is a human pathogen that produces two types of toxins, A and B, that cause a potentially lethal gastrointestinal syndrome termed pseudomembranous colitis. Virtually nothing is known about the mechanism of regulation of toxin production in this organism, and -regulatory regions of neither toxin have yet been identified, thus prompting this investigation. A motif homologous with the Shine-Dalgarno sequence of occurs upstream from the putative initiation codon of toxin B, making this region also a candidate to contain a promoter. Therefore, a subgenomic DNA library of in a plasmid vector was first constructed encompassing the 5′-end of the toxin B gene. A 450-bp DNA fragment was excised from the subgenomic DNA library clone and subcloned into a promoter-probe plasmid vector that contains two divergently oriented, promoterless genes to assay for promoter function. This subcloned DNA fragment directed the expression of alkaline phosphatase, a reporter gene product of the promoterless vector, thus indicating the presence of a functional promoter. To locate the promoter more precisely, a series of nested deletions of the toxin B promoter subclone was constructed with exonuclease III. The promoter that facilitates expression of the toxin B gene in was localised, based on alkaline phosphatase activity. The transcriptional initiation site of toxin B mRNA in was mapped by primer extension analysis, suggesting two closely associated tandem start sites directed by two similarly spaced promoters within this localised region.


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