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pubmed:abstractText |
Thiostrepton induced the expression of four proteins (17, 19, 30, and 56 kilodaltons) of unknown function in Streptomyces lividans. The chromosomal gene which encoded the 19-kilodalton protein (tipA) was cloned and sequenced. Transcription of the tipA promoter was induced at least 200-fold by thiostrepton. The tipA 200-fold by thiostrepton. The tipA transcriptional start site (located by S1 mapping and primer extension experiments) was preceded by a 45-base-pair imperfect inverted-repeat sequence which included the -10 and -35 regions of the promoter. Under noninducing conditions in vivo, this might form a cruciform structure which is not recognized by RNA polymerase. A 143-base-pair fragment including this region was cloned into a promoter probe vector, pIJ486. In this plasmid, pAK114, the thiostrepton-inducible tipA promoter controlled the expression of a kanamycin resistance gene encoding an aminoglycoside phosphotransferase. As little as 1 ng of thiostrepton spotted on a lawn of S. lividans(pAK114) induced kanamycin-resistant growth. Other thiostreptonlike antibiotics also induced tipA, but structurally unrelated antibiotics which inhibit translation had no effect. In S. lividans, the promoter could be induced by thiostrepton during either growth or stationary phase. The tipA promoter should be a valuable tool for expression studies in streptomycetes.
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