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.     

A cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

In order to determine the mechanism of action of the 27 kDa mosquitocidal delta-endotoxin of Bacillus thuringiensis var. israelensis we have studied its effects on the conductance of planar lipid bilayers. The toxin formed cation-selective channels in the bilayers, permeable to K+ and Na+ but not to N-methylglucamine or Cl-, showing very fast, cooperative opening and closing. Channel opening was greatly reduced in the presence of divalent cations (Ca2+, Mg2+) and the effect was reversed when these ions were removed. These results are consistent with our proposal that B. thuringiensis toxins act by a mechanism of colloid-osmotic lysis.     

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.     

Influence of media composition on the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis

Powders of edible leguminous seeds, greengram (Vigna radiata) or soybean (Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/liter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against fifth-instar Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 x 10(6)/mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 x 10(6)/mg, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced delta-endotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard.     

Effect of the media composition on the growth parameters and biological properties ofBacillus thuringiensis var.israelensis delta-endotoxin

The effects of media composition on growth parameters, total protein production (including delta-endotoxin) and its relation to the biological properties ofBacillus thuringiensis var.israelensis was investigated. The replacement of glucose by glycerol as the carbon source yielded higher concentrations of delta-endotoxin. This increase in toxicity was associated with increased amounts of the 130 kDa polypeptide fraction. The biocide activity was not related to haemolytic activity. specific growth rate nor spore count.

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Résumé On a étudié les effets de la composition du milieu sur les paramètres de croissance, sur la production de protéines totales, ce qui inclut la delta-endotoxine, ainsi que sur sa relation avec les propriétés biologiques deBacillus thuringiensis var.israelensis. Le remplacement du glucose par le glycerol comme source de carbone resulte en des concentrations plus élevées de delta-endotoxine. Cet accroissement en toxicité est associé avec une quantité croissante de la fraction polypeptidique de 130 kDa. L'activité biocide n'est liée ni à l'activité hémolytique, ni à la vitesse specifique de croissance ni à l'énumération des spores.

     

A new medium for growth and delta-endotoxin production by Bacillus thuringiensis var. kurstaki

Summary The influence of composition of media used for growth and delta-endotoxin production by B. thuringiensis var. kurstaki was studied with the idea of finding a cheap medium for attaining high yields of spore-crystal preparations. A new medium, based on malt sprouts is proposed. Data on growth and bioinsecticidal activity are given. A concentration of 2.1 × 10⁹ spores.ml^-1 was attained in a 48 h process. The spore-crystal preparations obtained present a LC₅₀ of 2.18 × 10⁸ spores.g^-1 against larvae of Galleria mellonella.     

Cloning and expression of Bacillus thuringiensis israelensis delta-endotoxin DNA in B. sphaericus

Bacillus thuringiensis israelensis delta-endotoxin genes were cloned into Bacillus sphaericus 2362, producing stable transformants reacting with antibody to the 28- and 65-kDa B. thuringiensis israelensis crystal proteins and approximately 10 times more toxic to Aedes mosquito larvae than the original host strain. The LC50 after 48 hr of exposure of Aedes larvae to the most active transformed clone was 0.19 microgram/ml, compared with an LC50 of 1.9 microgram/ml for B. sphaericus 2362 and less than 0.1 microgram/ml for B. thuringiensis israelensis. The cloning vector, plasmid pPL603E, was also effective in transforming B. subtilis 1E20 with B. thuringiensis israelensis DNA, producing highly toxic clones with less stable gene expression than the clones of B. sphaericus.     

The use of coconut wastes for the production ofBacillus thuringiensis var.israelensis

Summary Good yields of crystal/spores and larvicidal activity were obtained whenBacillus thuringiensis var.israelensis was grown in coconut water and endosperm extracts. Coconut water contained 2.2 mg protein/ml, while the endosperm extracts contained 1–4 mg protein/ml extract. The carbohydrate content of the media ranged from 6–38.4 mg/ml.

