Parasporal Protein Research Articles

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus.

Dose Response of Omnivorous Leafroller to Commercial Formulations of Bacillus Thuringiensis Var. Kurstaki in the Laboratory, 1992

Abstract We compared three formulations of B. thuringiensis var. kurstaki (Btk) for their insecticidal activity against first-instar OLR. Products tested included: (1) NOVO Laboratories Biobit, a flowable concentrate containing live bacteria with spores and parasporal crystal protein bodies; (2) Abbott Laboratories Dipel 2X, a wettable powder also containing active bacteria with spores and parasporal crystal protein bodies; and (3) Mycogen Corporations MVP Bioinsecticide, a flowable concentrate containing dead bacterial cells which bioencapsulate the Btk proteins. The AI in each of these products is the insecticidal protein, or delta-endotoxin, of Btk. Dose-response was determined by feeding trials exposing first-instar OLR to each product. Serial dilutions of each formulation were made and 10 μ1 of each dilution was applied to the surface of an agar-based diet (Bioserv No. 9370) and then allowed to air dry. One larva was placed in each vial containing ca. 1 ml of surface-contaminated diet (surface area = 1 cm2). Thirty to 60 larvae were observed for each dilution and formulation tested. Doses were expressed in mg AI per sq. cm. Mortality evaluations were made after 3, 5, and 7 days. Response data were analyzed by the POLO-PC PROBIT procedure (LeOra Software, 1987).

Bacillus thuringiensis crystal protein (δ-endotoxin) gene expression is independent of early sporulation specific functions

Bacillus thuringiensis produces a parasporal insecticidal crystal protein. The correlation between sporulation and crystal protein production inBacillus thuringiensis var.israelensis was studied. The strain was made resistant’against streptomycin (StR)-Acrystalliferous (Cry-) cured derivatives and asporogenous acrystalliferous (Spo− Cry−) mutants blocked at an early stage of sporulation were isolated. Plasmid transfer experiments were performed between StR Spo+ Cry+ (streptomycin sensitive sporogeneous crystalliferous) and StRR Spo+ Cry− and also between Sts Spo+ Cry+ and StR Spo− Cry− strains. StR colonies were selected. Insect toxicity was exhibited by the StR isolates in both the cases. The process of crystal formation is, therefore, independent of early sporulative events.

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: Comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.

Affinity purification of a 65-kilodalton parasporal protein from Bacillus thuringiensis PG-14 that shows mosquitocidal activity.

By using antibody-mediated affinity chromatography, a highly mosquito larvicidal but nonhemolytic fraction was obtained from alkali-solubilized, silkworm (Bombyx mori) larval gut juice-treated parasporal inclusions of Bacillus thuringiensis strain PG-14 (serotype 8a:8b). This fraction contained a 65-kDa protein only but not a 25-kDa protein, the main component in the flow through fraction unbound to the affinity column. The 25-kDa protein purified from the unbound fraction by CM-cellulose chromatography demonstrated a high hemolytic activity against sheep red blood cells but very low mosquito larvicidal activity.

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.

Characterization of monoclonal antibodies to a crystal protein of Bacillus thuringiensis subsp. kurstaki.

Ten monoclonal antibodies were produced against a k-1-type crystal protein of Bacillus thuringiensis subsp. kurstaki. Eight of the antibodies belong to the immunoglobulin G1 (IgG1) subclass, with pI values ranging from 5.5 to 8.6, one could be assigned to the IgG2b subclass, and one could be assigned to the IgM class. Competitive antibody-binding assays and analysis of antibody specificity indicated that the 10 antibodies recognized at least nine distinct antigenic determinants. Eight antibodies bound to both protoxin and toxin, whereas the other two interacted with protoxin only. One antibody completely inhibited the biological activity of the delta-endotoxin, five antibodies reduced it by 15 to 82%, and four antibodies did not affect it at all. Based on cross-reaction studies, homologies and differences in the crystal protein structures of different B. thuringiensis subspecies were revealed. All of the monoclonal antibodies strongly cross-reacted with crystal proteins from strains of B. thuringiensis subsp. tolworthi, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. dendrolimus, B. thuringiensis subsp. sotto, and B. thuringiensis subsp. subtoxicus. Some antibodies interacted only weakly with crystal proteins from strains of B. thuringiensis subsp. morrisoni and B. thuringiensis subsp. entomocidus, and some of these did not interact with B. thuringiensis subsp. kenyae and B. thuringiensis subsp. darmstadiensis. No cross-reaction was found with the parasporal inclusion protein of B. thuringiensis subsp. israelensis. Studies with the monoclonal antibodies also disclosed that crystal proteins from strains of the same subspecies can exhibit substantial differences in antigenic structure. In particular, striking strain-specific differences in the protoxins of B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. thuringiensis were observed.

