Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

An index for determining the culture time of inoculum in cultivation of Bacillus thuringiensis

The intensity of motility of Bacillus thuringiensis was used to determine the optimal culture time of the inoculum being used for thuringiensin production. Thuringiensin production with an inoculum taken at its maximum motility was about 3 times higher than that with an inoculum taken in the late exponential phase.     

On the Adaptive Nature of the Dissociation Process in Bacillus thuringiensis

The process of dissociation into variants that differ in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40°C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.     

Distribution of Bacillus thuringiensis strains in Southern Sweden

Bacillus thuringiensis strains were found to be naturally present in the soils of southern Sweden, being isolated from nine out of 12 sites examined. Forest soil samples were more rich in B. thuringiensis strains than soil samples collected from cultivated areas. A wide diversity of B. thuringiensis strains, representing different biochemical groups, was isolated; samples from Aspö and Fogdö regions showed the highest degree of diversity.R. Landén and M. Bryne are with the Department of Microbiology, Stockholm University, S-10691 Stockholm, Sweden. A. Abdel-Hameed is with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt. A. Abdel-Hameed's present address is the Department of Applied Chemistry and Microbiology, PO Box 27, Viikki, Building B, SF-00014 University of Helsinki, Helsinki, Finland     

Bacillus thuringiensis: ecology,the significance of natural genetic modification,and regulation

Legislation and regulation in respect of genetically modified microorganisms must be based on rick-assessment principles rather than on the current European concept of pathogenicity, which is a measure of hazard. Natural genetic modification has commerical attractions where the processes can be exempted from regulation. Bacillus thuringiensis is used to illustrate this in the context of the European Directives and UK regulation.     

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera)

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.     

Developmental polymorphism: a major factor for understanding sublethal effects of Bacillus thuringiensis

Developmental polymorphism in Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae) was induced by Bacillus thuringiensis (Berliner) in the laboratory. The B.t. formulation concentration induced a quadratic response on the incidence of developmental polymorphism. A double application of Bacillus thuringiensis at fourth and fifth instar respectively resulted in a higher occurrence of developmental polymorphism than a single application at fifth instar. Smaller larval weight prior to B.t. exposure resulted in higher incidence of developmental polymorphism. However, larvae exhibiting developmental polymorphism exhibited larger pupae and an extended larval period. Thus, developmental polymorphism and Bacillus thuringiensis ingestion together prolonged the development time but induced opposite effects on pupal weight. These findings suggest that the calculation of sublethal effects on pupal weight and development time that include developmental polymorphism as an uncontrolled source of variation could lead to an inadequate understanding of insect response in any stress-induced experiments.     

Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae)

In this study, interactions between Cry1Ac, a toxic crystal protein produced by Bacillus thuringiensis (Berliner), and Beauveria bassiana on the mortality and survival of Ostrinia furnacalis was evaluated in the laboratory. The results showed that Cry1Ac is toxic to O. furnacalis. Not only were larval growth and development delayed, but pupation, pupal weight and adult emergency also decreased when larvae were fed on artificial diet containing purified Cry1Ac toxin. When third instars O. furnacalis were exposed to combination of B. bassiana (1.8 × 10⁵, 1.8 × 10⁶ or 1.8 × 10⁷ conidia ml⁻¹) and Cry1Ac, (0.2 or 0.8 μg g⁻¹), the effect on mortality was additive, however, the combinations of sublethal concentrations showed antagonism between Cry1Ac (3.2 or 13 μg g⁻¹) and B. bassiana (1.8 × 10⁵ or 1.8 × 10⁶ conidia ml⁻¹). When neonates were reared on sublethal concentrations of Cry1AC until the third instar, and survivors exposed B. bassiana conidial suspension, such treatments showed additive effect on mortality of O. furnacalis except for the combination of Cry1Ac (0.2 μg g⁻¹) and B. bassiana (1.8 × 10⁶ conidia ml⁻¹) that showed antagonism.     

Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins

Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred into corn plants (event MON 89034) for controlling lepidopteran pests. This new generation of Bt corn with stacked-genes of Cry1A.105 and Cry2Ab2 will become commercially available in 2009. Susceptibility of Cry1Ab-susceptible and -resistant strains of D. saccharalis were evaluated on four selected Bt proteins including Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. The Cry1Ab-resistant strain is capable of completing its larval development on commercial Cry1Ab-expressing corn plants. Neonates of D. saccharalis were assayed on a meridic diet containing one of the four Cry proteins. Larval mortality, body weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded after 7 days. Cry1Aa was the most toxic protein against both insect strains, followed in decreasing potency by Cry1A.105, Cry1Ac, and Cry2Ab2. Using practical mortality (larvae either died or no significant weight gain after 7 days), the median lethal concentration (LC₅₀) of the Cry1Ab-resistant strain was estimated to be >80-, 45-, 4.1-, and −0.5-fold greater than that of the susceptible strain to Cry1Aa, Cry1Ac, Cry1A.105 and Cry2Ab2 proteins, respectively. This information should be useful to support the commercialization of the new Bt corn event MON 89034 for managing D. saccharalis in the mid-southern region of the United States.     

