Evidence supporting an operon model for the expression of toxins A and B by Clostridium difficile

Clostridium difficile is a human pathogen which produces two protein toxins, A and B, that cause a potentially lethal gastrointestinal syndrome termed pseudomembranous colitis. Production of both toxins correlates with the onset of sporulation of this organism. In addition, although C. difficile strains vary significantly in how much of both toxins they produce, a 1:1 ratio is maintained by all strains, suggesting that the production of both toxins is coordinately regulated. Moreover, both toxin genes are adjacent on the bacterial chromosome with the toxin B gene being upstream of the toxin A gene. Nevertheless, nothing is known about the regulation of toxin production, and cis-regulatory region(s) of neither toxin gene have yet been identified.

Therefore, the hypothesis being examined here is whether these toxin genes are under operon control, thereby implying a single promoter for both toxin genes. To test this hypothesis, a putative promoter region upstream of toxin B was localized to a 245-bp DNA fragment and characterized in an Escherichia coli system by nested deletion, gel retardation and binding competition analyses. Further studies of this promoter region revealed two sets of overlapping promoters that showed homology to both the consensus sequences oiE. coli and Gram (+) bacteria. This promoter region upstream of the toxin B gene was also footprinted with the purified E. coli RNA polymerase for additional characterization.