Isolation of botulinolysin, a thiol-activated hemolysin, from serotype D Clostridium botulinum: A species-specific gene duplication in Clostridia

Botulinolysin (BLY) is a toxin produced by Clostridium botulinum that belongs to a group of thiol-activated hemolysins. In this study, a protein exhibiting hemolytic activity was purified from the culture supernatant of C. botulinum serotype D strain 4947. The purified protein displayed a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of 55 kDa, and its N-terminal and internal amino acid sequences exhibited high similarity to a group of thiol-activated hemolysins produced by gram-positive bacteria. Thus, the purified protein was identified as the BLY. Using the nucleotide sequences of previously cloned genes for hemolysins, two types of genes encoding BLY-like proteins were cloned unexpectedly. Molecular modeling analysis indicated that the products of both genes displayed very similar structures, despite the low sequence similarity. In silico screening revealed a specific duplication of the hemolysin gene restricted to serotypes C and D of C. botulinum and their related species among thiol-activated hemolysin-producing bacteria. Our findings provide important insights into the genetic characteristics of pathogenic bacteria.