Plasmid Transfer between Bacillus thuringiensis subsp. israelensis Strains in Laboratory Culture, River Water, and Dipteran Larvae

Plasmid transfer between strains of Bacillus thuringiensis subsp. israelensis was studied under a range of environmentally relevant laboratory conditions in vitro, in river water, and in mosquito larvae. Mobilization of pBC16 was detected in vitro at a range of temperatures, pH values, and available water conditions, and the maximum transfer ratio was 10⁻³ transconjugant per recipient under optimal conditions. Transfer of conjugative plasmid pXO16Tn5401 was also detected under this range of conditions. However, a maximum transfer ratio of 1.0 transconjugant per recipient was attained, and every recipient became a transconjugant. In river water, transfer of pBC16 was not detected, probably as a result of the low transfer frequency for this plasmid and the formation of spores by the introduced donor and recipient strains. In contrast, transfer of plasmid pXO16Tn5401 was detected in water, but at a lower transfer ratio (ca. 10⁻² transconjugant per donor). The number of transconjugants increased over the first 7 days, probably as a result of new transfer events between cells, since growth of both donor and recipient cells in water was not detected. Mobilization of pBC16 was not detected in killed mosquito larvae, but transfer of plasmid pXO16::Tn5401 was evident, with a maximum rate of 10⁻³ transconjugant per donor. The reduced transfer rate in insects compared to broth cultures may be accounted for by competition from the background bacterial population present in the mosquito gut and diet or by the maintenance of a large population of B. thuringiensis spores in the insects.     

Plasmid transfer between the Bacillus thuringiensis subspecies kurstaki and tenebrionis in laboratory culture and soil and in lepidopteran and coleopteran larvae

Plasmid transfer between Bacillus thuringiensis subsp. kurstaki HD1 and B. thuringiensis subsp. tenebrionis donor strains and a streptomycin-resistant B. thuringiensis subsp. kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid transfer was detected in vitro at temperatures of 5 to 37 degrees C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10(-1) transconjugant/donor) were detected within 4 h. In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed. When a B. thuringiensis subsp. kurstaki strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae. When a B. thuringiensis subsp. tenerbrionis strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B. thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment.     

Characterization of Tunisian Bacillus thuringiensis Strains with Abundance of kurstaki Subspecies Harbouring Insecticidal Activities Against the Lepidopteran Insect Ephestia kuehniella

The study of 257 crystal-producing Bacillus thuringiensis isolates from bioinsecticide free soil samples collected from different sites in Tunisia, was performed by PCR amplification, using six primer pairs specific for cry1, cry2, cry3, cry4, and vip3A genes, by the investigation of strain plasmid pattern, crystal morphology and delta-endotoxin content and by the assessment of insecticidal activities against the lepidopteran insect Ephestia kuehniella. Based on plasmid pattern study, 11 representative strains of the different classes were subjected to morphological and molecular analyses. The comparison of the PFGE fingerprints confirmed the heterogeneity of these strains. B. thuringiensis kurstaki strains, harbouring at the same time the genes cry1A, cry2, cry1Ia, and vip3A, were the most abundant (65.4%). 33.34% of the new isolates showed particular delta-endotoxin profiles but no PCR products with the used primer sets. B. thuringiensis israelensis was shown to be also very rare among the Tunisian B. thuringiensis isolates diversity. These findings could have considerable impacts for the set up of new pest control biological agents.     

Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays

The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays.     

Intergeneric protoplast fusion between agrobacterium tumefaciens and Bacillus thuringiensis subsp. kurstaki

Summary Intergeneric protoplast fusion betweenA.tumefaciens andB.thuringiensis was performed. The fusants exhibited some properties of both the parental strains. One of the Gram positive fusants with most of theBacillus properties showed tumor inducing capacity in pigeonpea (Cajanas cajan).     

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.     

