Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.     

Importance of spores and δ-endotoxin protein crystals of Bacillus thuringiensis in Galleria mellonella

Larvae of Galleria mellonella were fed on a honey-rich artificial food containing live spores or toxic crystals of Bacillus thuringiensis serotype V or various combinations of both. In this food; 1:1 combinations were 10 times more potent than live spores alone and about 10⁴ times more potent than crystals alone. Reduction in the proportion of spores, but not in that of crystals, decreased the slope of probit lines from 3.4 to 0.6. One or more factors in the spore are at least partly responsible for the potency of serotype V in G. mellonella. The results suggest than an observed gross loss of potency of this serotype in beehives is more likely to be due to death of spores than to deterioration of crystals. The reaction of G. mellonella to serotype V is nearest to that of a type 3 host species. Spores of serotype I are almost inactive in this host.     

Expression of insecticidal activity in Rhizobium containing the δ-endotoxin gene cloned from Bacillus thuringiensis subsp. tenebrionis

Bacillus thuringiensis subsp. tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the -endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.Abbreviations B.t.t. Bacillus thuringiensis subsp. tenebrionis - IPTG isopropyl--D-thiogalactoside     

The main features of Bacillus thuringiensis δ-endotoxin molecular structure

The crystal-forming proteins (-endotoxins) produced by various serotypes of Bacillus thuringiensis and toxic for Lepidoptera reveal the same pattern of molecular organisation. These proteins (M _r of ca. 145,000–130,000) contain an N-terminal domain (M _r of 85,000–65,000) resistant to proteolysis whereas their C-terminal moieties (M _r of 65,000) undergo an extensive degradation by trypsin that leads to stepwise cleavage off the fragments with M _r of 15,000–35,000.The N-terminal domain from serotype V -endotoxin is active when introduced into the hemocoel of Galleria mellonella larvae. Hence, it correponds to the true toxin normally formed by larva proteases action on the crystalforming protein (protoxin). Some differences were found in the properties of the N-terminal domains isolated from the crystal-forming proteins of III, V and IX serotypes, e.g., in their solubility, digestion by subtilisin, molecular weights and the distribution of methionine residues along the polypeptide chains.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacryl amide gel electrophoresis - CFP crystal-forming protein - DNS 5-dimethylamino-1-naphthalene-sulphonyl     

Relationship between Poly- β-hydroxybutyrate Production and δ-endotoxin for Bacillus thuringiensis var. kurstaki

A linear relationship between total solid concentration (TSC), δ-endotoxin production [Cry = 0.2795(TSC)−0.2472, R² = 0.8644] and poly-β-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R² = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between δ-endotoxin and PHB accumulation [Cry = 2.1573(PHB)−1.1248, R² = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l⁻¹ was required before the onset of δ-endotoxin production. Revisions requested 28 September 2005 and 4 November 2005; Revisions received 28 October 2005 and 1 February 2006     

Influence of some plant phenolics on the activity of δ-endotoxin of Bacillus thuringiensis var. galleriae on Heliothis armigera

Bioassays with Bacillus thuringiensis var. galleriae Berliner -endotoxin and plant phenolics on Heliothis armigera Hübner enhanced the activity of B.t. var. galleriae endotoxin. The presence of plant phenolics with B.t var. galleria endotoxin not only reduced feeding potential and weight gain by the larvae, but also enhanced the LC50 value of the toxin. Our study demonstrates the effect of phytochemicals from resistant crop plants on the biocidal activity of B. thuringiensis strains in laboratory conditions.     

Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis

A novel δ-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF486523","term_id":"1121787749","term_text":"EF486523"}}EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel β-sheets (domain II); and 134 residues forming a β-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC₅₀s) were 8.98 μg/ml for the expressed Cry7Ca1, 0.87 μg/ml for the activated toxin 1, and 4.43 μg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.     

