Specific Features of Accumulation Kinetics of Entomocidal Exotoxins of Bacillus thuringiensis Subsp. thuringiensisduring the Fermentation Stage of Bitoxibacillin Production

Accumulation of Bacillus thuringiensis subsp. thuringiensis -exotoxin (BET) in the course of industrial fermentation (a stage in the production of the entomocidal biopreparation bitoxibacillin) has been studied. It has been demonstrated in model experiments that the decrease in the content of BET in the culture fluid is accounted for by the toxin interaction with an attendant product, the exogenous metabolite (EM). EM has been isolated from the culture fluid and characterized. EM causes alkalization of the medium, exerts entomocidal effects (in Musca domestica), and fails to form salts on treatment with BaCl₂. The absorption spectrum of EM is similar to that of BET, showing a maximum at = 259 nm. The light-absorbing chromophore is a pyrimidine or purine base. A method for quantitative determination of both exotoxins (BET and EM) in bacterial preparations has been developed.     

Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki

The gene encoding the 66-kDa entomocidal protein (P2 protein or mosquito factor) from Bacillus thuringiensis var. kurstaki has been isolated by the use of a 62-mer oligonucleotide probe that encoded 21 amino acids of the P2 protein NH2 terminus. The DNA sequence of the gene, designated cryBI, was unique from the published sequences of other B. thuringiensis genes. However, the amino acid sequence of the P2 protein, as deduced from the DNA sequence of the cryBI gene, was found to contain a sequence of 100 amino acids having 37% homology to a group of B. thuringiensis entomocidal proteins, the P1 proteins. Late stationary phase Bacillus megaterium cells harboring the cloned B. thuringiensis cryBI gene contained large aggregates of the P2 protein, and the cells were highly toxic to both lepidopteran and dipteran larvae. In contrast, Escherichia coli cells harboring the cloned cryBI gene contained very low levels of the P2 protein. DNA blot hybridization experiments showed that certain B. thuringiensis strains contained at least one cryBI-related DNA sequence in addition to the cryBI gene itself.     

The construction of Bacillus thuringiensis strains expressing novel entomocidal delta-endotoxin combinations.

Using our recently reported method of electroporation to transform Bacillus thuringiensis [Bone & Ellar (1989) FEMS Microbiol. Lett. 58, 171-178], cloned B. thuringiensis entomocidal delta-endotoxin genes have been introduced into several native B. thuringiensis strains. In many cases the resulting transformants expressed both their native toxins and the cloned toxin, producing strains with broader toxicity spectra. The introduction of the var. tenebrionis toxin gene into B. thuringiensis var. israelensis resulted in a strain with activity against Pieris brassicae (cabbage white butterfly), an activity which neither parent strain possesses. We discuss further the possibility of synergism and also the problems associated with introducing cloned DNA by this method.     

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.     

The biotechnology of Bacillus thuringiensis

One of the challenges in the application of biotechnology to pest control is the identification of agents found in nature which can be used effectively. Biotechnology offers the potential of developing pesticides based on such agents which will provide environmentally sound and economically feasible insect control alternatives. Such an agent, the insect pathogen Bacillus thuringiensis, is the subject of intense investigations in several laboratories. Insecticides which use the entomocidal properties of B. thuringiensis are currently produced and sold worldwide; new products are currently in the development stage. Herein, the biology and genetics of B. thuringiensis and the problems associated with current products are critically reviewed with respect to biotechnology. Moreover, the economic and regulatory implications of technologically advanced products are evaluated.     

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

Identification and characterization of a novel Bacillus thuringiensis δ-endotoxin entomocidal to coleopteran and lepidopteran larvae

A new class of Bacillus thuringiensis delta-endotoxins, or insecticidal control proteins (ICPs), is defined by an apparently cryptic protein with a unique primary structure and novel entomocidal specificity for certain coleopteran and lepidopteran species. The discovery of a new group of ICPs will extend the use of this natural insecticide in integrated pest-management systems.     

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.     

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.     

Directed mutagenesis of selected regions of a bacillus thuringiensis entomocidal protein

Comparison of the sequences of Bacillus thuringiensis entomocidal toxins of widely differing specificity reveals six conserved domains. The role of one of the most highly conserved domains (D1) located near the N-terminus has been investigated by site directed mutagenesis at two positions. Although preliminary results indicate that the capacity of the mutants to bind to putative receptors on the plasma membrane of susceptible cells was unaffected, toxicity in vivo was reduced by 70-80%. The role of the highly hydrophobic segment exposed at the N-terminus of the toxin after proteolytic activation was investigated by substituting two aspartate residues for phenylalanine and valine located adjacent to each other in the centre of this segment. The toxicity of the resulting mutant protein was only 40% of the unmutated toxin but again preliminary results suggest that binding to putative receptors was unaffected. These results suggest that regions close to the N-terminus of this and similar toxins may play an important role in the membrane insertion event which is believed to follow receptor recognition.     

Relative toxicity of native Chilean Bacillus thuringiensis strains against Scrobipalpuloides absoluta (Lepidoptera: Gelechiidae)

The larva of Scrobipalpuloides absoluta , a South American moth, is the most devastating insect pest of tomato production in Chile. The potential for using bacterial insecticides was studied analysing the relative toxicity of native Bacillus thuringiensis (BT) isolates belonging to the Chilean collection. The polymerase chain reaction (PCR) technique was used in order to facilitate the prescreening. Mixtures of homologous specific primers to regions within genes encoding CryI , CryIII and CryIV crystal proteins were employed to generate a PCR product profile of each BT isolate. Four isolates were selected and further characterized by means of SDS-PAGE, Western blot and bioassays on fourth-instar S. absoluta larvae. Relative toxicities were evaluated by LD₅₀ determinations. The entomocidal activity of isolate 121e, an autoagglutinating strain, was threefold higher than toxin synthesized by B. thuringiensis var. kurstaki . This native strain was also active against Culex pipiens larvae, although much less than towards S. absoluta .     

