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Regulation of Valanimycin Biosynthesis in Streptomyces viridifaciens: Characterization of VlmI as a Streptomyces Antibiotic Regulatory Protein (SARP)

Rice University

Streptomyces antibiotic regulatory proteins (SARPs) have been shown to activate transcription by binding to a tandemly arrayed set of heptameric, direct repeats located around the -35 region of their cognate promoters. Experimental evidence showing that VlmI is a regulatory gene in the valanimycin biosynthetic gene cluster of Streptomyces viridifaciens and encodes for a protein belonging to the SARP family is presented here. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, vlmHORBCD, vlmJKL, and vlmA. Disruption of vlmI abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the vlmI mutant and the production of VlmK is severely diminished. These results demonstrate that the expression of these genes from the three potential transcripts is under the positive control of VlmI. The vlmA-vlmH and the vlmI-vlmJ intergenic regions both exhibit a pattern of heptameric, direct repeats. Gel shift assays with VlmI overproduced in E. coli as a C-terminal FLAG-tagged protein clearly demonstrated that VlmI binds to DNA fragments from both regions that contain these heptameric repeats. When a high-copy vlmI expression plasmid was introduced into S. coelicolor M512, which contains mutations in the undecylprodigiosin and actinorhodin activators redD and actII-orf4, undecylprodigiosin production was restored, showing that vlmI can complement a redD mutation. Introduction of the same vlmI expression plasmid into the S. viridifaciens vlmI mutant restored valanimycin production to wild type levels.

¹ E-mail: parry{at}rice.edu