Development of a cost-effective medium for the large scale production of Bacillus thuringiensis var israelensis

Bacillus thuringiensis var israelensis (B.t.i.) has been widely used in mosquito control programs, but the large scale production of this bacillus is expensive because of the high cost of the medium. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw materials including soybean flour (Glycine max), groundnut cake powder (Arachis hypogea), and wheat bran extract (Triticum aestivum) by using 100-L fermentor. Sporulation, toxicity, and biomass were satisfactory after B.t.i. was produced on all the three media. Use of the soybean culture medium resulted in maximum toxicity (LC₅₀ 8.89 ng/ml against Culex quinquefasciatus IIIrd instar larvae), highest spore count (0.48 × 10¹¹ c.f.u./ml), and maximum biomass (7.8 g/L) within a short fermentation time of 24 h. Hence, this soybean-based culture medium was considered most economical for the large scale industrial production of B.t.i.     

The use of coconut wastes for the production ofBacillus thuringiensis var.israelensis

Summary Good yields of crystal/spores and larvicidal activity were obtained whenBacillus thuringiensis var.israelensis was grown in coconut water and endosperm extracts. Coconut water contained 2.2 mg protein/ml, while the endosperm extracts contained 1–4 mg protein/ml extract. The carbohydrate content of the media ranged from 6–38.4 mg/ml.

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Resumen Utilización de desechos de coco para la producción de Bacillus thuringiensisvar. israelensis Agua de coco y extractos de endosperma se han utilizado para el cultivo deBacillus thuringiensis var.israelensis obteniendo un buen rendimiento en cristales/esporas y una buena actividad larvicida. El agua de coco contenía 2.2 mg de proteína/ml y los extractos de endosperma 1–4 mg/protei'na/ml de extracto. El contenido en hidratos de carbono de los medios utilizados varió entre 6–38.4 mg/ml.

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Résumé Utilisation des déchets de noix de coco pour la production de Bacillus thurigiensisvar. israelensisDe bons rendements en cristaux, spores et activité larvicide ont été obtenus en cultivantBacillus thurigiensis var.israelensis dans l'eau et les extraits endospermiques de noix de coco. L'eau des noix contient 2,2 mg de protéines/ml et les extraits d'endosperme 1–4 mg. La teneur des milieux en carbohydrates était comprise entre 6–38,4 mg/ml.

     

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. Part of the work was carried out at Biotechnology Programme, Jadavpur University, Calcutta 700 032, India.     

DNA homology between the crystal toxin genes of several mosquito pathogenicBacillus thuringiensis strains

With the recombinant pVB131 plasmid, which encodes the mosquitocidal 130 kilodalton peptide ofBacillus thuringiensis var.israelensis as a probe, DNA homology between crystal toxin genes of several dipteran-toxic strains was tested. Results from this study indicate that, while the crystal toxin genes ofB. thuringiensis var.kyushuensis and var.morrisoni isolate PG-14 share homology to the crystal toxin gene of var.israelensis, the -endotoxin genes of other dipteran-active strains tested (i.e., var.colmeri and var.kurstaki) do not exhibit any homology. The crystal toxin genes of vars.kyushuensis andmorrisoni isolate PG-14 were found to be located on plasmids of 60 and 94 megadaltons, respectively.     

Structural features of crystal-forming proteins produced by Bacillus thuringiensis subspecies israelensis

Entomocidal crystals produced by Bacillus thuringiensis ssp. israelensis are formed by two proteins with molecular masses of 130 and 28 kDa, whereas the protein with a molecular mass of 70 kDa appears as a result of 130 kDa protein limited proteolysis by admixtures of bacterial proteinases in the course of its dissolution. The comparison of the N-terminal sequences of the protein with molecular mass of 70 kDa (Met-Glu-Asn-Xaa-Pro-Leu-Asp-Thr-Leu-Ser-Ile-Val-Asn-Glu-Thr-Asp) and that of 28 kDa (Met-Glu-Asn-Leu-Asn-[Phe]-[Trp]-Pro-Leu-Gln-Asp-Ile-Lys-Val-Asn-Pro) reveals only marginal similarity between them (only 4 identical residues among 16 aligned). Both B. thuringiensis israelensis crystal-forming proteins appear hardly related to those contained in the crystal produced by other B. thuringiensis subspecies, e.g. kurstaki. It might be concluded that at least some of the entomocidal proteins found in the crystalline inclusion bodies of various B. thuringiensis subspecies revealed rather strong variations in their primary structures that facilitate their adaptation to different hosts.Bacillus thuringiensis ssp. israelensis δ-EndotoxinEntomocidal crystalInsecticideMosquito     

Hyper-Production of Insecticidal Crystal Protein (δ-Endotoxin)by Bacillus thuringiensis var. israelensis Is Not Related toSporulation-Specific Biochemical Functions

