An improved enzyme-linked immunoassay for the detection and quantification of the entomocidal parasporal crystal proteins of Bacillus thuringiensis subsp. kurstaki and israelensis

An improved and simplified enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of parasporal crystalline toxins from Bacillus thuringiensis subsp. kurstaki. The improved procedure involved pretreatment of the polystyrene cuvettes with glutaraldehyde before antibody coating. A direct comparison of treated and untreated cuvettes is provided. ELISAs were then used for the analysis of the entomocidal crystalline proteins in commercial and experimental formulations of B. thuringiensis subspp. kurstaki and israelensis.     

Two types of entomocidal toxins in the parasporal crystals of Bacillus thuringiensis kurstaki

Two types of entomocidal proteins of Bacillus thuringiensis kurstaki were isolated from the parasporal bodies (crystals), and their structures were compared with each other in relation to the toxic activity. When the crystals were dissociated in 2% 2-mercaptoethanol at pH 10, a protein of Mr = 135,000, called delta-endotoxin, was liberated. The crystals of a strain of B. thuringiensis kurstaki, the HD-1 strain, also released another protein in small quantities. This minor component of HD-1, which had been discovered and named mosquito factor by Yamamoto and McLaughlin (T. Yamamoto and R. E. McLaughlin (1981) Biochem. Biophys. Res. Commun. 103, 414-421) because of its toxicity to mosquito larvae, could be liberated selectively from the crystals by alkali treatment without any thiol reagent at pH 11. Electron microscopic observation suggested that the bipyramidal crystal is composed of a homogeneous component, presumably the delta-endotoxin, and the mosquito factor is not within the crystal matrix. The liberated toxins, including the mosquito factor, were purified by Sephacryl S-300 column chromatography and activated by proteinases obtained from gut juice of the cabbage looper (Trichoplusia ni). The activated toxins were characterized by peptide mapping using techniques of HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping revealed that the mosquito factor is a protein distinctly different from the delta-endotoxin. Furthermore, a comparison between two strains of B. thuringiensis kurstaki indicated that minor differences in the structure of the delta-endotoxins, in particular the differences in their proteinase-resistant region, caused significant variations in their toxicity to susceptible insects.     

Degradation of the parasporal crystal produced by Bacillus thuringiensis var. kurstaki

Degradation products of the parasporal crystals of Bacillus thuringiensis var. kurstaki obtained by treatment with alkali, gut juice from larvae of Bombyx mori, and various plant and mammalian enzymes were compared for elution pattern, approximate molecular weight (MW), and toxicity. The results indicated that with alkaline treatment the most toxic extract was obtained with 0.05–0.1 M NaOH. Toxicity was found associated mainly with a protein peak of 230,000 MW although other toxic peaks were found in the tailing. Heat-treated midgut juice from larval B. mori gave similar results. After digestion of parasporal crystals with clarified midgut juice, five peaks causing toxicity and having MW of approximately 235,000, 67,000, 30,200, 5000, and 1000, respectively, were identified. Treatment of B. thuringiensis δ-endotoxin with α-chymotrypsin gave peaks causing mortality of approximate MW 235,000, 34,000, 5000, and 1000. Trypsin, pronase, carboxypeptidase, and enterokinase digests of the B. thuringiensis δ-endotoxin gave toxic components ranging from 235,000 to 30,000 MW. The protein protoxin molecules are digested to give small toxic subunits that may be of practical value for structural determinations and for molecular mode of action studies.     

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.     

Purification of the insecticidal toxin from the parasporal crystal of Bacillus thuringiensis subsp. kurstaki

The insecticidal toxin of $\text{Bacillus}\text{thuringiensis}$ subsp. $\text{kurstaki}$ was isolated from parasporal crystals. The toxin, which is stable for several months, is a glycoprotein with an apparent molecular weight of 68,000 that is generated upon solubilization and activation of a higher molecular weight protoxin (MW_app = 1.3 × 10⁵) at alkaline pH. The toxin was purified by gel filtation and anion exchange chromatography and its molecular weight was established by gel filtration chromatography and SDS polyacrylamide gel electrophoresis.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains.

Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins.     

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73

The secondary structure of the toxin fromBacillus thuringiensis subsp.kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33–40% -helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32–40% -sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both -helical and -sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.     

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.     

