Isolation of alpha-toxin, theta-toxin and kappa-toxin mutants of Clostridium perfringens by Tn916 mutagenesis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and mediates infection and disease by producing numerous extracellular toxins, including alpha-toxin, theta-toxin and kappa-toxin. Tn916-mutagenesis was used to isolate mutants defective in their ability to produce either alpha-toxin or theta-toxin. Nine independently derived mutants were isolated. In four of these mutants Tn916 had inserted at sites located 193 bp or 198 bp upstream of the theta-toxin structural gene, pfoA. Four mutants contained large deletions, three in regions which encompassed the theta-toxin structural and regulatory genes pfoA and pfoR, respectively, and the K-toxin structural gene, colA, and one in a region encompassing the a-toxin structural gene, pie. These mutants should prove to be invaluable for further genetic studies aimed at determining the role of these toxins in virulence. (C) 1997 Academic Press Limited.