Additional file 1:

Detected sequence similarities between CBO0798 (flagellin) and CNT sequences. SSEARCH was used to screen the C. botulinum A protein database (3615 sequences) plus the target CNTs using default parameters. E-values were calculated within SSEARCH using randomly reshuffled copies of the library sequences, as described in Methods. CBO0798's rank relative to all 3615 C. botulinum proteins, associated E-value, and the alignment regions are reported for nine search cases (BoNT/A-G, TeNT, and NTNHA). The flagellin aligned to two separate regions in the CNTs, suggesting an ancestral duplication.

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Additional file 2:

Key alignments between CBO0798 and CNTs produced by SSEARCH and PSI-BLAST. Smith-Waterman alignments between BoNT/A-CBO0798 and NTNH-CBO0798 were performed using SSEARCH (default parameters used with -z 11 flag). The listed E-values are based on a single pairwise alignment of both sequences rather than a database search. The alignment between CBO0798 and BoNT/E from C. butyricum is the result of a PSI-BLAST search of CBO0798 (restricted to Clostridia) using default parameters with composition-based statistics.

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Additional file 3:

Multiple alignment of clostridial flagellins. The N- and C-terminal domains are indicated, and the intermediate section represents the flagellin hypervariable region. The collagenase-like insert identified within the hypervariable region of FliA(H) is boxed in red. CBO0798 is underlined in black. Additional clostridial flagellins containing large hypervariable region inserts are grouped with CBO0798 and FliA(H) at the beginning of the alignment.

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