The primary goal of the Johnson lab for this project is to purify the novel neurotoxins for structural and mechanistic studies, and for development of countermeasures. During the past 12 months we have expanded the Multi Locus Sequence Typing (MLST) method developed in our laboratory for Clostridium botulinum to include serotypes B, E, and F. The results indicated considerable phylogenetic diversity of strain lineages and identified novel strains. In addition, diversity was investigated using a microarray system for the discovery of novel genes among serotypes and subtypes. Initial array analysis has shown that the diversity is effectively delineated by this approach. Our laboratory also discovered that the genes encoding BoNT/A3 and BoNT/A4 reside on large plasmids of about 260-280kb. This is the first demonstration of genes for BoNT/A being present on plasmids. Toxin production in C. botulinum A3 and A4 was increased about 10-fold by modifying the medium and culture conditions. New methods are in progress for purification of BoNT/A3, BoNT/A4 and non-toxic proteins associated with the neurotoxins. Gene components for nontoxic non-hemagglutinin (NTNH) and p47 proteins have been cloned, expressed, and antibodies produced for affinity purification of the BoNTs. The successful production and purification of the neurotoxins and nontoxic proteins will enable biochemical, mechanistic, and structural studies of the proteins, and afford materials for countermeasure development.