Relationship of the syntheses of spore coat protein and parasporal crystal protein in Bacillus thuringiensis.

Two major classes of polypeptides were extracted from the spore surface of Bacillus thuringiensis subsp. kurstaki: the 134,000-dalton protoxin that is the major component of the crystalline inclusion and spore coat polypeptides very similar to those found on Bacillus cereus spores. The quantity of spore coat polypeptides produced was reduced when compared with that produced by certain acrystalliferous mutants or by B. thuringiensis subsp. israelensis. The latter organism produced an inclusion toxic to mosquito larvae, but deposited very little of the inclusion protein on the spore surface. The reduction in spore coat protein in B. thuringiensis subsp. kurstaki was also seen in freeze-etched electron micrographs of spores. B. thuringiensis subsp. kurstaki spores germinated rather slowly when compared with related species, a property previously correlated with a deficiency or defect of the spore coat. Many mutants of B. thuringiensis subsp. kurstaki unable to form a crystalline inclusion were nontoxic and lacked a well-defined spore coat. Other mutants isolated either directly from the wild type or from coat-deficient mutants produced spores that were identical to those produced by the closely related species. Bacillus cereus, on the basis of morphology, germination rate, and the size and antigenicity of the spore coat polypeptides. Most of the protein extractable from the inclusion produced by B. thuringiensis subsp. israelensis was about 26,000 daltons, considerably smaller than the major polypeptide extractable from other inclusions. Some of the B. thuringiensis subsp. israelensis inclusion protein was found on the spore surface, but the majority of the extractable spore coat protein was the same size and antigenicity as that found on B. cereus spores. The B. thuringiensis subsp. israelensis spores germinated at a rate close to that of B. cereus, especially when the spores were formed at 37 degrees C, and the morphology of the spore surface was very similar to that of B. cereus.

Structure and function of the Bacillus thuringiensis protein crystal

AbstractThe structural chemistry of the Bacillus thuringiensis parasporal protein crystal is discussed in terms of purification techniques, removal of contaminating proteases, crystal subunit size, crystal shape, interchain crosslinks, the ultimate toxin, and lysinoalanine. The alkaline pH cleavage of disulfide bonds is stressed in relationship to this role in crystal solubilization and toxin formation. The future implication s of plasmid‐coded crystal formation and B. thuringiensis var. israelensis (effective against mosquitoes and black flies) are also discussed.

Parasporal crystals produced by oligosporogenous mutans of Bacillus thuringiensis (Spo-Cr+).

Six oligosporogenic (Spo-) mutant strains of Bacillus thuringiensis were selected from survivors of treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Each strain was blocked at or before stage II of spore development, but all produced typical bipyramidal-shaped crystalline inclusion bodies. Toxicity of the paraporad typical bipyramidal-shaped crystalline inclusion bodies. Toxicity of the parasporal endotoxin isolated from the mutant strains was assayed by an in vitro technique using cultured insect cells, and was comparable with that of normal wild-type parasporal protein. Multiple parasporal inclusion bodies per cell were often produced, and smaller embedded particles were numerous and distinct.

Isolation and partial characterization of the parasporal body of Bacillus popilliae.

The sporangial wall of Bacillus popilliae encloses a spore and parasporal body and is very resistant to breakage. High-energy sonication can disrupt this envelope and release free spores and parasporal bodies which retain refractility. Parasporal bodies, free spores, and sporangial debris were separated by centrifugation in linear sucrose gradients. Purified parasporal bodies are proteinaceous. Of the 17 amino acids detected, glutamic and aspartic acids are highest in concentration and account for the low isoelectric point of 4.5; a lipid moiety was not found. Parasporal bodies are soluble in 0.1N NaOH, 8M guanidine hydrochloride, 8M urea, or alkaline thioglycollate. Paper electrophoresis at 3000 V separated soluble parasporal protein into three cathodic components. Solubilized or whole intact parasporal bodies fed to third-instar larvae of the Japanese beetle were not detectable toxic. However, solubilized parasporal protein was twice as toxic as the intact parasporal bodies when injected into the hemocoel.

Arguments based on hybridization-competition experiments in favor of the [formula omitted] synthesis of sporulation-specific mRNAs by the RNA polymerase of [formula omitted

Qualitative differences between the RNA transcripts synthesized in-vitro by the vegetative or by the two sporulation RNA-polymerases of B. thuringiensis were demonstrated, using the hybridization-competition technique. On the other hand, the in-vitro RNA preparations obtained with the sporulation Form II enzyme, were shown to present large homologies with “stable” RNA fractions extracted from wild-type sporulating cells. Comparison of these data with those obtained with in-vivo RNAs from two asporogenous mutants strongly suggests the involvement of this Form II in the synthesis of sporulation-specific RNAs, among them the mRNA coding for the parasporal protein.

