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Clostridium difficile 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 cis-regulatory regions of neither toxin have yet been identified, thus prompting this investigation. A motif homologous with the Shine-Dalgarno sequence of Escherichia coli 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 C. difficile 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 E. coli was localised, based on alkaline phosphatase activity. The transcriptional initiation site of toxin B mRNA in E. coli was mapped by primer extension analysis, suggesting two closely associated tandem start sites directed by two similarly spaced promoters within this localised region.

Molecular analysis of the promoter region of the Clostridium difficile toxin B gene that is functional in Escherichia coli