Expression of three genes coding for 135-kilodalton entomocidal proteins inBacillus thuringiensis kurstaki

The HD-1 strain ofBacillus thuringiensis (B.t.)kurstaki contains three homologous genes coding for 130–134-kilodalton entomocidal proteins [13]. In the present study, expression levels of these genes in strains of B.t.kurstaki were determined. In attempts to isolate a protein coded by a single gene, a number of variants were derived from strains of B.t.kurstaki, such as HD-263 and HD-1, by plasmid curing. The entomocidal proteins produced by the parental strains and their plasmid-cured variants were isolated by Sephacryl S-300 column chromatography and peptide-mapped by high performance liquid chromatography (HPLC). The results indicated that HD-263 produced two distinctive proteins, one identical with the protein of HD-73, which contains only a 6.6 kb Hind III class gene, and the other protein presumably coded by a 4.5 kb Hind III class gene. HPLC analysis revealed that 70% of the total protein in the HD-263 crystals consisted of the product of the 6.6 kb gene (6.6-kb protein), and the remaining 30% was the 4.5-kb protein. In the case of HD-1, the crystal consisted of at least two different proteins in equal amounts (50% each). The gene coding for one of these proteins was presumed to be a 5.3 kb Hind III class gene. The remaining 50% of the HD-1 crystal was accounted for by a protein similar to the 4.5-kb protein identified in HD-263. It appeared that the 6.6-kb protein was expressed poorly, if it was indeed expressed, in the HD-1 strain.     

Separation of three biologically distinct activities from the parasporal crystal ofBacillus thuringiensis var.israelensis

Three fractions showing biologically distinct activities were isolated from theBacillus thruingiensis var.israelensis crystal -endotoxin. Using a shallow sodium chloride gradient on a DEAE anion-exchange column, three pooled fractions were obtained consisting of the 25Kd peptide; 25,26Kd peptides; and 31,34,35Kd peptides, respectively. The 25Kd peptide was hemolytic against human erythrocytes but not mosquitocidal. The 25,26Kd peptide mixture was hemolytic, not mosquitocidal, but toxic to larvae ofTrichoplusia ni when injected into the hemocoel of these animals. The 31,34,35Kd peptide mixture was toxic to mosquito larvae, but neither hemolytic nor toxic to larvae ofT. ni upon injection. In addition, teratogenic effects were observed when wandering larvae ofT. ni were injected with each of these three fractions. All of the biological activities described were destroyed when peptides were treated for 15 s at 95°C or greater. The hemolytic activity was elevated by heating the peptide(s) at 70°C for 15 s. On the other hand, this elevation was not observed with the two insecticidal activities.     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

Lack of mosquitocidal activity by the cytolytic protein of theBacillus thuringiensis subsp.israelensis parasporal crystal

A 25,000-dalton cytolytic protein was isolated from the parasporal crystal ofBacillus thuringiensis subsp.israelensis. Hemolytic activity of this protein decreased with increasing pHs and was totally inhibited at pH 10.0. No mosquito larvacidal activity was observed with this protein either in the solubilized form or when the protein was adsorbed to latex beads.     

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis

Three crystalliferous (Cry⁺) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry^?) mutants, either induced or spontaneously derived from a single Cry⁺ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry⁺ and Cry^? variants were analyzed by both a cleared lysate- and a modified Eckhardt lysateelectrophoresis technique. All of the Cry^? mutants derived from the Cry⁺ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry^? variants. All three Cry⁺ strains, including the parent of the Cry^? strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry⁺ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry^? derivatives differed in other respects from the pattern for their parent strain. The various Cry⁺ and Cry^? strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.     

Secondary structure of the entomocidal toxin fromBacillus thuringiensis subsp.kurstaki HD-73

The secondary structure of the toxin fromBacillus thuringiensis subsp.kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33–40% α-helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32–40% β-sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both α-helical and β-sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.     

Characterization of the anti-cancer-cell parasporal proteins of a Bacillus thuringiensis isolate

An unusual activity, associated with non-insecticidal and non-haemolytic parasporal inclusion proteins of a Bacillus thuringiensis soil isolate, designated 89-T-26-17, was characterized. The parasporal inclusion of this isolate was bipyramidal, rounded at both ends, containing proteins of 180, 150, 120, 100, and 88 kDa. No homologies with the Cry and Cyt proteins of B. thuringiensis were detected based on N-terminal sequences. Proteolytic processing of the inclusion proteins by proteinase K, trypsin, and chymotrypsin produced a major protein of 64 kDa exhibiting cytocidal activity against human leukaemic T cells and uterus cervix cancer (HeLa) cells. The protease-activated proteins showed no cytotoxicity to normal T cells.     

Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var,kurstaki toxic to the mosquito larva,Aedes taeniorhynchus

The parasporal crystal produced by a strain of Bacillus thuringiensis var. kurstaki (HD-1) contains two serologically distinct proteins. These proteins were isolated from a preparation of the parasporal crystal by Sephacryl S-300 column chromatography. Their molecular weights were estimated as 135,000 and 65,000. Both proteins were toxic to the cabbage looper, Trichoplusia ni, but only the 65,000-dalton protein was toxic to larvae of the mosquito, Aedes taeniorhynchus. Biochemical comparisons based on isoelectric focusing and peptide mapping by two-dimensional gel electrophoresis indicated that the two toxins were distinctly different.     

Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal.

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 X 10(-8) mol of free sulfhydryls and 3.24 X 10(-8) mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Nonreduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.