Physical Mapping of the Bacillus thuringiensis subsp. kurstaki and alesti Chromosomes

Two strains of the well-known insect pathogen and biopesticide, Bacillus thuringiensis (Bt), belonging to subspecies alesti (strain Bt5) and kurstaki (strain Bt213), were chosen for genetic characterization. The two strains belong to different serotypes and are currently classified into different subspecies, although their insecticidal activity is similar. Physical maps were constructed of Bt alesti and Bt kurstaki using Pulsed Field Gel Electrophoreses (PFGE), and the map positions of several genes were determined. The 5.5 Mb combined genetic and physical chromosome maps of the two strains were found to be indistinguishable, and the only differences detected between the strains were of extrachromosomal origin. A cryIA toxin gene probe hybridised to a chromosome fragment and to two extrachromosomal elements in both strains, migrating as 100 kb and 350 kb, respectively. In addition a cry hybridizing extrachromosomal element migrating as 80 kb was present only in Bt alesti. Both strains were also found to contain sequences hybridizing to an enterotoxin (hbla) gene probe. Such sequences were positioned on the 350 kb extrachromosomal element, as well as on the chromosome. Received: 20 April 2001 / Accepted: 29 May 2001     

Conjugal Plasmid Transfer in Bacillus subtilis under Conditions of Soil Microcosms

Conjugal transfer of the small plasmid pUB110 betweenBacillus subtilis strains was studied under conditions of microcosms with sterile and nonsterile soil. Plasmid transfer proved to be possible after soil inoculation with vegetative partner cells or with their spores. Plasmid transfer occurred at temperatures of 30 and 22–23°C.     

Facile autoplast generation and transformation in Bacillus thuringiensis subsp. kurstaki.

We describe a method for maximizing the rate of conversion of Bacillus thuringiensis subsp. kurstaki vegetative cells to osmotically fragile forms in the absence of exogenously added enzymes. Optimal generation of autoplasts occurred in 50 mM sodium acetate buffer (pH 7.0) at 37 degrees C with 10% (wt/vol) polyethylene glycol as an osmotic stabilizer. The maximum autolytic rate resulted in a conversion of greater than 90% of bacilli to spherical autoplasts in 6 min. Autoplasts regained bacillary morphology upon plating on DM3-G regeneration medium, with reversion frequencies ranging from 1.2 x 10(-1) to 5.3 x 10(-3). The autoplasts could efficiently take up exogenously added plasmid DNA. The presence of plasmids was verified by Southern hybridization analysis.     

Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb Toxicities to Coleopteran Larvae by a Toxin-Binding Fragment of an Insect Cadherin