Toxicity of several delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain

Toxicity and larval growth inhibition of 11 insecticidal proteins of Bacillus thuringiensis were evaluated against neonate larvae of Helicoverpa armigera, a major pest of important crops in Spain and other countries, by a whole-diet contamination method. The most active toxins were Cry1Ac4 and Cry2Aa1, with LC50 values of 3.5 and 6.3 microg/ml, respectively. At the concentrations tested, Cry1Ac4, Cry2Aa1, Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1, produced a significant growth inhibition, whereas Cry1Aa3, Cry1Ca2, and Cry1Ea had no effect.     

Influence of pH Control Agents on Entomotoxicity Potency of Bacillus thuringiensis using Different Raw Materials

Summary In this investigation, ammonium hydroxide and acetic acid were used as pH control agents during Bacillus thuringiensis (Bt) fermentation in a pilot scale fermentor (150-l) employing two secondary wastewater sludges from two different wastewater treatment plants (CUQS and JQS) and semi-synthetic soybean meal medium as raw materials. Regardless of the cultivation medium, a substantial increase in total cell count, spore count, protease activity and entomotoxicity was achieved when the pH of the culture was controlled using NH₄OH/CH₃COOH. At harvest, total cell count increased by almost 17%, 33% and 25%; protease activity was enhanced by 12%, 33% and 53% and maximal spore count augmented by almost 28%, 48% and 33% in CUQS, JQS and soybean medium, respectively. Entomotoxicity potency was improved by 22%, 21% and 14% in CUQS, JQS and soybean medium, respectively compared to results obtained with NaOH/H₂SO₄ as pH control agents. A higher entomotoxicity was also observed using sludge compared to the soybean medium. This improvement of the Bt process performance was a consequence of the addition of rapidly utilizable carbon and nitrogen source through pH control, which stimulated endotoxin production in the crystal and enhanced sporulation.     

Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton

The susceptibilities of the major pests of cotton in Australia, Helicoverpa armigera and Helicoverpa punctigera, to some insecticidal proteins from Bacillus thuringiensis were tested by bioassay. A commercial formulation, DiPel, and individual purified insecticidal proteins were tested. H. armigera was consistently more tolerant to B. thuringiensis insecticidal proteins than was H. punctigera, although both were susceptible to only a limited range of these proteins. Only Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Vip3A killed H. armigera at dosages that could be considered acceptable. There was no significant difference in the toxicities of Cry1Fa and Cry1Ac for H. punctigera but Cry1Fa had little toxicity for H. armigera. The five instars of H. armigera did not differ significantly in their susceptibility to DiPel on the basis of LC(50). However, there were significant differences in the susceptibility to Cry1Ac and Cry2Aa of three strains of H. armigera. Bioassays conducted with Cry1Ac and Cry2Aa showed that there was a small but significant negative interaction between these delta-endotoxins.     

Production of thuringiensin from Bacillus thuringiensis using a net-draft-tube,modified airlift reactor

A net-draft-tube, modified airlift reactor and a stirred-tank reactor were used for thuringiensin production by Bacillus thuringiensis subsp. darmstadiensis growing with various concentrations of molasses. The optimum concentration of molasses for thuringiensin production in both reactors was 15 g/l. There was a 6 h delay in sporulation in the modified airlift reactor compared with that in the stirred-tank reactor. Thuringiensin yield in the modified airlift reactor (2.2 g/l) was consequently higher than that in the stirred-tank reactor (1.1 g/l).     

