Construction and application of a promoter-probe plasmid that allows chromogenic identification in Streptomyces lividans.
We cloned a Streptomyces coelicolor A3(2) DNA fragment which directed synthesis of a brown pigment, presumably a shunt product in the actinorhodin biosynthetic pathway, on the plasmid vector pIJ41 in Streptomyces lividans. The pigment production was observed only when the DNA fragment was inserted downstream from a functional promoter sequence. By subcloning the fragment together with in vitro manipulation, a promoter-probe plasmid vector (pARC1) with a unique BamHI cloning site was constructed that allows chromogenic identification of transcriptional control signals in Streptomyces lividans based on the expression of the cloned pigment gene(s). The Escherichia coli tac (trp-lac hybrid) promoter, consisting of 92 base pairs and a promoter region including the leader sequence of erythromycin resistance gene (ermC) on staphylococcal plasmid pE194, when ligated in the correct orientation in the BamHI site of pARC1, promoted expression of the cloned pigment gene(s) in Streptomyces lividans, whereas the Saccharomyces cerevisiae GAL7 promoter did not. In the case of the ermC, induction of the pigment production by the addition of either erythromycin or lincomycin, but not virginiamycin, was observed. The system was also shown to be useful and convenient in isolating transcriptional control signals of Streptomyces chromosomal DNA and estimating their activities.
Horinouchi
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Beppu
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D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004917:Erythromycin / Q000494:pharmacology; D005822:Genetic Vectors; D009876:Operon; D010860:Pigments, Biological / Q000096:biosynthesis*; D010957:Plasmids; D013302:Streptomyces / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D010860:Pigments, Biological; D004917:Erythromycin