MOLECULAR ANALYSIS OF THE PROMOTER REGION OF THE CLOSTRIDIUM-DIFFICILE TOXIN B-GENE THAT IS FUNCTIONAL IN ESCHERICHIA-COLI

Clostridium difficile is a human pathogen that produces two types of t oxins, A and B, that cause a potentially lethal gastrointestinal syndr ome 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-Dalga rno sequence of Escherichia coli occurs upstream from the putative ini tiation codon of toxin B, making this region also a candidate to conta in 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 DN A library clone and subcloned into a promoter-probe plasmid vector tha t contains two divergently oriented, promoterless genes to assay for p romoter function. This subcloned DNA fragment directed the expression of alkaline phosphatase, a reporter gene product of the promoterless v ector, thus indicating the presence of a functional promoter. To locat e the promoter more precisely, a series of nested deletions of the tox in B promoter subclone was constructed with exonuclease III. The promo ter that facilitates expression of the toxin B gene in E. coli was loc alised, based on alkaline phosphatase activity. The transcriptional in itiation site of toxin B mRNA in E. coli was mapped by primer extensio n analysis, suggesting two closely associated tandem start sites direc ted by two similarly spaced promoters within this localised region.