Activation of phenoxazinone synthase expression in Streptomyces lividans by cloned DNA sequences from Streptomyces antibioticus.
Recombinant plasmids carrying Streptomyces antibioticus DNA (phenoxazinone synthase plasmids 1.8 and 4.3) have been used as templates in a streptomycete-coupled transcription-translation system. Analysis of the synthetic products from these experiments shows that PHS 1.8 and 4.3 appear to code for polypeptides which are not structurally related, as judged by immunoprecipitability, to the phenoxazinone synthase subunit. 35S-labeled cell extracts of Streptomyces lividans and S. lividans transformed with the phenoxazinone synthase plasmids each contain a protein which is precipitable with antiserum to S. antibioticus phenoxazinone synthase. These proteins have exactly the same electrophoretic mobility as the S. antibioticus phenoxazinone synthase subunit on one-and two-dimensional gels and the peptide maps of the proteins from the S. lividans clones are essentially indistinguishable from the map of the S. antibioticus phenoxazinone synthase subunit. Glycerol-gradient centrifugation of extracts of cells transformed with PHS 1.8 and 4.3 showed that both the large and small phenoxazinone synthase enzyme forms were present. Southern hybridization experiments demonstrated that pIJ2505, a pBR322 derivative containing the phenoxazinone synthase structural gene, hybridized to S. lividans DNA digested with BclI, SphI, SstI, and XhoI. The results obtained are discussed in terms of the hypothesis that the expression of phenoxazinone synthase activity in cells transformed with the PHS 1.8 and 4.3 plasmids results from the activation of an endogenous phenoxazinone synthase gene in S. lividans (and perhaps in other streptomycetes in the case of PHS 4.3) which is normally "silent" or expressed at very low levels.
D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004247:DNA / Q000494:pharmacology*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D046911:Macromolecular Substances; D010088:Oxidoreductases / Q000032:analysis / Q000235:genetics*; D010957:Plasmids; D013302:Streptomyces / Q000201:enzymology*; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D004247:DNA; D010088:Oxidoreductases; C023944:2-aminophenol oxidase; D004262:DNA Restriction Enzymes