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.     

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.     

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.     

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.     

Facile preparation and characterization of the toxin from Bacillus thuringiensis var. kurstaki.

We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.     

Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line

A cloned CryIVB toxin was purified from a cured strain of Bacillus thuringiensis (BT) containing the cryIVB gene on the recombinant plasmid Cam135. Solubilized protoxin was treated with Aedes gut extract or trypsin for varying times and tested for toxicity in vitro on three dipteran and one lepidopteran cell line. Treatment with the Aedes extract but not trypsin, produced an active toxin which lysed only Aedes aegypti cells out of those tested. This activation was time-dependent reaching a maximum after 6 h. Both the Aedes extract-treated and trypsin-treated toxin killed A. aegypti larvae, but this toxicity declined rapidly with increasing time of exposure to the proteolytic preparations.     

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.     

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 detergent-like mechanism of action of the cytolytic toxin Cyt1A from Bacillus thuringiensis var. israelensis

The cytolytic delta-endotoxin Cyt1A from Bacillus thuringiensis var. israelensis is used in commercial preparations of environmentally safe insecticides. The current hypothesis on its mode of action is that the toxin self-assembles into well-defined cation-selective channels or pores, which results in colloid-osmotic lysis of the cell. Recently, a new hypothesis has been put forward suggesting that Cyt1A rather nonspecifically aggregates on the membrane surface and acts in a detergent-like manner. To distinguish between these two hypotheses, we investigated whether in the presence of lipid Cyt1A self-assembles into stoichiometric oligomers, which are characteristic of pores or channels, or aggregates into nonstoichiometric complexes, which would support the detergent-like model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that in the presence of lipid Cyt1A forms protein aggregates with a broad range of molecular weights, some being too large to enter the gel. Cyt1A tryptophan (Trp) fluorescence in the presence of lipid exhibited a decrease in anisotropy and quantum yield, but an unchanged lifetime, which is consistent with the presence of toxin aggregates in the membrane. Electrostatic interactions between the charged amino acid residues and the lipid headgroups are responsible for bringing the protein to the membrane surface, while hydrophobic and/or van der Waals interactions make the membrane binding irreversible. Fluorescence photobleaching recovery, a technique that measures the diffusion coefficient of fluorescently labeled particles, and epifluorescence microscopy revealed that upon addition of Cyt1A lipid vesicles were broken into smaller, faster diffusing objects. Since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes, our results support the detergent-like mode of action of Cyt1A.     

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.     

Amino sugars in the glycoprotein toxin from Bacillus thuringiensis subsp. israelensis.

The carbohydrate content of purified Bacillus thuriniensis subsp. israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins. The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars. The amino sugars consisted of 70% glucosamine and 30% galactosamine. No N-acetylneuraminic acid (sialic acid) was detected. The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin. These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively. The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal. The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separate by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. This study demonstrates the covalent attachment of amino sugars and indicates that the B. thuringiensis subsp. israelensis protein toxins should be viewed as glycoprotein toxins. The crystals used in the present study were purified on sodium bromide density gradients. Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars.     

Bacillus thuringiensis var.israelensis crystal hemolysis as a possible basis for an assay of larval toxicity

Summary Optimal conditions for the extraction of a hemolysin from crystals ofBacillus thurin-giensis var.israelensis (B.t.i.) have been described. Evidence is given that the mechanism of hemoly-sin release from crystals involves proteolytic en-zymes. The hemolysin extracted from B.t.i. crys-tals has been shown to be antigenically different from the hemolysin excreted by vegetative cells of the same strain. As there is good correlation be-tween the hemolytic and larvicidal properties of various B.t.i. preparations it is suggested that the hemolytic assay should be tried as a rapid assay for preliminary determinations of the activity of B.t.i. preparations.     

Parameters affecting attachment ofBacillus thuringiensis var.israelensis toxin to latex beads

Summary Bacillus thuringiensis var.israelensis protein toxin radiolabeled with¹⁴C-iodoacetamide was bound to latex beads. The efficiency of attachment was not affected by the temperature or length of incubation or by the presence of several different buffers, salts, or organic solvents. However, attachment decreased dramatically at pH values >8.5 or in the presence of detergents (anionic, neutral, or cationic). Protein concentrations 2–3 mg/m² of bead surface led to a progressively decreasing efficiency of protein adsorption. With minor variations, these findings should also be applicable to the attachment of numerous other proteins of diagnostic significance.Abbreviations BSA bovine serum albumin - Bti Bacillus thuringiensis var.israelensis - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - LAT latex agglutination test - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate - TCA trichloroacetic acid - Tris Tris(hydroxymethyl)aminomethane - TTAB tetradecyltrimethylammonium bromide     

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).     

A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis

Bacillus thuringiensis var. israelensis (BTI), serotype 14, which produces parasporal crystals toxic to certain dipteran larvae, was analyzed by agarose gel electrophoresis and found to contain a complex plasmid array. Eight plasmids were detected, with approximate sizes of 3.3, 4.2, 4.9, 10.6, 68, 75, 105, and 135 MDa, as well as a plasmidlike linear DNA element of ~10 MDa. Partially cured mutants of BTI implicated the 75-MDa plasmid in crystal production. Fifteen independently isolated acrystalliferous (Cry^?) mutants were found to lack this plasmid. In plasmid transfer experiments, several of the BTI plasmids transferred into a plasmid-free, Cry^? BTI recipient, but only transfer of the 75-MDa plasmid converted the recipient to crystal toxin production. The presence or absence of mosquito-toxic activity in all Cry⁺ and Cry^? variants of BTI was confirmed by bioassay of sporulated cultures against larvae of Aedes aegypti. Southern blot analyses revealed that in one unusual Cry⁺ variant in which no 75-MDa plasmid band was detectable, plasmid sequences were still present, possibly integrated into the chromosome. The 75-MDa plasmid could also apparently recombine with the 68-MDa plasmid, to which it was partially homologous.