An enzyme linked immunosorbent assay for monitoring Bacillus sphaericus toxin.

Larvicidal proteins of B. sphaericus H5a5b (strain VCRC B42), purified by ion-exchange chromatography were used to raise antibodies in rabbits. The antibodies were specific in reacting to alkali-solubilized fractions from whole cells of toxic strains only. Ouchterlony immunodiffusion showed homology in toxin structure between strains of different serotype. A sandwich ELISA using avidin-biotin amplification was standardized. The lowest detectable limit was 6.25 ng/ml. Near linear quantitative binding of the antigen was found in the range 25-200 ng/ml. The growth, toxin level and LC50 values during various stages of fermentation of B. sphaericus strains 1593 and B42 were compared. There was significant correlation between LC50 values and toxin levels as measured by ELISA.     

球形芽孢杆菌Ts—1毒蛋白的分离纯化

Bacillus sphaericus strain Ts-1 is highly insecticidal to larvae of the mosquito. It's insecticidal component is toxic proteins. The toxin was extracted from spore-crystal complexes by disruption in a Sonicator Cell Disruptor Model W-220F followed by treatment with 0.05 mol/L NaOH. Fraction recovered from chromatography of the spore-crystal complexes on column of Sephadex G-200 were assayed against mosquito larvae and the toxic fractions from gel chromatography were subjected to SDS-PAGE. The toxic proteins in B. sphaericus Ts-1 spore-crystal complex migrated in position corresponding to 42kD and 43kD. Bioassay of the two purified proteins prepared by PAGE indicated that they were all toxic to mosquito larvae. Toxic protein was further purified by DEAE-cellulose chromatography. The toxic protein with a molecular weight of 42kD was obtained.     

Screening of Brazilian Bacillus sphaericus strains for high toxicity against Culex quinquefasciatus and Aedes aegypti

Abstract:  In this work, 246 Bacillus sphaericus strains were evaluated against Aedes aegypti and Culex quinquefasciatus larvae to select the most effective ones to be used as the basis of a national product. All strains were isolated from different regions of Brazil and they are stored in a Bacillus spp. collection at Embrapa Genetic Resources and Biotechnology. The selected strains were characterized by biochemical and molecular methods. Based on selective bioassays, 87 strains were identified as toxic to one or both target species. All of these strains contain genes that encode the 42, 51 kDa proteins that constitute the binary toxin and the 100 kDa Mtx1 toxin. All toxic strains presented a very high LC₅₀ against A. aegypti , so, a product based on any of these B. sphaericus strains would not be recommended for use in programmes to control A. aegypti . S201 had highest activity against C. quinquefasciatus , presenting the lowest LC₅₀ and LC₉₀ in bioassays.     

Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high-molecular-weight precursors

Crystals were purified from spore-crystal complexes of Bacillus sphaericus 2362 by disruption in a French pressure cell followed by centrifugation through 48% (wt/vol) NaBr. Crystals from such preparations had a 50% lethal concentration of 6 ng of protein per ml for the larvae of the mosquito Culex pipiens. When subjected to polyacrylamide gel electrophoresis under denaturing conditions, the proteins in B. sphaericus crystals migrated in positions corresponding to 43, 63, 98, 110, and 125 kilodaltons (kDa); solubilization of the crystal at pH 12 with NaOH eliminated all but the bands at 43 and 63 kDa. Since NaOH-solubilized preparations were toxic to mosquito larvae, these proteins were purified to electrophoretic homogeneity and antiserum was obtained to each. Analysis of the two purified proteins indicated that the 43-kDa protein was toxic to mosquito larvae (50% lethal concentration, 35 ng of protein per ml), whereas the 63-kDa protein was not. Further differences between them were their amino acid compositions, their lack of immunological cross-reactivity, their opposite net charges at pH 7.5, and their susceptibility to digestion by larval midgut proteases (the 63-kDa protein was highly susceptible, whereas the 43-kDa protein was not). The sequence of the 40 N-terminal residues of the 43-kDa protein was determined and found to contain a high percentage of hydrophobic amino acids. The sequence of the 63-kDa protein could not be determined, since it had multiple N termini. By electrophoretically separating the crystal proteins and then electroblotting onto nitrocellulose paper and visualizing the bands with antisera to the 43- and 63-kDa proteins in conjunction with an immunoblot assay, it was found that the high-molecular-mass crystal proteins (98 to 125 kDa) contained antigenic determinants of both proteins. These results suggested that the lower-molecular-weight crystal proteins detected in polyacrylamide gels after electrophoresis under denaturing conditions were derivatives of one or more of the higher-molecular-weight crystal proteins. In vivo studies of the products of crystal degradation by larvae of Culex pipiens indicated that the high-molecular-weight proteins and the 63-kDa antigenic determinants were rapidly degraded and that a 40-kDa protein related to the 43-kDa toxin persisted for the duration of the experiment (4 h). Some of the studies performed with B.sphaericus 2362 were extended to strains 1593, 1691, and 2297 of this species with results which indicated a high degree of similarity between the crystal proteins of all these larvicidal strains.     

