Daniel J. Wilson, Yongquan Xue, K. Reynolds
Jun 1, 2001
Citations
6
Influential Citations
80
Citations
Quality indicators
Journal
Journal of Bacteriology
Abstract
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 frompik cluster loci were linked to the xylEreporter and placed in S. venezuelae wild-type andpikD mutant strains. This analysis demonstrated that PikD-mediated transcriptional regulation occurs at promoters controlling expression of pikRII, pikAI, anddesI but not those controlling pikRI orpikC.