Quantification in soil of Bacillus thuringiensis var. kurstakiδ-endotoxin from transgenic plants

Transgenic plants that produce pesticidal proteins have the potential to release these products into the environment when the plants are incorporated into soil. This could result in novel exposure of soil organisms to these pesticidal proteins. There is a lack of knowledge about the fate and persistence of transgenic pesticidal products in the soil. A model system of transgenic cotton, which produces Bacillus thuringiensis kurstakiδ-endotoxin (Bt toxin), was used to address this issue. Methods were developed to quantify Btk toxin in soil and soil/plant litter by extraction of the Btk toxin with an aqueous buffer and quantification by ELISA. The highest recovery of Btk toxin from soil was obtained with a high salt, high pH buffer. In addition, for certain soil types, addition of a non-ionic detergent, Tween-20, was needed for optimal recovery. Recovery of Btk toxin from soil ranged from 60% for a low clay content, low organic matter soil to 27% for a high clay content, high organic matter soil. The limit of detection of this method is 0.5 ng of extractable toxin per g dry weight soil. The method was shown to be useful in tracking over time the persistence of both purified and transgenic Btk toxin in laboratory experiments.     

Dual production of amylase and δ-endotoxin by Bacillus thuringiensis subsp. kurstaki during biphasic fermentation

This study examined the efficacy of a Bacillus thuringiensis (Bt) strain in producing amylase (EC 3.2.1.1) as a by-product without affecting its unique ability for producing δ-endotoxin, thus to establish a cultivation strategy for the dual production and recovery of both δ-endotoxin and amylase from the fermented medium with an industrial perspective. LB medium was individually supplemented (5 to 100%, wt/vol) with flour from six naturally available starchy stored foods (banana, Bengal gram, jack seed, potato, taro or tapioca); after initial fermentation (12 h), the supernatant in the medium obtained by centrifugation (1000 g, 10 min) was used for harvesting amylase and the resultant pellet was further incubated aseptically for the production of endospores and δ-endotoxin by solid-state fermentation. Maximum crude amylase activity (867 U/gram dry substrate, 12 h) was observed in potato flour-supplemented medium (10% wt/vol, 12 h), while the activity in LB control was only 4.36 U/mL. SDS-PAGE profile of the crude (supernatant), as well as partially purified (40–60% (NH₄)₂SO₄ fractionation) amylase showed that its apparent molecular mass was 51 kDa, which was further confirmed by native PAGE. The harvest of industrially significant extracellular amylase (probably α-amylase) produced as a byproduct during early growth phase would boost the economics of the Bt-based bio-industry engaged in δ-endotoxin production.     

The effect of oxygen on the sporulation, δ-endotoxin synthesis and toxicity of Bacillus thuringiensis H14

Summary The sporulation and toxicity of Bacillus thuringiensis H14 were studied as a function of aeration. The fed-batch cultures carried out in the similar aeration conditions were followed in four different oxygen transfer rates containing 0, 20, 100 and 250 mmol/l/h. The percentage of total cells which had formed refractile spores in these four oxygen transfer rates were 100, 93, 84 and 48%, respectively. The highest rate of sporulation was observed in the absence of oxygen and the mature spores were the only population present under this condition at the end of culture. Sporulation in a large portion of cells failed under saturated oxygenation and either mature spores or vegetative cells were present at the end of culture. In the intermediate conditions, cells in different physiological states could be observed at the end of culture. It was found that the optimal conditions for spore yield and for δ-endotoxin yield were not the same, even though sporulation and δ-endotoxin formation proceed simultaneously during the fermentation process. The 130-kDa δ-endotoxin seemed to be more sensitive to aeration conditions. The higher toxicity against Culex pipiens was obtained under the saturated condition.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal -endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis δ-endotoxins

Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry 1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectagular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.     

