Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)

Microbiology
2010.0

Abstract

<jats:p> <jats:italic>Streptomyces</jats:italic> 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 is presented here showing that <jats:italic>vlmI</jats:italic> is a regulatory gene in the valanimycin biosynthetic gene cluster of <jats:italic>Streptomyces viridifaciens</jats:italic> and encodes a protein belonging to the SARP family. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, <jats:italic>vlmHORBCD</jats:italic>, <jats:italic>vlmJKL</jats:italic> and <jats:italic>vlmA</jats:italic>. Disruption of <jats:italic>vlmI</jats:italic> abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the <jats:italic>vlmI</jats:italic> mutant and that 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 <jats:italic>vlmA–vlmH</jats:italic> and <jats:italic>vlmI</jats:italic>–<jats:italic>vlmJ</jats:italic> intergenic regions both exhibit a pattern of heptameric direct repeats. Gel shift assays with VlmI overproduced in <jats:italic>Escherichia coli</jats:italic> 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-number <jats:italic>vlmI</jats:italic> expression plasmid was introduced into <jats:italic>Streptomyces coelicolor</jats:italic> M512, which contains mutations in the undecylprodigiosin and actinorhodin activators <jats:italic>redD</jats:italic> and <jats:italic>actII-orf4</jats:italic>, undecylprodigiosin production was restored, showing that <jats:italic>vlmI</jats:italic> can complement a <jats:italic>redD</jats:italic> mutation. Introduction of the same <jats:italic>vlmI</jats:italic> expression plasmid into an <jats:italic>S. viridifaciens vlmI</jats:italic> mutant restored valanimycin production to wild-type levels.

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