Cytotoxicity of two proteins isolated from Bacillus thuringiensis var. israelensis crystals to insect and mammalian cell lines

Abstract Two toxic proteins (proteins A and B) were isolated from Bacillus thuringiensis var. israelensis crystals using ion-exchange chromatography. A cell cytotoxicity assay based on the uptake of [³H]uridine by tissue culture cells following exposure to the toxic proteins, was used to measure the viability of the cells. Proteins A and B were cytotoxic to Aedes aegypti and MA104 cells. Protein B was also cytotoxic to Spodoptera frugiperda cells.     

Restriction endonuclease mapping of three plasmids from Bacillus thuringiensis var. israelensis

Restriction endonuclease cleavage site maps have been constructed of plasmids pTX14-1, pTX14-2, and pTX14-3 from Bacillus thuringiensis var. israelensis (Bti).     

Toxicity of Bacillus thuringiensis var. israelensis crystals to Aedes aegypti larvae: carbonate reversal

The toxicity of purified Bacillus thuringiensis var. israelensis crystals to larvae of Aedes aegypti could be reversed 100-fold by levels of K(2)CO(3) as low as 0.15%.     

Novel antibacterial proteins from entomocidal crystals of Bacillus thuringiensis ssp. israelensis

Proteins with molecular masses of 36 and 34 kDa (Bti36 and Bti34) were isolated from entomocidal crystals formed by Bacillus thuringiensis ssp. israelensis cells. The samples of Bti36 contained the admixture of a protein with a molecular mass of 33 kDa (Bti33), apparently a product of proteolysis of Bti36. These 3 proteins are significantly different in N-terminal sequences from known delta-endotoxins of B. thuringiensis and show antibacterial activity toward Micrococcus luteus. The combination of Bti36 and Bti33 also suppresses the growth of some other microorganisms including Streptomyces chrysomallus. The effects of the mixture of Bti36 and Bti33 on the M. luteus cell surface and on the surface of S. chrysomallus cells and exospores are similar, but they are different from the effect of endotoxin Cry11A on micrococcal cells.     

Isolation and characterization of mutants of Bacillus thuringiensis var. israelensis

Several mutants of Bacillus thuringiensis var. israelensis HD-500 (Bti) were obtained after treatment with N -methyl-n'-nitro- N -nitrosoguanidine. On the basis of the production (+) or absence (-) of spore (Spo) and crystal (Cry) the strains were grouped into three categories: Spo + Cry +, Spo + Cry - and Spo - Cry + . NGI-22, a Spo + Cry - mutant lacked all crystal proteins. Both NGI-23 and NGI-23-1 were Spo - Cry + . NGI-23-1, however, produced multiple crystals per cell. These mutants could prove useful not only for analysing sporulation and crystal formation as well as the linkage between the two genetic processes but also for improving the potential of Bti as a microbial insecticide.     

Development of selective media for Bacillus sphaericus and Bacillus thuringiensis var. israelensis

Summary The resistance of 8 strains of Bacillus sphaericus and of 2 strains of Bacillus thuringiensis var. israelensis (B.t.i.) to various antibiotics and antibiotic combinations were tested. All B. sphaericus strains were resistant to streptomycin, lincomycin and bacitracin, and six strains were resistant to combinations of these antibiotics. This antibiotic resistance could be utilised to establish selective media to identify and follow the fate of B. sphaericus and of B.t.i. in the field.     

Identification of the peptides of the crystals of Bacillus thuringiensis var israelensis involved in the mosquito larvicidal activity

Tryptic digestion of the proteins from the purified crystals of B.thuringiensis var israelensis resulted in the decline of high molecular weight peptides without the loss of mosquito larvicidal activity, measured after immobilization of the digests with DEAE- Sephadex A 50 beads. Amongst the peptides generated (less than 44 kDa), a 21 kDa peptide was immunoreactive to the crystal antiserum. Analysis of the peptides released from spores of the toxic (Cry+) and non-toxic (Cry-) strains has revealed a pattern in which only the 26kDa peptide was missing in the Cry-strain. Sporulation and crystal formation were dissociated by the addition of the antibiotic netropsin, which could also inhibit the crystal assembly, without considerable decrease of the larvicidal activity and retention of the 26kDa peptide. These results implicate the 26kDa peptide in the larvicidal action.     

