The Nucleotide and Deduced Amino Acid Sequences of EcoRI Fragment Containing the 5′‐Terminal Region of Clostridium botulinum Type E Toxin Gene Cloned from Mashike, Iwanai and Otaru Strains

Abstract

Chromosomal

The complete nucleotide sequence of the gene encoding the nontoxic component of Clostridium botulinum type E progenitor toxin

“…Therefore, it is likely that some nucleotide sequence in the nontoxic component gene may work as a promoter for the neurotoxin gene in E. coli. Such a promoter region has been proposed by Fujii et al (1990) and Whelan et al (1992 b) : the ' -10' region located between nucleotides 3535 and 3540. However, such a sequence may not function as a promoter in C. botulinum.…”

Cloning and Whole Nucleotide Sequence of the Gene for the Light Chain Component of Botulinum Type E Toxin from Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

“…1). These bands were then hybridized with the 1.0 Kbp EcoRI-gene fragment previously cloned (data not shown) (5,6). From these results, it was concluded that toxin gene was presented in 2.…”

“…Extraction of DNA from cells. Cellular DNAs from toxigenic C. butyricum strain BL6340 (isolated from a case of infant botulism and kindly provided by Dr. Hatheway, Centers for Disease Control, Atlanta, Ga., U.S.A.) and C. botulinum type E strain Mashike (isolated from food-borne botulism in Hokkaido Prefecture) were prepared according to the procedure reported previously (5,6). Briefly, both strains were cultured in 250 ml LYG medium [1% (w/v) lactalbumin hydrolysate (Difco, Detroit, Mich., U.S.A.) ; 2% (w/v) yeast extract (Difco) ; 0.5% (w/v) glucose; 0.15% (w/v) cysteine hydrochloride, pH 7.2] at 30 C for 12 hr, the cells were collected by centrifugation at 11,000 •~ g, and resuspended in 20 ml LYG medium containing 8% (w/v) polyethylene glycol 4,000, 10 U/ml penicillin G (Banyu Pharmaceutical Co., Ltd., Tokyo), and 10 jig/ml lysozyme (Wako Pure Chemical Industries, Ltd., Osaka, Japan).…”

“…4 in Fig. 3) between botulinum type A, Cl, D, E, and tetanus toxins (5). This time, it became clear that L components of these toxins possessed one more homologous region though the homogeneity of only type Cl toxin was slightly low (Box No.…”

“…The complete nucleotide sequences of toxin genes have been already determined in C. botulinum types A, Cl and D, and C. tetani (1,2,4,8). As for the toxin gene of C. botulinum type E, Binz et al (1) and we (5) have succeeded to clone the 1 Kbp EcoRI-fragment coding for 252 amino acid residues of the N-terminus of the toxin. We also succeeded to clone the 1 Kbp EcoRI-toxin gene fragment from type E toxin-producing C. butyricum BL6340, and found that eight bases in the untranslated region (227 bases), and 27 bases in the open reading frame (756 bases) were different between these cloned type E and butyricum toxin gene fragments (6).…”