Plasmids of Bacillus thuringiensis var. galleriae strain 612

We studied Bacillus thuringiensis var galleriae, strain 612 plasmids. B. thuringiensis cells contain double-stranded plasmid DNA molecules (ranging of about 12% from total DNA content) with buoyant density 1.59 g/cm3. Plasmid DNA content was constant during the exponential and stationary phases of bacterial growth. The plasmid fractions consist of DNA molecules with molecular weights of 5.9 x 10(6), 10.0 x 10(6), and 110.9 x 10(6) daltons (pVD1, pVD2 pVD3, respectively). Endonuclease EcoRI cuts the plasmids pVD2 and pVD3 into two and four fragments, respectivelyy, but pVDI seemed to be resistent to EcoRI treatment. We found that pVD2 and pVD3 plasmids contain a common DNA fragment with the molecular weight of 6.7 x 10(6) dalton as it was shown by restriction analysis. In contrast, the same plasmids contain the common fragment with molecular weight of 7.5 x 10(6) dalton as shown by heteroduplex analysis. Plasmid pVD3 has a transposon-like structure.     

Transport of Bacillus thuringiensis var. kurstaki via fomites

The intentional and controlled release of an aerosolized bacterium provides an opportunity to investigate the implications of a biological attack. Since 2006, Los Alamos National Laboratory has worked with several urban areas, including Fairfax County, VA, to design experiments to evaluate biodefense concepts of operations using routine spraying of Bacillus thuringiensis var. kurstaki (Btk). Btk is dispersed in large quantities as a slurry to control the gypsy moth, Lymantria dispar. Understanding whether personnel and equipment pick up residual contamination during sampling activities and transport it to other areas is critical for the formulation of appropriate response and recovery plans. While there is a growing body of literature surrounding the transmission of viral diseases via fomites, there is limited information on the transport of Bacillus species via this route. In 2008, LANL investigated whether field sampling activities conducted near sprayed areas, post-spray, resulted in measurable cross-contamination of sampling personnel, equipment, vehicles, and hotel rooms. Viable Btk was detected in all sample types, indicating transport of the agent occurred via fomites.     

Occurrence of two serologically distinct groups within Bacillus thuringiensis serotype 3 ab var. kurstaki

Two groups distinguishable on the basis of crystal serology have been identified within Bacillus thuringiensis var kurstaki (serotype 3 ab). The toxicities of these two groups to Trichoplusia ni and Heliothis virescens expressed as $\text{T}\text{H}$ ratio also differed. It is suggested that serotype 3 ab be separated into two subgroups designated as K-1 and K-73.     

Degradation of the parasporal crystal produced by Bacillus thuringiensis var. kurstaki

Degradation products of the parasporal crystals of Bacillus thuringiensis var. kurstaki obtained by treatment with alkali, gut juice from larvae of Bombyx mori, and various plant and mammalian enzymes were compared for elution pattern, approximate molecular weight (MW), and toxicity. The results indicated that with alkaline treatment the most toxic extract was obtained with 0.05–0.1 M NaOH. Toxicity was found associated mainly with a protein peak of 230,000 MW although other toxic peaks were found in the tailing. Heat-treated midgut juice from larval B. mori gave similar results. After digestion of parasporal crystals with clarified midgut juice, five peaks causing toxicity and having MW of approximately 235,000, 67,000, 30,200, 5000, and 1000, respectively, were identified. Treatment of B. thuringiensis δ-endotoxin with α-chymotrypsin gave peaks causing mortality of approximate MW 235,000, 34,000, 5000, and 1000. Trypsin, pronase, carboxypeptidase, and enterokinase digests of the B. thuringiensis δ-endotoxin gave toxic components ranging from 235,000 to 30,000 MW. The protein protoxin molecules are digested to give small toxic subunits that may be of practical value for structural determinations and for molecular mode of action studies.     

Production of Bacillus thuringiensis Berliner var. kurstaki Grown in Alternative Media

Agro - industrial residues and by - products available in southeastern Brazil were used as ingredients for low - cost culture media for liquid fermentation of Bacillus thuringiensis var. kurstaki. Highest spore yield was obtained with a medium containing cheese whey , soya bean milk and molasses (WSM) . Crystals and spores were produced in all media and potency of the final product was highest for nutrient broth + yeast extract medium (NBY) . There was no correlation between the number of spores in the fermented media and the potency of the preparations . Considering all three factors , the potencies , costs and yields of the final products , lowest relative cost was obtained with BMM medium ( Bombyx mori pupae + molasses) . NBY and WSM had intermediate relative cost approximately nine times higher than BMM . The cost analysis suggests that BMM medium should be preferred for local production of B. thuringiensis var . kurstaki in comparison to other media tested . The results also demonstrate the importance of considering yields , cost and potency of the B. thuringiensis preparations in selecting the production medium .     

Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus.

DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.     

Facile preparation and characterization of the toxin from Bacillus thuringiensis var. kurstaki.

We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.     

Effects of Bacillus thuringiensis var. kurstaki δ-endotoxin on isolated lepidopteran mitochondria

An investigation was undertaken to determine the effects of Bacillus thuringiensis var. kurstaki δ-endotoxin on mitochondria isolated from Bombyx mori midgut epithelium. Using manometric and colorimetric techniques, the investigation revealed that toxic polypeptides had stimulatory effects on mitochondria oxygen uptake and inhibitory effects on ATP production. These results indicated that B. thuringiensis δ-endotoxin could act as an uncoupler of oxidative phosphorylation. Loss of ATP production caused by the action of the δ-endotoxin would lead to metabolic imbalance and possible cell death.     

Long-term survival and germination of Bacillus thuringiensis var. kurstaki in a field trial

Long-term survival, dispersal, and germination of Bacillus thuringiensis var. kurstaki DMU67R has been investigated in a field trial. An experimental cabbage plot was sprayed with DMU67R in 1993 and allowed to lie fallow since. The investigations reported here were carried out from 1997 to 2000 in this plot. High persistence of DMU67R for 7 years in the bulk soil of the plot has been demonstrated. The numbers have not significantly reduced since 1994, stabilizing around 6.6 x 10(2) cfu/g from 1996 to 2000. Horizontal dispersal of DMU67R in the 1994-1999 period was limited. Vertical dispersal occurred from 1994 to 1999, as 77% of the population of DMU67R occurred in the 0-2 cm layer in 1994, while only 22% of the population was found there in 1999. Most of the population in 1999 was present homogeneously in the upper 6 cm of the soil profile. Germination, as evidenced by the ratio of DMU67R cfu before and after heat treatment, was not observed in the bulk soil. However, in the rhizospheres of dandelion (Taraxacum officinalis) and quackgrass (Agropyron repens), 40 and 50% of DMU67R was present as vegetative germinated cells, respectively. No germination occurred in the rhizosphere of red fescue (Festuca rubra). The material from the gut of the earthworm species Lumbricus rubellus, Lumbricus terrestris, and Apporrectodea caliginosa and from a tipulid larvae from the plot also contained vegetative cells of DMU67R. Further investigations of A. caliginosa showed that germination seemed to be restricted to the gut and that sporulation occurred after defecation. The germination of DMU67R in rhizospheres and in the gut of nontarget invertebrates suggests that survival in the soil of B. thuringiensis is a dynamic process involving germination, cell divisions, and sporulation in specific microhabitats.     

Correlation between alkaline activation of Bacillus thuringiensis var. kurstaki spores and crystal production

The spores of crystal-forming (Cry⁺) and non-crystal-forming (Cry^-) strains of Bacillus thuringiensis var. kurstaki and Bacillus cereus were tested for the ability to be activated by 0.1 m K₂CO₃ (pH 10). Only the spores of crystal-forming strains could be activated, and this phenotype was independent of whether crystals were present with the spores in the activation solution. The spores of a B. thuringiensis var. kurstaki strain that is temperature sensitive for protoxin accumulation could be activated by the alkaline solution when produced at the permissive temperature, whereas spores produced at the nonpermissive temperature were not activated. The results indicate that protoxin in the spore coat is responsible for the alkaline-activation phenotype and may serve an ecological function for the organism.     

Wastewater Sludge Pre-treatment for Enhancing Entomotoxicity Produced by Bacillus thuringiensis var. kurstaki

Summary Wastewater sludge is a complex raw material for fermentation and requires pre-treatment in order to transform less biodegradable compounds into more easily degradable ones. In this study, sludge was treated by thermo-alkaline and oxidative pre-treatment methods and subjected to Bacillus thuringiensis fermentation in bench scale fermentors under controlled conditions. Thermo-alkaline treatment was found to be an effective process in order to enhance the entomotoxicity tested against spruce budworm. The total cell and spore counts were improved by 40 and 46%, respectively as compared to that using the untreated sludge. The final entomotoxicity potency increased from 12.3 × 10⁹ SBU/l of the raw sludge to 16.6 × 10⁹ SBU/l of the thermo-alkaline pre-treated sludge. The improvement of the process performance was attributed to a better oxygen transfer due to decrease in media viscosity and an improvement of nutrient availability due to the sludge solubilization and biodegradability.     