Hypertoxic mutant strains of Bacillus thuringiensis var. israelensis were isolated by mutagenesis of the parent strain. The correlation, if any, between hyper-production of insecticidal crystal protein (δ-endotoxin) by hypertoxic mutant strains of Bacillus thuringiensis var. israelensis and sporulation-specific biochemical functions was studied. No increase in sporulation-specific biochemical markers was observed in the hypertoxic mutant strains. Asporogenous mutants of hypertoxic mutant strains blocked at different stages of sporulation were isolated, and larvicidal activity was studied. The hypertoxic parent strains and the sporulation-deficient, hypertoxic mutant strains showed almost identical larvicidal activity. Therefore, the increased production of toxin is not related to sporulation-specific biochemical changes. Received: 14 February 2000 / Accepted: 21 March 2000     

Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis

Summary The gene (cytA) coding for the 27 kDa polypeptide of the Bacillus thuringiensis var. israelensis mosquito larvicidal -endotoxin, was cloned into a plasmid containing the T7 bacteriophage promoter. The plasmid was used to transform an Escherichia coli strain containing the T7 RNA polymerase gene 1, under the control of lacP. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of transformed cells. The cytA gene product may kill E. colicells by disrupting their chromosome replicating apparatus.     

Individual and combined effects of Bacillus thuringiensis var. israelensis, temephos and Leptolegniachapmanii on the larval mortality of Aedes aegypti

Larvicidal effects of interaction between Bacillus thuringiensis var. israelensis (Bti), temephos and Leptolegnia chapmanii zoospores on larvae of Aedes aegypti were determined under laboratory and seminatural conditions. In laboratory bioassays, two concentrations of Bti (0.012, 0.027 ppm), two of temephos (0.00035, 0.001 ppm), and a single concentration of L. chapmanii zoospores (6.1 × 10⁴ zoospores ml⁻¹) were evaluated. Trials under field-like conditions were performed in a single container and then placed either in the shade or in direct exposure to sunlight. We evaluated concentrations of Bti and temephos at 3-fold those normally used in laboratory tests: 0.09 and 0.003 ppm, respectively, plus 1.8 × 10⁵ zoospores ml⁻¹ of L. chapmanii. The combined effect of sublethal concentrations of Bti, temephos, and L. chapmanii zoospores thus indicated that this fungus is not inhibited by the larvicides and also demonstrated the synergistic effect of the action of L. chapmanii when used together with Bti and temephos.     

Un nouveau candidat a la lutte biologique contre les moustiques:Bacillus thuringiensis var.Israelensis

Résumé Le nouveau sérotype H₁₄ individualisé dans le groupe desBacillus thuringiensis Berliner, sous le nom de variétéisraelensis est un pathogène puissant et essentiel des larves de moustiques. Son pouvoir larvicide n'a aucun équivalent chez les autres sérotypes connus deB. thuringiensis et appara?t très compétitif avec celui deB. sphaericus. A dose forte,B. thuringiensis var.israelensis tue les larves d'Aedes aegypti L. en 20 à 30 mn et celles d'Anopheles stephensi (Liston), en 100 à 110 mn, les DL 50 en 24 h étant de l'ordre de 2,4.10⁴ spores/ml pourAedes et de 9,8.10⁴ spores/ml pourAnopheles. La toxicité deB. thuringiensis var.israelensis pour les larves de moustiques est liée à une endotoxine protéique présente dans les cristaux, de nature et de mode d'action comparables à ceux des endotoxines des autres souches deB. thuringiensis pathogènes pour les lépidoptères. L'étude histopathologique surAedes aegypti montre que l'effet primaire correspond à une désintégration de l'épithélium intestinal par gonflement, distorsion puis éclatement des cellules. L'absence d'action deB. thuringiensis var.israelensis sur les lépidoptères étudiés:Anagasta kuehniella, Z.,Plutella maculipennis Curtis etProdenia litura F., ainsi que son innocuité ?per os? pour les mammifères tendent à prouver une certaine spécificité pour les diptères. Toutes ces qualités, jointes à la parfaite connaissance du groupethuringiensis découlant de sa longue utilisation en forêts et en cultures, devraient faire de la variétéisraelensis un candidat préférentiel pour la lutte biologique contre les moustiques.

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Summary The new serotype 14 which has been discovered in theBacillus thuringiensis Berliner group and named varietyisraelensis is a major pathogen for mosquito larvae. Its larvicidal power has been found without any equivalence in comparison with the other knownB. thuringiensis serotypes, and very competitive with the larvicidal activity ofBacillus sphaericus. At high doses,B. thuringiensis var.israelensis kills theAedes aegypti L. larvae in 20 to 30 mn, and theAnopheles stephensi (Liston) larvae in 100 to 110 mn. The DL₅₀ in 24 h are about 2,4.10⁴ spores/ml forA. aegypti and 9,8.10⁴ spores/ml forA. stephensi. The toxicity ofB. thuringiensis var.israelensis for mosquito larvae is linked with a proteic endotoxin in its crystals, the nature and mode of action of which look like these ones of the otherB. thuringiensis strains, pathogens for lepidoptera larvae. The histopathological study onA. aegypti has shown that the primary action consists in the loss of integrity of the gut epithelium, as a result of the swelling, distortion and finally bursting of the cells. The lack of activity ofB. thuringiensis var.israelensis on the tested Lepidoptera:Anagasta kuehniella Z.,Plutella maculipennis Curtis andProdenia litura F. and its innocuity “per os” for mammals lead to suggest some specificity for Diptera. All these qualities, which are enhanced by the detailed knowledge ofB. thuringiensis group coming from its long practical use on large scale in forests and cultures, ought to put theisraelensis variety as a preferential candidate for the biological control of mosquitoes.

