Botulinum toxin (BoNT) is an extremely potent Category A biological agent with potential implications in bioterrorism. As part of an overall project to develop countermeasures against BoNT, hundreds of strains of Clostridium botulinum from Dr. Eric Johnson's strain repository and other sources will be characterized phenotypically and genotypically. The repository will provide the foundation for a thorough characterization of genetic heterogeneity of BoNT genes, toxin gene complexes, and strain lineages of neurotoxigenic clostridia. This information will be critical for countermeasure development since related research has demonstrated variability of toxin gene complexes and inability of countermeasures for neutralization and inhibition of BoNTs. Specifically, strains will initially be analyzed for cultural and phenotypic properties and BoNT production. Culture lineages will be determined by PFGE and other genetic and phenotypic analyses. Thirdly, RFLP analysis of the toxin gene clusters will be performed to identify unique toxin sequences and cluster arrangements. The nucleotide sequences of the unique toxin genes will be determined. In addition, BoNTs and toxin complexes from the unique isolates differing from known prototype strains will be isolated and evaluated for neutralization experiments and inhibition by small molecule inhibitors generated within this RCE. BoNTs exhibiting distinct characteristics, particularly resistance to antibodies and inhibitors, will be produced in quantities sufficient for structural studies and novel antibody/inhibitor development. Finally, for select toxins, individual domains will be expressed for detailed structural studies. This will provide valuable information for development of antibodies and inhibitors for diagnostic and therapeutic purposes.