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis

The binary toxin produced from Bacillus sphaericus is highly toxic against larvae of Culex and Anopheles mosquitoes. The two major components of the binary toxin are 42-kDa BinA and 51-kDa BinB, which are produced as crystalline inclusions during sporulation. Currently, there is no detailed knowledge of the molecular mechanism of the binary toxin, mainly due to the lack of structural information. Herein, we describe an expression protocol with modified conditions allowing production of soluble, biologically active BinA and BinB for further structural analysis. The binA and binB genes from B. sphaericus 2297 strain were independently cloned and fused with a polyhistidine tag at their N-termini. Both (His)(6)-tagged BinA and (His)(6)-tagged BinB were expressed as soluble forms at low temperature. Highly pure proteins were obtained after two-step purification by Ni-NTA affinity and size exclusion chromatography. In vitro activation by trypsin digestion generated a resistant fragment, of 40kDa for BinA, and of 45kDa for BinB, and an oligomeric complex of BinA and BinB in solution was observed after proteolytic activation. Their functional and structural properties were confirmed by a biological assay and far-UV circular dichroism, respectively. The mixture of BinA and BinB, either as a protoxin or as a trypsin-activated form, exhibited high mosquito-larvicidal activity against Culex quinquefasciatus larvae with LC(50) of about 10ng/ml, while no toxicity was observed from the single binary toxin component. Results from far-UV circular dichroism of BinA and BinB suggest the presence of mainly β-structure. The expression and purification protocols reported here will be useful for the production of the active and homogeneous binary toxin to allow further detailed structural investigation.     

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains.

Highly larvicidal strains of Bacillus sphaericus produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefasciatus. To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in Escherichia coli of the toxin proteins and their NH2- and COOH-terminal deletion derivatives as fusions with glutathione S-transferase (GST). Toxin proteins were purified by affinity chromatography followed by cleavage from the GST carrier with thrombin. The LC50 values for the purified toxin proteins and their deletion derivatives were determined. The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42 kDa protein. Examination of the toxin deletion derivatives revealed that the NH2-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein. Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.     

Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins.

In the course of sporulation, Bacillus sphaericus produces an inclusion body which is toxic to a variety of mosquito larvae. In this review we discuss the general biology of this species and concentrate on the genetics and physiology of toxin production and its processing in the midgut of the larval host. The larvicide of B. sphaericus is unique in that it consists of two proteins of 51 and 42 kDa, both of which are required for toxicity to mosquito larvae. There is a low level of sequence similarity between these two proteins, which differ in their sequences from all the other known insecticidal proteins of Bacillus thuringiensis. Within the midgut the 51- and 42-kDa proteins are processed to proteins of 43 and 39 kDa, respectively. The conversion of the 42-kDa protein to a 39-kDa protein results in a major increase in toxicity; the significance of the processing of the 51-kDa protein is not known. In contrast to the results with mosquito larvae, the 39-kDa protein is alone toxic for mosquito-derived tissue culture-grown cells, and this toxicity is not affected by the 51-kDa protein or its derivative, the 43-kDa protein. Comparisons of larvae from species which differ in their susceptibility to the B. sphaericus toxin indicate that the probable difference resides in the nature of the target sites of the epithelial midgut cells and not in uptake or processing of the toxin. A similar conclusion is derived from experiments involving tissue culture-grown cells from mosquito species which differ in their susceptibility to the B. sphaericus toxin.     

Cloning of the gene for the larvicidal toxin of Bacillus sphaericus 2362: evidence for a family of related sequences.

During sporulation, Bacillus sphaericus 2362 produces a parasporal crystalline protein which is toxic for the larvae of a number of mosquito species. Using the Escherichia coli cloning vector lambda gt11, in which gene products of the inserts may be fused to beta-galactosidase, we isolated 29 bacteriophages which produced peptides-reacting with antiserum to crystal protein. On the basis of restriction enzyme analyses of the recombinants and Ouchterlony immunodiffusion experiments with induced lysogens as a source of antigens, the recombinants were assigned to three groups, designated A, B, and C. Group A consisted of three clones which appeared to express all or part of the B. sphaericus toxin gene from their own promoters and one clone producing a beta-galactosidase-toxin fusion protein. The host cells of two induced recombinant lysogens of this group were toxic to larvae of Culex pipiens. A cell suspension containing 174 ng (dry weight) of the more toxic recombinant per ml killed 50% of the larvae. Both recombinants formed peptides with molecular sizes of 27, 43, and 63 kilodaltons (kDa). The antigenically related 27- and 43-kDa peptides were distinct from the 63-kDa peptide, which resembled crystals from sporulating cells of B. sphaericus in which antigenically distinct 43- and 63-kDa proteins are derived from a 125-kDa precursor. A 3.5-kilobase HindIII fragment from recombinants having toxic activity against larvae was subcloned into pGEM-3-blue. E. coli cells harboring this fragment were toxic to mosquito larvae and produced peptides of 27, 43, and 63 kDa. The distribution of the A gene among strains of B. sphaericus of different toxicities suggested that it is the sole or principal gene encoding the larvicidal crystal protein. The two recombinants of group B and the 23 of group C were all beta-galactosidase fusion proteins, suggesting that in E. coli these genes were not readily expressed from their own promoters. The distribution of these two genes in different strains of B. sphaericus suggested that they do not have a role in the toxicity of this species to mosquito larvae.