Purification of a soil bacteria exotoxin using silkworm toxicity to measure specific activity.
Overnight cultures of 16 of 25 species of soil bacterium injected into silkworm hemolymph killed the silkworms. Injection of culture supernatants of five of these 16 bacterial species into the hemolymph also killed silkworms. Analysis of 16S rRNA indicated that the toxic species were Bacillus species. We purified an exotoxin produced by one of the Bacillus species by measuring the specific activity based on silkworm toxicity. The final step of purification revealed that the silkworm-killing activity was due to a 34-kDa protein. LC/MS/MS analysis revealed that the 34-kDa protein is sphingomyelinase C of Bacillus cereus. We propose that the silkworm infection model is useful for identifying pathogenic bacteria from soil and purifying bacterial toxins.
Usui
Kimihito
K
Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Miyazaki
Shinya
S
Kaito
Chikara
C
Sekimizu
Kazuhisa
K
D000818:Animals; D001407:Bacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D015373:Bacterial Typing Techniques; D012831:Bombyx / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis; D005098:Exotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D006458:Hemolymph / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D005098:Exotoxins; D012336:RNA, Ribosomal, 16S; D013108:Sphingomyelin Phosphodiesterase