A new medium for growth and delta-endotoxin production by Bacillus thuringiensis var. kurstaki

Summary The influence of composition of media used for growth and delta-endotoxin production by B. thuringiensis var. kurstaki was studied with the idea of finding a cheap medium for attaining high yields of spore-crystal preparations. A new medium, based on malt sprouts is proposed. Data on growth and bioinsecticidal activity are given. A concentration of 2.1 × 10⁹ spores.ml^-1 was attained in a 48 h process. The spore-crystal preparations obtained present a LC₅₀ of 2.18 × 10⁸ spores.g^-1 against larvae of Galleria mellonella.     

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.     

Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R · Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.     

Behavior of Bacillus thuringiensis var. kurstaki under continuous phased cultivation in a cyclone fermentor

Summary Growth, sporulation, insecticidal crystalline protein (ICP) production and plasmids of Bacillus thuringiensis var. kurstaki (HD-1) were investigated during batch and continuous phased cultivation using a laboratory scale cyclone fermentor. When grown in batch culture at 28°C, 93% of the cells sporulated and produced ICP within 10 h of commencement of stationary phase. The batch culture runs were completed within 50 h of inoculation. A predominantly sporogenous and crystalliferous cell population was also obtained by second, stage processing of culture harvested from the first 10 to 25 cycles of continuous phased cultivation. In contrast, after 25 or more cycles of cultivation the population in the continuous phased culture shifted towards a predominantly asporogenous and acrystalliferous one. Culture conditions in continuous phased cultivation did not affect the plasmid content of B. thuringiensis (HD-1), yet influenced sporulation and plasmid-coded ICP production.     

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.     

Biological characteristics of the variants of Bacillus thuringiensis subsp. galleriae formed during continuous culture

Bacillus thuringiensis subsp. galleriae S. variants formed during continuous cultivation differ from the parent culture in certain properties. In contrast to the parent R form, their growth in the chemostat does not yield virulent mutants which can cause their lysis on solid media. The chemostat S forms are resistant against virulent phage mutants produced when the R variants are grown under the conditions of continuous cultivation and against a virulent phage obtained from the parent culture 69-6 under the action of vancomycin. The R forms are sensitive to these phages. When the S forms are grown under the chemostat conditions, they do not revert to the R forms. The R and S forms do not differ noticeably in the character of their growth, formation of spores and crystals, and biological activity.     

Evaluating a dual microbial agent biopesticide with Bacillus thuringiensis var. kurstaki and Beauveria bassiana blastospores

A biopesticide with a mixture of entomopathogenic microbial agents was studied for improvements in efficacy. Recently developed liquid fermentation techniques were used to produce blastospores of Beauveria bassiana (Balsamo) Vuillemin strain GHA (Bb), which were mixed with traditional fermentation of Bacillus thuringiensis Berliner kurstaki (Bt) spores and crystals. Based on a dosage response bioassay using Trichoplusia ni (Hübner) neonates exposed to treated leaf disks, the LC₅₀ values were determined to be 3.85?×?10⁷ spores mL^?1 for the unformulated Bt and 3.58?×?10⁷ blastospores mL^?1 for the unformulated Bb. Using the same bioassay conditions, mixtures of Bt:Bb at ratios of 1:0, 0.75:0.25. 0.5:0.5, 0.25:0.75, and 0:1 indicated that the greatest mortality was caused by the 0.5:0.5 mixture ratio of the microbial agents. Based on these results, the 0.5:0.5 mixture was formulated as a dry powder by spray drying and applied to field-grown cabbage for comparison with commercial Bt and Bb products. Overall, the mixed agent treatment caused equal or greater mortality of exposed insects when compared with commercial products. The insecticidal interaction was determined to be synergistic when combining these two microbial agents into a single product, providing greater mortality of target insects. Assuming similar fermentation costs, the combined treatment could provide improved efficacy without increasing production costs. These benefits support the concept of formulating mixed microbial agents as an environmentally friendly pest control tool.