The Cry3Aa and Cry3Bb insecticidal proteins of Bacillus thuringiensis are used in biopesticides and transgenic crops to control larvae of leaf-feeding beetles and rootworms. Cadherins localized in the midgut epithelium are identified as receptors for Cry toxins in lepidopteran and dipteran larvae. Previously, we discovered that a peptide of a toxin-binding cadherin expressed in Escherichia coli functions as a synergist for Cry1A toxicity against lepidopteran larvae and Cry4 toxicity against dipteran larvae. Here we report that the fragment containing the three most C-terminal cadherin repeats (CR) from the cadherin of the western corn rootworm binds toxin and enhances Cry3 toxicity to larvae of naturally susceptible species. The cadherin fragment (CR8 to CR10 [CR8-10]) of western corn rootworm Diabrotica virgifera virgifera was expressed in E. coli as an inclusion body. By an enzyme-linked immunosorbent microplate assay, we demonstrated that the CR8-10 peptide binds α-chymotrypsin-treated Cry3Aa and Cry3Bb toxins at high affinity (11.8 nM and 1.4 nM, respectively). Coleopteran larvae ingesting CR8-10 inclusions had increased susceptibility to Cry3Aa or Cry3Bb toxin. The Cry3 toxin-enhancing effect of CR8-10 was demonstrated for Colorado potato beetle Leptinotarsa decemlineata, southern corn rootworm Diabrotica undecimpunctata howardi, and western corn rootworm. The extent of Cry3 toxin enhancement, which ranged from 3- to 13-fold, may have practical applications for insect control. Cry3-containing biopesticides that include a cadherin fragment could be more efficacious. And Bt corn (i.e., corn treated with B. thuringiensis to make it resistant to pests) coexpressing Cry3Bb and CR8-10 could increase the functional dose level of the insect toxic activity, reducing the overall resistance risk.The Cry3 class of Bacillus thuringiensis Cry proteins is known for toxicity to coleopteran larvae in the family Chrysomelidae. Cry3Aa and Cry3Bb proteins are highly toxic to Colorado potato beetle (CPB) Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), and both were used for the development of Bt crops (crops treated with B. thuringiensis to make them resistant to pests) and Bt biopesticides. Due to the limited efficacy of Cry3-based biopesticides/plants and the success of competing chemical pesticides, these biopesticides have had limited usage and sales (12). Cry3Bb is toxic to corn rootworms (8, 17), and a modified version is expressed in commercialized MON863 corn hybrids (26).Cry3 toxins have a mode of action that is similar to, yet distinct from, the action of lepidopteran-active Cry1 toxins. The Cry3A protoxin (73 kDa) lacks the large C-terminal region of the 130-kDa Cry1 protoxins, which is removed by proteases during activation to toxin. The Cry3A protoxin is activated to a 55-kDa toxin and then further cleaved within the toxin molecule (5, 18). Activated Cry3A toxin binds to brush border membrane vesicles with a K_d (dissociation constant) of ∼37 nM (19) and recognizes a 144-kDa binding protein in brush border membrane vesicles prepared from the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (2). Recently, Ochoa-Campuzano et al. (20) identified an ADAM metalloprotease as a receptor for Cry3Aa toxin in CPB larvae.Structural differences between Cry3Bb and Cry3Aa toxins must underlie the unique rootworm activities of Cry3Bb toxin. As noted by Galitsky et al. (11), differences in toxin solubility, oligomerization, and binding are reported for these Cry3 toxins. Recently, Cry3Aa was modified to have activity against western corn rootworm (WCRW) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) (27). Those authors introduced a chymotrypsin/cathepsin G cleavage site into domain 1 of Cry3Aa that allowed the processing of the 65-kDa form to a 55-kDa toxin that bound rootworm midgut.Cadherins function as receptors for Cry toxins in lepidopteran and dipteran larvae. A critical Cry1 toxin binding site is localized within the final cadherin repeat (CR), CR12, of cadherins from tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) and tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) (14, 28). Unexpectedly, a fragment of B. thuringiensis R₁ cadherin, the Cry1A receptor from M. sexta, not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (6). If the binding residues within CR12 were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist. Recently, we identified a cadherin from mosquito Anopheles gambiae (Diptera: Culicidae) that binds Cry4Ba toxin and probably functions as a receptor. We discovered a similar effect where a fragment of a cadherin from A. gambiae enhanced the toxicity of the mosquitocidal toxin Cry4Ba to mosquito larvae (15). Sayed et al. (22) identified a novel cadherin-like gene in WCRW and proposed this protein as a candidate Bt toxin receptor. The cadherin-like gene is highly expressed in the midgut tissue of larval stages. The encoded protein is conserved in structure relative to that of other insect midgut cadherins.In this study, we hypothesized that a fragment from a beetle cadherin that contains a putative Bt toxin binding region might enhance the insecticidal toxicities of Cry3Aa and Cry3Bb toxins. The region spanning CR8 to CR10 (CR8-10) of the WCRW cadherin (22) was cloned and expressed in E. coli. This cadherin fragment significantly enhanced the toxicities of Cry3Aa and Cry3Bb toxins to CPB and rootworms.     

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.     

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.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

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.     

Bacillus thuringiensis Cry1Ac Toxin-Binding and Pore-Forming Activity in Brush Border Membrane Vesicles Prepared from Anterior and Posterior Midgut Regions of Lepidopteran Larvae

It is generally accepted that Bacillus thuringiensis Cry toxins insert into the apical membrane of the larval midgut after binding to specific receptors, and there is evidence that the distribution of binding molecules along the midgut is not uniform. By use of the voltage-sensitive dye DiSC₃(5) and ¹²⁵I-labeled Cry1Ac, we have measured the effect of Cry1Ac in terms of permeabilization capacity and of binding parameters on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior regions of the larval midgut from two insect species, Manduca sexta and Helicoverpa armigera. The permeabilizing activity was significantly higher with BBMV from the posterior region than with the one observed in the anterior region in both insect species. Instead, ¹²⁵I-Cry1Ac bound specifically to BBMV from the two midgut regions, with no significant differences in the binding parameters between the anterior and posterior regions within an insect species. N-acetylgalactosamine inhibition patterns on pore formation and binding differed between anterior and posterior midgut regions and between species, providing evidence of a multifaceted involvement of the sugar in the Cry1Ac mode of action. The analysis of binding and pore formation in different midgut regions could be an effective method to study differences in the mode of action of Cry1Ac toxin in different species.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis.

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.