An improved bioassay for the detection of Bacillus thuringiensis β-exotoxin

Some Bacillus thuringiensis strains secrete β-exotoxin, which is an insecticidal, thermostable adenine nucleotide analogue. Discrepancies between detection of β-exotoxin by high-performance liquid chromatography and insect bioassays have shown the importance of bioassays in the determination of β-exotoxin production. With the aim of improving the fly β-exotoxin bioassay, a range of fly diets were evaluated and the best performing diet was incorporated into a novel β-exotoxin bioassay. The improved bioassay is characterised by good control pupation percentages, low variability, easy setup and monitoring. The bioassay allowed unambiguous differentiation between β-exotoxin producing and non-producing strains, and is suitable for the routine screening of B. thuringiensis strains for β-exotoxins.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Bacillus thuringiensis subsp. Aizawai δ-endotoxin inhibits the k+/amino acid cotransporters of lepidopteran larval midgut

1. The δ-endotoxin of Bacillus thuringiensis subsp. aizawai inhibits in a dose-dependent manner the K⁺-driven accumulation of histidine and lysine as well as their equilibrative uptake into brush border membrane vesicles from the midgut of Bombyx mori larvae.2. The decrease of uptake in the absence of a K⁺-gradient is neither due to a leakage of the labelled substrate from the vesicles nor to a reduction of the osmotically active space available, as a result of a detergent-like effect of the toxin.3. The toxin acts as a non competitive inhibitor of the K⁺/amino acid cotransporters of the larval midgut of Lepidoptera, with no preference for a specific transport system.     

Bacillus thuringiensis growth,sporulation and δ-endotoxin production in oxygen limited and non-limited cultures

The production of crystals and spores ofBacillus thuringiensis var.israelensis was studied under different aeration conditions. The results with 4 l batch cultures showed that for O₂ non-limited, cultures cell yield, toxin production and spore count were constant for all oxygen transfer rates (OTR). Under O₂ limitation, °-endotoxin concentrations and spore counts were lower than those obtained in non-limited cultures. In addition, -endotoxin yields diminished under O₂ limitation, suggesting that the toxin synthesis mechanism could have been affected.     

Stirred tank culture of Bacillus thuringiensis H-14 for production of the mosquitocidal δ-endotoxin: mathematical modelling and scaling-up studies

The effect of culture conditions on -endotoxin production by strain S128 of Bacillus thuringiensis H-14 (locally isolated in Egypt) was investigated using a 10l working volume fermenter. Fermentation medium formulated from the inexpensive locally available soya (as a nitrogen source) and molasses (as a carbon source) was used. Aeration, agitation, pH and initial concentration of molasses were chosen as experimental factors and their influence on toxin yield was investigated using 2⁴ central composite experimental design. The mathematical model obtained revealed that the optimal batch cultivation conditions with respect to agitation, pH, and initial concentration of molasses were 325 rev min^–1, 7.1 and 2.1% (w/v) respectively. The mathematical model obtained indicated that by increasing the aeration rate over 0.89 v/v per minute the productivity could still be increased. A simulated scaling-up study, in which the Simplex method was applied, is also presented. The results of this investigation could be of great help for large-scale production of a cheap mosquito-larvicide in developing countries where mosquito-borne diseases are still a serious health and economic problem.     

Studies onBacillus thuringiensis H-14 strains isolated in Egypt—III. Selection of media for δ-endotoxin production

Several medium ingredients locally available in Egypt were investigated for their ability to support -endotoxin production by two mosquito-toxic strains ofBacillus thuringiensis H-14 usingCulex pipiens for the bioassay of -endotoxin. Soya beans, black-eyed beans, common peas and lentils supported good production of toxin whereas peanuts, fodder yeast, cheese whey and corn steep liquor gave only low amounts of toxin. Molasses as the sole carbon source at 2 and 3% (w/v) with soya (or Proflo) as the sole nitrogen source at 3% gave the best yields of toxin for strain M1 and S128 respectively.

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Résumé On a étudié plusieurs ingrédients de milieu de culture, disponibles en Egypte pour leur capacité de favoriser la production d'endotoxine par deux souches deBacillus thurigiensis H-14, toxiques pour les moustiques, en utilisantCulex pipiens pour l'essaiin vivo d'endotoxine . Les fèves de soja, les haricots à taches noires, les pois communs et les lentilles favorisent la production de toxine, tandis que les arachides, la levure fourragère, le petit lait et la liqueur de trempage de maïs ne produisent que de faibles quantités de toxines. La mélasse comme seule source de carbone à 2% (p/v) avec la soja (ou le Proflo) comme seule source d'azote de 2 à 3% donnent les meilleurs rendements en toxine.