Akiko Uemori

Ubiquity of parasporin-1 producers in Bacillus thuringiensis natural populations of Japan

Parasporin, a Bacillus thuringiensis parasporal protein, is unique in having a strong cytocidal activity preferential for human cancer cells. In this study, we characterized parasporin activities associated with three novel geographical isolates of B.thuringiensis. Parasporal inclusion proteins of the three isolates were highly toxic to human uterus cervix cancer cells (HeLa), but not to non-cancer uterine smooth muscle cells (UtSMC). Inclusions of the isolates lacked insect toxicity and hemolytic activity against sheep erythrocytes. Ouchterlony immunodiffusion tests revealed that the proteins of the three isolates are immunologically closely related to parasporin-1 (Cry31A), but dissimilar to the three other existing parasporin groups. Our results provide evidence that the parasporin-1-producing organism is a common member in B. thuringiensis populations occurring in natural environments of Japan.

Cloning and Characterization of Two Novel Genes, cry24B and s1orf2, from a Mosquitocidal Strain of Bacillus thuringiensis serovar sotto

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.

Identification of Parasporin Genes in Vietnamese Isolates of Bacillus thuringiensis

Four genes encoding parasporins, cytotoxins preferentially killing human cancer cells in vitro, were isolated from four Vietnamese strains of Bacillus thuringiensis. Nucleotide sequence analysis revealed that: (1) three genes fall into the two known classes, ps1Aa and ps1Ab, and (2) another one belongs to ps1Ac, a novel gene class established in this study. Upon proteolytic activation, parasporal protein of the organism with ps1Ac exhibited strong cytocidal activity against human cancer cells, HeLa and Hep G2, but not to non-cancer normal cells, UtSMC and HC.

Occurrence of Bacillus thuringiensis in canopies of a natural lucidophyllous forest in Japan.

A total of 39 Bacillus thuringiensis isolates were recovered from 38 leaves collected from 5- to 10-m-high canopies of 8 micro-/meso-phanerophyte species in a lucidophyllous forest of Japan. B.thuringiensis-positive leaves accounted for 1.4% of a total of 2805 leaves from 15 tree species. The frequency of the organism was 0.8% among the Bacillus cereus/B. thuringiensis group. Of 39 isolates obtained, 27 (69.2%) were allocated to 11 H serovars, and 12 isolates remained unidentified: 11 were motile but lacked reactivity to the 55 reference antisera, and 1 isolate was not flagellated. Two H serovars, kurstaki (H3abc) and tohokuensis (H17), occurred predominantly on canopy phylloplanes. Larvicidal activities against Bombyx mori and/or Aedes aegypti were associated with 49% of the canopy isolates. Strong hemolysis was induced by parasporal inclusion proteins of the two isolates of serovar israelensis (H14). Hemagglutinating (lectin) activity was associated with parasporal proteins of nine isolates. There was little correlation between insecticidal activity and lectin activity.

Identification of two haemolysins in larvicidal activity of Bacillus thuringiensis against the bean bug, Riptortus clavatus

Abstract:  This study characterized larvicidal activity against the bean bug, Riptortus clavatus (Hem., Alydidae), associated with a strain of Bacillus thuringiensis serovar morrisoni (H8ab). Purified crystals and solubilized crystal proteins exhibited only low‐level activities, while the supernatant of broth culture contained rapid and strong larvicidal activity. Heating at 100°C for 10 min destroyed the activity. Two extracellular vegetative proteins, with molecular masses of 40 and 45 kDa, were obtained by diethylaminoethyl (DEAE)‐cellulose column chromatography from the bacterial culture fluid. Both proteins were related to the known haemolysins of Bacillus cereus, showing strong cytolytic activity against sheep erythrocytes. The bean bug‐killing activity was not associated with individual proteins; however, strong activity was induced when two proteins were combined. The combined proteins were toxic to larvae in the early stage of first instar but not against larvae of later instars and adults. Larvae of the diamondbackmoth, Plutella xylostella, were not killed by these proteins.