[11] encoded an E3 subtype toxin. Figure 1 Dendrogram of bont/E nucleotide sequences. Shown is a neighbor-joining find more tree of bont/E nucleotide sequences with bootstrap values (based on 100 replications) and genetic distance (bar) shown. BoNT/E subtypes (E1-E9) encoded by Selleckchem Small molecule library clusters of genes are also shown. Accession numbers for bont/E genes not sequenced in this study are indicated with an asterisk. Strain CDC66177 harbored a significantly divergent bont/E gene which formed a unique clade when compared to other bont/E genes. Comparison of the translated amino acid sequence of this gene with the gene encoding BoNT/E1 in strain Beluga indicated that the sequences differed by ~11%. Since previous comparisons of BoNT/E subtypes resulted in

differences of up to 6% amino acid sequence variation, the BoNT/E produced by strain CDC66177 can be considered a unique subtype (E9) [10, 11]. Comparison of the amino acid sequence of BoNT/E9 with representatives of BoNT/E subtypes E1-E8 demonstrated that the most divergent region

of the toxin was located in the last ~200 residues (Figure 2) which corresponds to the C-terminal part of the heavy chain (Hc-C) that is involved with binding to neuronal cells [14]. BLAST analysis of this region indicated < 75% amino acid sequence identity with other BoNT/E sequences. Figure 2 Comparative analysis of representative BoNT/E subtypes. Shown is a similarity plot comparing representative BoNT/E subtype amino acid sequences Casein kinase 1 to BoNT/E9 (from strain CDC66177). The most divergent region of the amino acid sequence is shaded. Sequences from representative strains examined in this study ��-Nicotinamide supplier or accession numbers retrieved from Genbank are compared in the plot as follows: E1, Beluga; E2, Alaska; E3, CDC40329; E4, AB088207 E5, AB037704; E6, AM695752; E7, Minnesota; E8, JN695730. BLAST analysis of the 16S rRNA nucleotide sequence from strain CDC66177 shared > 99.8% identity with strains Alaska E43 and 17B indicating that the strain clusters with other Group II C. botulinum strains [9]. Mass spectrometric analysis of BoNT/E produced by strain CDC66177 Since the BoNT/E produced by strain CDC66177 appeared to

be a previously unreported toxin subtype, the enzymatic light chain activity of the toxin was assessed in culture supernatants generated from the strain. The light chain of BoNT/E cleaves the synaptosomal-associated protein, SNAP-25, and the Endopep-MS method was used to measure this activity upon a specific peptide substrate mimic of SNAP-25 (IIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKT). Endopep-MS analysis revealed that the toxin cleaved the peptide substrate for BoNT/E in the expected location, resulting in products with peaks at m/z 1136.8 and 2924.2 [15] (Figure 3A). Figure 3 Mass spectral analysis of BoNT/E9. Panel A shows the products of endopeptidase cleavage of a type E specific peptide substrate detected by mass spectrometry. Peaks indicating the cleavage of the substrate by the toxin are marked with asterisks.