Isolation of botulinolysin, a thiol-activated hemolysin, from serotype D Clostridium botulinum: A species-specific gene duplication in Clostridia.
Botulinolysin (BLY) is a toxin produced by Clostridium botulinum that belongs to a group of thiol-activated hemolysins. In this study, a protein exhibiting hemolytic activity was purified from the culture supernatant of C. botulinum serotype D strain 4947. The purified protein displayed a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of 55kDa, and its N-terminal and internal amino acid sequences exhibited high similarity to a group of thiol-activated hemolysins produced by gram-positive bacteria. Thus, the purified protein was identified as the BLY. Using the nucleotide sequences of previously cloned genes for hemolysins, two types of genes encoding BLY-like proteins were cloned unexpectedly. Molecular modeling analysis indicated that the products of both genes displayed very similar structures, despite the low sequence similarity. In silico screening revealed a specific duplication of the hemolysin gene restricted to serotypes C and D of C. botulinum and their related species among thiol-activated hemolysin-producing bacteria. Our findings provide important insights into the genetic characteristics of pathogenic bacteria.
Department of Nutritional Science and Food Safety, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan. Electronic address: ts200588@nodai.ac.jp.
Tomonori
T
Suzuki
Tokyo International Clinic, 4-18-19 Takanawa, Minato-ku, Tokyo, 108-0074, Japan.
Thomas
T
Nagano
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, 099-2493, Japan.
Koichi
K
Niwa
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, 099-2493, Japan.
Shingo
S
Mutoh
Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan.
Masataka
M
Uchino
Department of Nutritional Science and Food Safety, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan.
Motohiro
M
Tomizawa
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, 099-2493, Japan.
Yoshimasa
Y
Sagane
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, 099-2493, Japan.
Toshihiro
T
Watanabe
D000595:Amino Acid Sequence; D001483:Base Sequence; D003014:Clostridium botulinum / Q000145:classification / Q000235:genetics*; D016000:Cluster Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D020440:Gene Duplication; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D053878:Hemolytic Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D065288:Serogroup
D006460:Hemolysin Proteins; D053878:Hemolytic Agents; C112565:botulinolysin
Clostridia; Clostridium botulinum; Gene duplication; Thiol-activated hemolysin