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A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

Botulinum neurotoxin (BoNT) binds to nontoxic nonhemagglutinin (NTNHA) protein in a pH-dependent manner, and yields the protease-resistant BoNT/NTNHA complex. Here, we screened short peptides that bind to the serotype D NTNHA (NTNHA-D) using random phage display technique. NTNHA was fixed onto electrode of quartz crystal microbalance (QCM) apparatus, and then the phages displaying random heptapeptides were exposed to the NTNHA-D under the acidic condition. After rinsing with acidic buffer, the released phages under the alkaline condition were collected. The binding and release of the phage were monitored by the frequency shift on the QCM. As a result of the screening, 16 were selected as peptides that bind to NTNHA-D. The selected peptides do not share any conserved sequence, but tend to be rich in basic and/or hydrophobic amino acid. This would explain the binding manner of the BoNT to the NTNHA protein.

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA-DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.