The comparative pathogenicity of some Bacillus thuringiensis varieties for larvae of the spruce budworm, Choristoneura fumiferana

In a laboratory assay in which synthetic media were used, several varieties of Bacillus thuringiensis have been tested for pathogenicity for larvae of the spruce budworm, Choristoneura fumiferana. The toxicity of nearly pure parasporal inclusions (crystals) and precipitated parasporal inclusion protein was also investigated. The varieties alesti, berliner, entomocidus, sotto, and galleriae were highly to moderately pathogenic; the varieties kenyae and dendrolimus were less so, and the variety finitimus was only slightly pathogenic. Although ingestion of crystals and precpitated crystal protein led to inhibition of feeding by larvae, the most pathogenic preparations were those that contained both spores and crystals.

Serological investigations of parasporal inclusion protein from Bacillus thuringiensis strain 562-5A

The toxic parasporal protein of Bacillus thuringiensis strain 562-5A, solubilized both in gut juice and under alkaline conditions, has been studied by serological methods and compared with similar preparations from B. thuringiensis var. thuringiensis. It was found that strain 562-5A differs in at least one antigen from B. thuringiensis var. thuringiensis but shares at least four antigens with it. Crystals dissolved in Bombyx mori larval gut juice and subjected to electrophoretic separation gave fractions that were serologically similar but of different toxicity.

Similarity of effect of valinomycin and Bacillus thuringiensis parasporal protein in larvae of Bombyx mori

Similarity of effect of valinomycin and Bacillus thuringiensis parasporal protein in larvae of Bombyx mori

Serological studies of the protein parasporal inclusions of Bacillus entomocidus var. entomocidus

The toxic parasporal protein of Bacillus entomocidus var. entomocidus solubilized in vivo in gut juice, and in vitro under alkaline conditions, has been studied by serological methods and compared with similar preparations from Bacillus thuringiensis var. sotto. It was found that B. entomocidus var. entomocidus shares at least one common toxic component with B. thuringiensis var. sotto, and also possesses an additional toxic component. Differences in electrophoretic mobility were found depending on whether the crystals were dissolved in gut juice or in mild alkali.

Serological studies of the protein parasporal inclusions of Bacillus thuringiensis var. sotto Ishiwata

The toxic parasporal protein of Bacillus thuringiensis var. sotto Ishiwata solubilized in vivo in gut juice, and in vitro under alkaline conditions, has been studied by serological methods. Antibodies elicited by alkali-dissolved protein from the crystals of Bacillus thuringiensis var. thuringiensis Berliner, Bacillus entomocidus var. entomocidus Heimpel and Angus, and B. thuringiensis var. sotto, inactivate the sotto protein in vitro and in the gut of larvae of the silkworm, Bombyx mori (linnaeus). Injections of toxin-specific antibody into the hemocoel do not protect larvae against the toxic effects of ingested crystals.

Chemical and morphological studies of bacterial spore formation. II. Spore and parasporal protein formation in Bacillus cereus var. alesti.

The development of both the spore and parasporal protein crystal of Bacillus cereus var. alesti was followed using chemical and cytological techniques. The changes which led to the formation of the fore-spore were similar to those already described for Bacillus cereus. However, adjacent to the developing fore-spore a small inclusion became discernible in phase contrast. This protein inclusion during its growth was differentiated from the chromatin and lipid-containing inclusions by sequential staining techniques. During spore and crystal formation no net synthesis of either nucleic acid was detected. Tracer studies with radioactive phosphorus confirmed that the spore chromatin was derived from that in the vegetative cell. These same studies also indicated that a turnover of ribonucleic acid occurred during the sporulation process. During their formation both the spore and crystal incorporated methionine-(35)S from the medium and from cellular material into a bound form. Sequential extractions with alkali and with alkaline-thioglycollate reagent revealed that the solubility characteristics of the mature crystal were possibly related to the presence of intermolecular disulphide bonds which developed after the major synthesis of the crystal was complete. The synthetic nature of sporogenesis and crystal formation is discussed with reference to the concept of "endotrophic" sporulation.

Localization of toxicity for silkworm larvae in the parasporal inclusion of Bacillus cereus var. alesti.

The parasporal protein inclusion of Bacillus cereus var. alesti accounts for one third of the dry weight of the washed products of sporulation. Although the spore:crystal ratio is 1:1, the weight ratio is 1.5:1. Like other parasporal crystals, those of var. alesti are swollen but not dissolved by alkalinity up to a critical pH (11.5–11.8); above this pH, dissolution occurs. Toxicity tests on extracts made at increasing pH levels indicated that the crystal protein itself is the toxic agent and not the carrier of it. The protein of actively dividing cells and of spores was non-toxic. Crystal-forming cells contained a toxic protein which would dissolve at a lower pH than would the free crystal.