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     

Influence of the Chitosan Oligomer on the Phage Particles and Reproduction of Phage 1-97A in the Culture of Bacillus thuringiensis

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensissubsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the basal plate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain

Bacillus thuringiensis is a spore-forming bacterium showing the unusual ability to produce endogenous crystals during sporulation that are toxic for some pest insects. This work was performed to study the composition, ecological distribution and insecticidal activity of isolates of this entomopathogenic bacterium from the Spanish territory. Using a standard isolation method, B. thuringiensis was isolated from 115 out of 493 soil samples collected in the Iberian Peninsula and the Canary and Balearic Archipelagos. The percentages of samples with B. thuringiensis were 31.7, 27.6 and 18.5 and the B. thuringiensis index 0.065, 0.067 and 0.11 for the Iberian Peninsula, Canary and Balearic Archipelagos, respectively. The prairies were shown to be the worst source of B. thuringiensis while forests, urban and agricultural habitats showed similar percentages. Strain classification based on H-antigen agglutination showed a great diversity among the Spanish isolates, which were distributed among 24 subspecies, including three new ones andaluciensis, asturiensis and palmanyolensis. We differentiated 65 different protein profiles of spore-crystal mixtures by sulfate-polyacrylamide gel electrophoresis and we selected 109 isolates representative of these profiles to evaluate their insecticidal activity against insects from the Orders Orthoptera, Dictyoptera, Coleoptera, Lepidoptera and Diptera. We found variable percentages of isolates active against Coleoptera and Lepidoptera, one isolate highly active against mosquito larvae and for the first time, three isolates active against cockroaches and locusts.     

Effects of individual Bacillus thuringiensis insecticidal crystal proteins on adult Heliothis virescens (F.) and Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Bacillus thuringiensis (Bt) is a grampositive, spore forming bacterium, which is principally distinguishedfrom other bacilli by the production of large, insecticidal,protein crystals (Insecticidal Crystal Proteins, or ICPs). Theseproteins are usually thought to act only on the actively feedinglarvae of susceptible species by a mechanism which involvesconsumption and proteolytic processing of the protein followed bybinding to, and lysis of, midgut epithelial cells. However, few authorshave reported Bt toxicity to adult insects. In the followingpaper, we expand on previous reports of toxicity to adult insects andpresent data which demonstrate that: (1) proteolytically activated ICPssignificantly reduce the lifespans of adult Heliothis virescensand Spodoptera exigua at concentrations of 500 g/ml, butnot 167 or 25 g/ml, (2) individual activated ICPs are differentiallytoxic to adult H. virescens and S. exigua, and (3)adult S. exigua are sensitive to Cry1C protoxin at aconcentration of 1 mg/ml.Deceased     

Studies on Bacillus thuringiensis strains isolated from Swedish soils: insect toxicity and production of B. cereus-diarrhoeal-type enterotoxin

At moderate concentration, 23 of 40 strains of Bacillus thuringiensis isolated from Sweden were toxic to Trichoplusia ni and five were toxic to Aedes aegypti. Five of the strains were toxic to Diabrotica undecimpunctata at high concentration, two were toxic to Heliothis virescens at low concentration and five produced thuringiensin (formerly called -exotoxin). No strain was toxic towards the beet armyworm Spodoptera exigua at low concentration. Twenty-three of the strains produced a B. cereus-diarrhoeal-type enterotoxin.A. Abdel-Hameed is with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt. R. Landén is with the Department of Microbiology, Stockholm University, S-10691 Stockholm, Sweden. A. Abdel-Hameed's present address is the Department of Applied Chemistry and Microbiology, P. O. Box 27, Viikki, Building B, FIN-00014 University of Helsinki;     

Evaluation of Bacillus thuringiensis bioinsecticidal protein effects on soil microorganisms

The effect of B. thuringiensis and its crystal protein on plant growth and on functional groups of microorganisms is not well understood. Soybean (Glycine max) var. Br 322 was grown in non-sterile soil infested with three B. thuringiensis (Bt) inocula: insecticidal crystal protein producer (Cry+), a mutant non-producer (Cry–), or insecticidal crystal protein (ICP), at a rate of 10⁷ cells g^–1 dry soil or 1.25 mg of protein g^–1 dry soil. Non-inoculated plants were maintained as control. Measurements were carried out on soil samples before sowing (time zero) and after sowing and inoculation (5, 15, 25, 35 and 45 d) on samples of rhizosphere soil. The effect of spore and crystal protein produced by B. thuringiensis on the populations of functional groups of microorganisms (bacteria including actinomycetes and fungi) involved in the biogeochemical cycling of carbon (cellulolytic, amylolytic and proteolytic), phosphorus (arbuscular mycorrhizal fungi), and nitrogen (number of nodules and proteolytic) were evaluated. Population sizes of culturable heterotrophic bacteria and saprophytic fungi were also evaluated. No difference was found in heterotrophic bacterial populations inoculated with B. thuringiensis. Difference was observed in functional groups of C-cycling microorganisms. Nodule formation and plant growth were increased by Cry+ strain and ICP when compared with uninoculated plants. Crystal protein did not show any effect on arbuscular mycorrhiza (AM) colonization. However, a deleterious effect was observed with Cry+ and Cry– strains that inhibited colonization of AM fungi when compared with uninoculated plants.