Although the structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages, the regulation of lox expression is controfle, to a large extent, by its bacterial host Corynebacleium diphtheriae. Optimal yields oftox gene products are obtained only when iron becomes the growth-rate-limiting substrate. Previous studies suggest that regulation oftox expression Is mediated through an iron-binding aporepressor. To facilitate molecular cloning of the tox regulatory element from genomic libraries of C. diphtheriae, we constructed a tox promoter/operator (toxPO)-acZ transcriptional fusion in Escherichia coli strain DH5a. We report the molecular cloning and nucleic acid sequence of a diphtheria tox iron-dependent regulatory element, dxR, and demonstrate that expression of f-galactosidase from the toxPO-4acZ fusion is regulated by dlxR-encoded protein in an iron-sensitive manner. In addition, we show that expression of the toxPO-acZ fusion is not affected by the E. coUl ironregulatory protein Fur and that the dlxR protein does not inhibit expression offur-regulated outer-membrane proteins.Diphtheria toxin is synthesized by Corynebacterium diphtheriae lysogenic for one of a family of corynebacteriophages that carries the structural gene for the toxin, tox (1, 2). Optimal yields of tox gene products have long been known to be obtained only from C. diphtheriae grown under conditions where iron becomes the growth-rate-limiting substrate (3, 4). In 1936, Pappenheimer and Johnson (5) showed that adding iron in low concentration to the growth medium inhibited the production of diphtheria toxin. Both biochemical and genetic evidence support the hypothesis that the corynebacteriophage tox gene is regulated by a corynebacterial-determined iron-binding repressor as postulated by . This model predicted an aporepressor that in the presence of iron forms a complex; this complex then binds to the tox operator and blocks transcription. Under conditions of iron limitation, the iron-repressor complex dissociates, derepressing the tox gene.The nucleic acid base sequence of tox revealed a 9-basepair (bp) inverted repeat that overlaps the " -10" region of the promoter (13). Because many operators exhibit dyad symmetry and are positioned near their respective promoters, this region was designated the putative tox operator.We here describe the genetic construction of an Escherichia coli host strain that carries a chromosomal diphtheria tox promoter/operator (toxPO)-lacZ transcriptional fusion in single copy. Because this strain constitutively expresses j-galactosidase and is phenotypically blue on 5-bromo-4-chloro-3-indolyl /3-D-galactoside (X-Gal)-containing agar medium, we have used it to screen genomic libraries of nontoxinogenic nonlysogenic C. diphtheriae for determinants that repress lacZ expression. We report the molecular cloning and deduced amino acid sequence (25,316 molecular weight) of a diphtheria tox iron-dependent regulatory element, dtxR.* We show that this factor acts as a negati...

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