Bacillus thuringiensis var. israelensis production involving re-use of the supernatant

The supernatant arising after biomass separation of Bacillus thuringiensis var. israelensis by flocculation/sedimentation was re-used after being supplemented with 25, 50 and 75% (w/v) of the original culture medium, based on corn steep liquor, glucose and mineral salts. Supplementation at 75% gave a spore concentration (1 x 10(10) c.f.u. ml(-1)) five times greater than that obtained with the other supplements.     

Antibacterial activity of Cry- and Cyt-proteins from Bacillus thuringiensis ssp. israelensis

Mosquitocidal endotoxins Cry4B, Cry11A, and CytA from Bacillus thuringiensis ssp. israelensis as well as the products of their limited proteolysis display antibacterial activity relative to Micrococcus luteus. The endotoxin Cry11A also induces the lysis of the micrococcus protoplasts. Potassium and sodium ions and N-acetylgalactosamine increased the antibacterial effect of Cry11A, whereas glucose and N-acetylglucosamine inhibited it. The endotoxin Cry11A displays the antibacterial effect on some other microorganisms.     

Development of a self floating slow release formulation of Bacillus thuringiensis var. israelensis and its larvicidal activity

Alginate encapsulated B. thuringiensis var. israelensis (B. t. i.) self floating type formulations were prepared. Its spore release rate, floating efficacy and larvicidal activity against Culex quinquefasiatus were tested in the laboratory. The larval mortality of 91-100% was induced by the floating formulation with a mean spore release of 3.04 x 10(4)/ml/day from 6th day to 27th day. From day 28 to 33 the mean number of spores released were 1.16 x 10(4)/ml/day which caused 72.2-88.2% mortality. From 34th day to 40th day the mean number of spores released were 4.97 x 10(3)/ml/day which caused 42.2-67.2% mortality. However, the self floating alginate encapsulated beads were intact and found to float upto 40 days.     

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 micrograms vs 83 micrograms, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC50 of about 600 spores/ml.     

Isolation and assay of the toxic component from the crystals ofBacillus thuringiensis var.israelensis

The 25-Kdal fragment of the 28-Kdal toxic protein extracted fromBacillus thuringiensis var.israelensis crystals was found to be responsible for the insecticidal, cytolytic, hemolytic, and mouse-lethal activities of the crude toxin extract. This activity was found to have no relation to the hemolysin produced by other strains ofB. thuringiensis. This protein was rich in the amino acids Asp and Glu, but did not contain Cys.     

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.     

Biological control of mosquitoes by the larvicidal activity of Bacillus thuringiensis var. israelensis delta endotoxin

Bacillus thuringiensis var. israelensis (B.t.i.) is a promising, safe toxic agent for control of mosquitoes, but the rapid disappearance of its toxicity makes its use in practice economically unattractive. The lack of evidence for B.t.i. multiplication in water also makes the natural ecology of B.t.i. puzzling. The observation that mosquito larvae readily cannibalize carcasses of B.t.i.-killed larvae, and that the carcasses become toxic to scavenging larvae provides a possible solution to this puzzle. Several experimental techniques have been developed to study these phenomena (e.g., quantitative determination of spore numbers despite aggregation, protocols for following toxicity development, etc.). Results suggest a cycle involving larval poisoning and death due to delta-endotoxin of ca. 1,000 spores, germination, vegetative growth and sporulation (with toxin production) of B.t.i. after multiplication to several million in the carcass. Implications of these results for the ecology of B.t.i., and for practical applications in mosquito control, are discussed.     

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.