Two types of entomocidal crystals of Bacillus thuringiensis ssp. finitimus have the same set of unique delta-endotoxins

The strain B-1166 differs from the other strains of Bacillus thuringiensis ssp. finitimus because it has two crystal types with different localization in the sporulating cell, i.e., inside and outside of exosporium membrane. Two dissociants of the strain were obtained containing only one of the crystal types. The initial strain produces at least three various delta-endotoxins (Fin2, Fin3, and Fin5) differing from all other known entomocidal proteins; Fin2 and Fin3 are similar to each other but differ from Fin5. Both crystal types contain the same endotoxins (Fin2, Fin3, and Fin5). In the B-1166 strain the site of crystal deposition is not determined by their protein composition.     

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.     

The role of bitoxibacillin components in the manifestation of its biological activity

New methods were developed and applied to quantitative determination of beta-exotoxin and antibiotic activity of delta-endotoxin with respect to Micrococcus spp. in bitoxibacillin (BTB) and the fermentation broths prepared under industrial conditions. The biosynthesis of beta-exotoxin in the period of its maximum accumulation during the fermentation was estimated. It was shown that the primary biological effect of BTB on insects consisted in the actions of beta-exotoxin and delta-endotoxin. Biological activity of each of the entomocidal components of the entomocidal components of BTB did not practically correlated with the number of viable spores. There was a correlation between the antibiotic activity of crystalline B. thuringiensis subsp. thuringiensis solutions and the insecticidal activity of the entomopathogenic preparations. Determination of beta-exotoxin and antibiotic activity of delta-endotoxin might be used as a complex procedure for testing the quality of BTB. The method for estimating antibiotic activity of the crystal solutions allowed one to assay the biological activity of other preparations based on Bacillus thuringiensis non-synthesizing beta-exotoxin.     

Cloning and expression of an entomocidal protein gene from Bacillus thuringiensis galleriae toxic to both lepidoptera and diptera

Abstract A gene encoding a 61 kDa entomocidal (P2) protein from Bacillus thuringiensis galleriae was cloned in Escherichia coli using oligonucleotide probes corresponding to N- and C-terminal DNA sequences of a Kurstaki P2 gene. When the gene of a 5.8 kb Hin dIII fragment was transformed into B. subtilis on a shuttle vector, sporulation was completely inhibited and expression could not be detected. When B. megaterium was transformed with the same plasmid, only 10% of the cells sporulated and a 61 kDa P2 protein which cross-reacted with kurstaki P2 antiserum was synthesised. Cell lysates of the transformed B. megaterium were found to be toxic to both lepidopteran and dipteran larvae.     

Screening of the insecticidal activity of Bacillus thuringiensis strains against Lygus hesperus (Hemiptera: Miridae) nymphal population

Lygus hesperus Knight (Hemiptera: Miridae) is an economically important insect pest controlled primarily by chemical pesticides. Bacillus thuringiensis Berliner is a gram-positive bacterium that has been developed for the control of some insect pests in the orders Lepidoptera, Coleoptera, and Diptera. In this study, whole culture extracts of 94 B. thuringiensis strains from 83 serovars were added to an artificial diet and assayed against L. hesperus first and second instars. A total of five B. thuringiensis strains, B. thuringiensis variety thuringiensis, thuringiensis exotoxin +, morrisoni, tolworthi, and darmstadiensis generated > 98% mortality after 7 d of incubation. The screening was repeated with 117 alkali-solubilized trypsin-digested B. thuringiensis cultures and the same five B. thuringiensis strains showed nearly identical results. All five strains produce beta-exotoxin, which exhibits a wide host spectrum activity. No beta-exotoxin-minus B. thuringiensis strains showed significant toxicity against L. hesperus nymphs. The present work is one of the first thorough screenings of the wide diversity of the B. thuringiensis varieties for the control of L. hesperus nymphal populations.     

Fed-batch culture of Bacillus thuringiensis for thuringiensin production in a tower type bioreactor

Fed-batch culture of Bacillus thuringiensis in a modified airlift reactor has been developed by using adaptive control of glucose concentration in the reactor. The glucose concentration was estimated via a correlation equation between carbon dioxide production rate and glucose consumption rate. The estimated glucose concentration as the output variable was fed back to computer for calculation of substrate addition. The modified reactor was an airlift reactor with a net draft tube. The airlift reactor had high oxygen transfer rate and low shear stress which were important factors for production of thuringiensin. Fed-batch culture of Bacillus thuringiensis in the modified airlift reactor provided significant improvement of thuringiensin production. (c) 1995 John Wiley & Sons, Inc.     

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.     

Pathogenicity of intrathoracically administrated Bacillus thuringiensis spores in Blatta orientalis

The ability of Bacillus thuringiensis to produce septicaemia in Periplaneta americana and Blatta orientalis has been investigated. Spores and crystals from several wild-type strains as well as spores of a B. thuringiensis crystal-deficient mutant, were first orally administrated at high doses, and no significant mortality was recorded. Intrathoracic injection of spore suspensions in P. americana revealed that this species is not very susceptible to B. thuringiensis spores. B. orientalis, by contrast, was found to be very susceptible to B. thuringiensis, with a LD(50) of about 35,000 spores, that is similar to that reported on Lepidoptera challenged with parenterally injected B. thuringiensis.