title: Genetic and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2).
abstract: Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA 'complemented' several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin: S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (greater than 5 MDal) and a 49 kDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of 'secondary metabolites' whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the 'late exponential' phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to 'secondary' metabolism in general.
mesh_terms: D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D008780:Methyltransferases / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D011353:Prodigiosin / Q000031:analogs & derivatives / Q000096:biosynthesis; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; C047347:undecylprodigiosin; D008780:Methyltransferases; D004262:DNA Restriction Enzymes; D011353:Prodigiosin