Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae

The Streptomyces venezuelae pikD gene from the pikromycin biosynthetic clus ter was analyzed, and its deduced product (PikD) was found to have amino ac id sequence homology with a small family of bacterial regulatory proteins. Database comparisons revealed two hypothetical domains, including an N-term inal triphosphate-binding domain and a C-terminal helix-turn-helix DNA-bind ing motif, Analysis of PikD was initiated by deletion of the corresponding gene (pikD) from the chromosome of S. venezuelae, resulting in complete los s of antibiotic production. Complementation by a plasmid carrying pikD rest ored macrolide biosynthesis, demonstrating that PikD is a positive regulato r. Mutations were made in the predicted nucleotide triphosphate-binding dom ain, confirming the active-site amino acid residues of the Walker A and B m otifs. Feeding of macrolide intermediates was carried out to gauge the poin ts of operon control by PikD. Although the pikD mutant strain was unable to convert macrolactones (10-deoxymethynolide and narbonolide) to glycosylate d products, macrolide intermediates (YC-17 and narbomycin) were hydroxylate d with high efficiency. To study further the control of biosynthesis, presu med promoter regions from pik cluster loci were linked to the xylE reporter and placed in S. venezuelae wild-type and pikD mutant strains. This analys is demonstrated that PikD-mediated transcriptional regulation occurs at pro moters controlling expression of pikRII, pikAI, and desI but not those cont rolling pikRI or pikC.