Characterization and role of a Streptomyces antibioticus DNA sequence in the activation of a silent gene for phenoxazinone synthase in Streptomyces lividans.

Abstract

Phenoxazinone synthase (PHS) catalyzes the penultimate step in the actinomycin D biosynthetic pathway of Streptomyces antibioticus. Earlier, a 4.3 kb DNA sequence derived from S. antibioticus was cloned and shown to activate a normally silent gene for PHS in S. lividans. Two mechanisms can be envisioned for the mode of activation of the silent gene. The 4.3 kb sequence could encode an activator that interacts with the genetic machinery of S. lividans. Alternatively, it could play the role of an operator sequence capable of titrating a repressor in the cells. To distinguish between the two possibilities, the strategy used involved deletion subcloning, DNA sequence analyses, coupled transcription-translation assays, total RNA dot blotting, and a gel mobility-shift assay. Evidence from these approaches favors the second mechanism and points to the presence of a protein in S. lividans that specifically binds to a 249 bp fragment from the S. antibioticus genome which retains the ability to activate the silent phs gene. Using primers that hybridize to the activating sequence, an S. lividans homologue was cloned and shown to be 95% identical to the S. antibioticus 249 bp insert. Upon reintroduction on a high copy number plasmid in S. lividans, this sequence was shown to activate the silent phs gene. Preliminary results indicate that this sequence is also able to bind to a protein from the S. lividans extract in a gel retardation assay.