Regulation of phenoxazinone synthase expression in Streptomyces antibioticus

Abstract

The cloned gene for the subunit of phenoxazinone synthase (PHS), an enzyme implicated in the biosynthesis of actinomycin in Streptomyces antibioticus, was used as a probe to study the regulation of the enzyme. The direction of transcription of the PHS gene was determined with end-labeled restriction fragments derived from the gene. Low-resolution S1 mapping revealed that transcription was initiated at a position which may lie within the SphI restriction site, which represents the limit of the cloned sequence. Northern blotting allowed the identification of the putative PHS message. This RNA appeared to be significantly larger than the size required to encode the PHS subunit. RNA dot blotting showed that the increase in PHS specific activity observed in cultures grown on antibiotic production medium, with galactose as a carbon source, was due in part to an increased production of PHS mRNA. PHS was also more stable than most cellular proteins and appeared to be protected against degradation under conditions in which most other proteins are broken down. This protective effect also contributed to the increase in PHS specific activity observed in S. antibioticus cultures grown on production medium. The repression of PHS synthesis by glucose was also reflective of a transcriptional control mechanism. At early time points postinoculation, PHS mRNA levels were lower in cultures grown on glucose as a carbon source than in cultures of the same age grown on galactose. mRNA levels presumably begin to increase only after all the glucose in the medium is utilized. The ability of 5-fluorouracil to stimulate PHS production in young cultures was also due to the synthesis of new mRNA for the enzyme.