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Journal of Bacteriology, June 2001, p. 3468-3475, Vol. 183, No. 11
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.11.3468-3475.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Daniel J. Wilson,1 Yongquan Xue,1 Kevin A. Reynolds,2 and David H. Sherman1,*

Department of Microbiology and Biological Process Technology Institute, University of Minnesota, Minneapolis, Minnesota 55455,1 and Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 232192

Received 9 January 2001/Accepted 19 March 2001

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.

* Corresponding author. Mailing address: Department of Microbiology, Mayo Mail Code 196, 420 Delaware Street S.E., Minneapolis, MN 55455-0312. Phone:(612) 626-0199. Fax: (612) 624-6641. E-mail: david-s{at}biosci.umn.edu.

Journal of Bacteriology, June 2001, p. 3468-3475, Vol. 183, No. 11
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.11.3468-3475.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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