nodosus) (144), NysRI and NysRIII (nystatin, Streptomyces noursei) (145), PikD (pikromycin, Streptomyces venezuelae) (146), and RapH (rapamycin, S. hygroscopicus) (147). None of the CSRs identified in S. coelicolor is a LAL, but 14 LAL-encoding genes unlinked to clusters were identified in the S. coelicolor genome (148).…”

Organization of the Biosynthetic Gene Cluster for the Polyketide Antitumor Macrolide, Pladienolide, inStreptomyces platensisMer-11107

“…Streptomyces platensis Mer-11107 and its mutants were cultured in SPCGB medium for 5 d at 25 C with shaking at 220 rpm. The cultures were extracted with 10 vol of acetonitrile and analyzed as described above.…”

“…The pldR gene encodes a transcription regulator that belongs to LAL (Large ATP-binding regulators of the LuxR family). Since almost all LAL are likely to have a role as transcriptional positive regulators of genes for type I PKSs, 25,26) the pldR gene might be a positive regulator in pld biosynthesis.…”