Mechanism of Bacillus thuringiensis Insecticidal δ-Endotoxin Action on Insect Cells in Vitro

Author(s):  
MICHIO HIMENO
1980 ◽  
Vol 26 (4) ◽  
pp. 486-491 ◽  
Author(s):  
Donovan E. Johnson ◽  
Debra M. Niezgodski ◽  
George M. Twaddle

Six oligosporogenic (Spo−) mutant strains of Bacillus thuringiensis were selected from survivors of treatment with N-methyl-N′-nitro-N-nitrosoguanidine. Each strain was blocked at or before stage II of spore development, but all produced typical bipyramidal-shaped crystalline inclusion bodies. Toxicity of the parasporal endotoxin isolated from the mutant strains was assayed by an in vitro technique using cultured insect cells, and was comparable with that of normal wild-type parasporal protein. Multiple parasporal inclusion bodies per cell were often produced, and smaller embedded particles were numerous and distinct.


1985 ◽  
Vol 49 (5) ◽  
pp. 1461-1468 ◽  
Author(s):  
Michio HIMENO ◽  
Naoto KOYAMA ◽  
Tomohiko FUNATO ◽  
Tohru KOMANO

2009 ◽  
Vol 75 (16) ◽  
pp. 5237-5243 ◽  
Author(s):  
Shangling Fang ◽  
Li Wang ◽  
Wei Guo ◽  
Xia Zhang ◽  
Donghai Peng ◽  
...  

ABSTRACT Bacillus thuringiensis has been used as a bioinsecticide to control agricultural insects. Bacillus cereus group genomes were found to have a Bacillus enhancin-like (bel) gene, encoding a peptide with 20 to 30% identity to viral enhancin protein, which can enhance viral infection by degradation of the peritrophic matrix (PM) of the insect midgut. In this study, the bel gene was found to have an activity similar to that of the viral enhancin gene. A bel knockout mutant was constructed by using a plasmid-free B. thuringiensis derivative, BMB171. The 50% lethal concentrations of this mutant plus the cry1Ac insecticidal protein gene were about 5.8-fold higher than those of the BMB171 strain. When purified Bel was mixed with the Cry1Ac protein and fed to Helicoverpa armigera larvae, 3 μg/ml Cry1Ac alone induced 34.2% mortality. Meanwhile, the mortality rate rose to 74.4% when the same amount of Cry1Ac was mixed with 0.8 μg/ml of Bel. Microscopic observation showed a significant disruption detected on the midgut PM of H. armigera larvae after they were fed Bel. In vitro degradation assays showed that Bel digested the intestinal mucin (IIM) of Trichoplusia ni and H. armigera larvae to various degrading products, similar to findings for viral enhancin. These results imply Bel toxicity enhancement depends on the destruction of midgut PM and IIM, similar to the case with viral enhancin. This discovery showed that Bel has the potential to enhance insecticidal activity of B. thuringiensis-based biopesticides and transgenic crops.


Parasitology ◽  
2008 ◽  
Vol 135 (12) ◽  
pp. 1355-1362 ◽  
Author(s):  
I. SIDÉN-KIAMOS ◽  
C. LOUIS

SUMMARYOokinetes are the motile and invasive stages of Plasmodium parasites in the mosquito host. Here we explore the role of intracellular Ca2+ in ookinete survival and motility as well as in the formation of oocysts in vitro in the rodent malaria parasite Plasmodium berghei. Treatment with the Ca2+ ionophore A23187 induced death of the parasite, an effect that could be prevented if the ookinetes were co-incubated with insect cells before incubation with the ionophore. Treatment with the intracellular calcium chelator BAPTA/AM resulted in increased formation of oocysts in vitro. Calcium imaging in the ookinete using fluorescent calcium indicators revealed that the purified ookinetes have an intracellular calcium concentration in the range of 100 nm. Intracellular calcium levels decreased substantially when the ookinetes were incubated with insect cells and their motility was concomitantly increased. Our results suggest a pleiotropic role for intracellular calcium in the ookinete.


2000 ◽  
Vol 66 (1) ◽  
pp. 118-124 ◽  
Author(s):  
D. John I. Thomas ◽  
J. Alun W. Morgan ◽  
John M. Whipps ◽  
Jon R. Saunders

ABSTRACT Plasmid transfer between Bacillus thuringiensis subsp.kurstaki HD1 and B. thuringiensis subsp.tenebrionis donor strains and a streptomycin-resistantB. thuringiensis subsp. kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid transfer was detected in vitro at temperatures of 5 to 37°C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10−1 transconjugant/donor) were detected within 4 h. In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed. When aB. thuringiensis subsp. kurstaki strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae. When a B. thuringiensis subsp.tenerbrionis strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B. thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment.


1980 ◽  
Vol 15 (2) ◽  
pp. 133-139 ◽  
Author(s):  
Junko NISHITSUTSUJI-UWO ◽  
Yasuhisa ENDO ◽  
Michio HIMENO

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.


Author(s):  
О. Sherstoboeva ◽  
А. Krzyhanivsky ◽  
А. Bunas

Antagonistic microorganisms, producing bactericidal and fungicidal substances, spread in space and occupy various ecological niches. Antagonistic action of microorganisms can be combined with a number of other properties that can be used by humans for their own purposes. Microorganisms — agents of biological products often have a multifunctional effect on the components of the agroecosystem, which under the influence of various environmental factors could have positive and negative consequences. The aim of the study was to study the effect of new highly active entomopathogenic strains of Bacillus thuringiensis on non-target objects — components of the biocenosis of the apple orchard. When developing technologies for biocontrol of the number of apple trees pests using B. thuringiensis revealed an additional positive protective effect — reducing the number of pathogens of fungal diseases on the leaf surface. Treatment of apple leaves with a liquid culture of strain B. thuringiensis 0376 after 72 hours of application reduced the number of scab of Fusicladium dendriticum by 7 times, and the pathogen of monolisis of Monilia fructigena — 9 times. Strain B. thuringiensis 0371 showed the highest level of antagonism, because in vitro completely inhibited the growth and development of micromycetes on the surface of the nutrient medium. On the leaf surface, the application of culture strain 0371 reduced the number of Fusicladium dendriticum by 92%, and Monilia fructigena — by 86% after 72 hours of treatment. In a field experiment when treating apple trees with a culture of this strain, scab development decreased by 7.1%. Strain B. thuringiensis 787 did not show antagonism against phytopathogenicmicromycetes during co-cultivation on nutrient medium, which may be explained by its inability to produce water-soluble exotoxin, in contrast to strains of B. thuringiensis, which are antagonists of scab and apple monolisis. Strain B. thuringiensis 0371 requires further research and development of its formulations, because it is promising for the creation of a complex biological product with insect-fungicidal properties for use in ecological and agriculture friendly technologies.


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