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Resumen Utilización de desechos de coco para la producción de Bacillus thuringiensisvar. israelensis Agua de coco y extractos de endosperma se han utilizado para el cultivo deBacillus thuringiensis var.israelensis obteniendo un buen rendimiento en cristales/esporas y una buena actividad larvicida. El agua de coco contenía 2.2 mg de proteína/ml y los extractos de endosperma 1–4 mg/protei'na/ml de extracto. El contenido en hidratos de carbono de los medios utilizados varió entre 6–38.4 mg/ml.

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Résumé Utilisation des déchets de noix de coco pour la production de Bacillus thurigiensisvar. israelensisDe bons rendements en cristaux, spores et activité larvicide ont été obtenus en cultivantBacillus thurigiensis var.israelensis dans l'eau et les extraits endospermiques de noix de coco. L'eau des noix contient 2,2 mg de protéines/ml et les extraits d'endosperme 1–4 mg. La teneur des milieux en carbohydrates était comprise entre 6–38,4 mg/ml.

     

Single amino acid changes in the Bacillus thuringiensis var. israelensis delta-endotoxin affect the toxicity and expression of the protein

Site-directed mutagenesis has been used to change individual amino acids of the larvicidal 27,000 Mr delta-endotoxin of Bacillus thuringiensis var. israelensis. Basic and acidic residues have been systematically replaced by alanine, and the resulting mutant polypeptides analysed for cytolytic and larvicidal activity, and binding to phosphatidyl choline liposomes. Replacement of residues at positions 154, 163, 164, 213 and 225 results in proteins which accumulate as inclusions in recombinant Bacillus subtilis cells similar to the wild-type, but have considerably reduced in-vitro and in-vivo toxicity. One mutant (Glu45 to Ala45) results in a protein that has reduced activity in vitro, but retains wild-type larvicidal toxicity. In addition, seven other mutations of charged residues result in proteins which form small or no inclusions in recombinant cells, despite being produced at levels similar to the wild-type in six out of seven cases. In most instances, the toxicity of these aberrantly expressed proteins is considerably less than the wild-type, although one (Lys124 to Ala124) results in a polypeptide with approximately threefold increased activity in vitro. A secondary structural model is proposed to explain these observations.     

Organic and inorganic nitrogen source effects on the metabolism of Clostridium acetobytulicum

Summary The effects of organic and inorganic nitrogen combinations on cell growth, solvent production and nitrogen utilization by Clostridium acetobutylicum ATCC 824 was studied in batch fermentations. Fermentations in media with 10 mM glutamic acid, as the organic nitrogen source, and 0 mM to 10 mM ammonium chloride, as the inorganic nitrogen source had a solvent yield of 0.8 to 1.08 mmol solvent/mmol glucose used, with a slow fermentation rate (2 mmol solvent/l h^-1). When media contained 20 mM or 30 mM glutamic acid as well as 2.5 to 7.5 mM ammonium chloride the fermentation rate increased (5.5 mmol/l h^-1) while the solvent yield remained constant (0.86 to 0.96 mmol solvent/mmol glucose used). Total solvent production was higher in media containing 20 mM or 30 mM glutamic acid than with 10 mM glutamic acid.     

The mosquito larvicidal activity of 130 kDa delta-endotoxin of Bacillus thuringiensis var. israelensis resides in the 72 kDa amino-terminal fragment

Bacillus thuringiensis var. israelensis produces 130 kDa delta-endotoxin which is highly toxic to mosquito-larvae. The mosquito-larvicidal activity was delineated by sequential deletions from ends of the 1136 amino acids delta-endotoxin. A maximum of 459 amino acids could be removed from the carboxy-terminal of the toxin without a significant loss of the larvicidal activity. However, no more than 38 amino acids could be deleted from the amino-terminal without losing the toxicity. The truncated peptide of 72 kDa exhibited similar toxicity to the 130 kDa toxin and was between 39th and 677th amino acids.     

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.     

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae.

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.     

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.     

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.