The Journal of Medical Microbiology, Vol 47, Issue 4 309-316, Copyright © 1998 by Society for General Microbiology
Molecular analysis of the promoter region of the Clostridium difficile toxin B gene that is functional in Escherichia coli
K. P. Song and C. Faust
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
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.