     

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene

Summary A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato bectle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73115 dalton protein and a flat, diamond-shaped crystal composed of a protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.     

A method for identifying colonies of bacteria possessing mosquito larvicidal effects

Summary The method described provides a new, relatively rapid tool for the tracing or identification of B. thuringiensis var. israelensis among indigenous bacteria and even closely related variants of B. thuringiensis.     

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 (St^R)-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 St^R Spo⁺ Cry⁺ (streptomycin sensitive sporogeneous crystalliferous) and St^RR Spo⁺ Cry⁻ and also between St^s Spo⁺ Cry⁺ and St^R Spo⁻ Cry⁻ strains. StR colonies were selected. Insect toxicity was exhibited by the St^R isolates in both the cases. The process of crystal formation is, therefore, independent of early sporulative events.     

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

Summary The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10^–9 to 9.8×10^–5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10^–9 to 3.7×10^–7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

Construction of a bifunctional genetically labelled plasmid for Bacillus thuringiensis subsp. israelensis

A small cryptic plasmid of Bacillus thuringiensis subsp. israelensis was labelled in vitro with two genetic markers. One of the recombinant plasmids was mapped and transformed in Escherichia coli, Bacillus subtilis and Bacillus thuringiensis. This and similar shuttle plasmids could be very useful as vectors for the investigation of the toxin genes in their own host.Abbreviations BTI Bacillus thuringiensis subsp. israelensis - MDal megadaltons     

Comparison of the activities of Bacillus thuringiensis mosquitocidal crystal proteins on mosquito cell lines (Diptera,Culicidae), using two colorimetric methods

Two colorimetric methods were compared for assaying the cell toxicity of soluble crystal proteins from five mosquitocidal serovarieties of Bacillus thuringiensis: israelensis, jegathesan, medellin, darmstadiensis and fukuokaensis. In mosquitoes cell lines from Culex quinquefasciatus, Aedes aegypti and Anopheles gambiae, all the crystal proteins were equally toxic. Both colorimetric methods MTT (tetrazolium salt) and Alamar Blue gave similar results, but the Alamar Blue method was simpler, faster, cheaper, and could be used to take readings from the same cell population at various time points. The most active crystal proteins were those of B. thuringiensis israelensis, followed by those of B. thuringiensis jegathesan, and B. thuringiensis medellin, which were much less active.     

Structure and function of the Bacillus thuringiensis protein crystal

The 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.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

Laser-diffusion measurements on δ-endotoxin crystals from Bacillus thuringiensis var. kurstaki HD1

Laser light scattering has been employed to investigate changes in the hydrodynamic properties of δ-endotoxin crystals of Bacillus thuringiensis var. kurstaki HD1. Crystals are polydisperse. Estimates of the mean hydrodynamic diameter agree with those obtained by light microscopy. Sodium dodecyl sulphate (SDS) at a final concentration of 1% results in swelling of the toxin crystals. Dithiothreitol-induced solubilization of the swollen crystals indicates the importance of disulphide bridges to the maintenance of the assembled crystal.     

An engineered Bacillus thuringiensis strain with insecticidal activity against Scarabaeidae (Anomala corpulenta) and Chrysomelidae (Leptinotarsa decemlineata and Colaphellus bowringi)

A genetically-engineered Bacillus thuringiensis (Bt) strain, 3A-HBF, with a broad insecticidal spectrum was constructed by introducing the recombinant plasmid pSTK-3A containing cry3Aa7 into the wild-type Bt strain HBF-1 containing the cry8Ca2 gene. The Cry3Aa7 protein produced by strain 3A-HBF was verified by SDS-PAGE and Western blotting. Flat rectangular crystals of Cry3Aa7 protein were observed besides spherical crystals (Cry8Ca2). The plasmid pSTK-3A was stable when strain 3A-HBF was grown in medium without antibiotics. The growth rate of 3A-HBF was not significantly different from that of the recipient strain, HBF-1. Strain 3A-HBF showed toxicity against two families of pests, Scarabaeidae and Chrysomelidae pests, which are susceptible to Cry8Ca (Anomala corpulenta) and Cry3Aa (Leptinotarsa decemlineata and Colaphellus bowringi). The 50% lethal concentrations of 3A-HBF against A. corpulenta, L. decemlineata and C. bowringi were 0.730 × 10⁸ c.f.u./g dry soil, 1.74 μg/ml and 1.15 μg/ml, respectively.