Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B

High titered Clostridium sordellii lethal toxin antiserum, cross-reactive with C. difficile cytotoxin B (ToxB), was used to isolate toxB fragments from a C. difficile expression library. Recombinant clones containing toxB fragments of the 5 and 3 end were isolate. A 2.5-kb HincII fragment of chromosomal DNA overlaps both groups of clones. A partial restriction map of the total toxB gene is presented. The gene is positioned upstream of utxA and toxA toxB has a size of 6.9kb, corresponding to a 250-kDa polypeptide. A partial sequence of the 5 end of toxB was determined. The sequence contains 398 bp upstream of toxB with a putative Shine-Dalgarno box (AGGAGA) and 609 bp of the toxB open reading frame. The N-terminal 203 amino acids of ToxB were compared with the N-terminal amino acids of the enterotoxin A (ToxA). A homology of 64% of the residues was detected, which proves the relatedness of ToxA and ToxB of C. difficile.Abbreviations PBS phosphate-buffered saline - ToxA C. difficile enterotoxin A - ToxB C. difficile cytotoxin B - toxB gene encoding ToxB - HT C. sordellii hemorrhagic toxin - LT C. sordellii lethal toxin