Molecular cloning of the gene encoding the mosaic neurotoxin, composed of parts of botulinum neurotoxin types C1 and D, and PCR detection of this gene from Clostridium botulinum type C organisms.
The DNA fragment common to the genes encoding botulinum neurotoxin types C1 (BN/C1) and D (BN/D) was amplified by PCR from the culture supernatant of Clostridium botulinum type C strain 6813 (C6813) that was treated with either DNase I or proteinase K but not from the supernatant that was treated with both DNase I and proteinase K, suggesting the neurotoxin gene is located on a certain bacteriophage DNA. Thus, to isolate the neurotoxin gene, we performed PCR with the culture supernatant of C6813 and seven primer pairs designed from the genes encoding BN/C1 and BN/D. The coding region in the connected sequence encodes a neurotoxin composed of 1,280 amino acids with a molecular weight of 147,817. The neurotoxin from C6813 has 95% amino acid identity to BN/C1, except for its C-terminal one-third, which is quite similar to the C-terminal one-third of BN/D (95% identity). When we performed PCRs with four primer pairs designed from the 5'-terminal two-thirds of the BN/C1 gene and two primers from the 3'-terminal one-third of the BN/D gene, DNA fragments of the expected sizes (0.5 to 1.3 kbp) could be amplified from C. botulinum type C strains 6812 and 6814. These results suggest that some strains of C. botulinum type C contain the gene encoding the mosaic neurotoxin composed of parts of BN/C1 and BN/D.
Moriishi
K
K
Department of Veterinary Science, National Institute of Health, Tokyo, Japan.
Koura
M
M
Fujii
N
N
Fujinaga
Y
Y
Inoue
K
K
Syuto
B
B
Oguma
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001905:Botulinum Toxins / Q000235:genetics*; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000145:classification / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009030:Mosaicism; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D001905:Botulinum Toxins