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.     

Use of crystal serology to differentiate among varieties of Bacillus thuringiensis

Rabbit antisera were developed to 10 different Bacillus thuringiensis crystal serotypes and their cross-reactivities were compared. Results from enzyme-linked immunosorbent assay, double diffusion immunoprecipitation, and Western blot analysis suggest that the 10 crystal types share more antigenic determinants than previously reported. Results indicate that the present classification of B. thuringiensis according to crystal serotype would be more accurately reduced from the 10 types investigated to only 2. Use of crystal serology for differentiating B. thuringiensis isolates on the basis of toxicity toward insects appears useful in detecting specificity among orders, but not within genera.     

Mode of Action of Bipyramidal δ-Endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1

The mode of action of the toxic fragment (P-59) derived from bipyramidal-shaped δ-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 on the silkworm Bombyx mori was investigated. An enzyme-linked immunosorbent assay showed that there was no translocation of P-59 from the gut lumen to the hemocoel. When membrane vesicles prepared from silkworm midgut were incubated with P-59, normally smooth surface of vesicles became rough, and patch formation was observed on the surface. Vesicles treated with P-59 tended to agglutinate. The vesicle-denaturing activity of a 130,000-dalton subunit protein of bipyramidal toxin was enhanced by treatment with a gut juice protease of the silkworm. P-59 did not cause any uncoupling effect on mitochondria of the silkworm midgut. These results suggest that the attacking site of this toxin is not the mitochondrion but the cell membrane of the susceptible cell.     

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.     

Diversity analysis and characterization of Coleoptera-, Hemiptera- and Nematode-active cry genes in native isolates of Bacillus thuringiensis

Applications to combat non-lepidopteran insects are not as common as applications against lepidopteran insects. The aim of the present work was to isolate and identify Bacillus thuringiensis isolates from soil samples using five approaches, viz., analysis of crystal protein production by microscopy; detection of cry gene content by PCR, SDS-PAGE profiling; cloning and sequencing; phylogenetic analysis; and toxicity testing. Two hundred soil samples were used for isolation of B. thuringiensis and a total of 69 putative isolates of B. thuringiensis that produce parasporal crystalline inclusions were isolated from 5,267 Bacillus-like colonies. A bipyramidal inclusion was predominant in 32.2 % of the B. thuringiensis isolates compared to other shapes. Crystal protein profiling of B. thuringiensis isolates by SDS-PAGE analysis showed the presence of bands of 130, 73, 34, 25 and 13 kDa, among which 50–60 kDa bands were present abundantly. PCR analysis revealed the predominance of Coleopteran-active cry genes in these isolates. Variation in nucleotide sequences, crystal morphology and mass of crystal protein(s) purified from the isolates of B. thuringiensis revealed genetic and molecular diversity. Four strains containing Coleopteran-active cry genes showed higher toxicity against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) adults when compared with B. thuringiensis subsp. morrisoni pathovar tenebrionis. These results are useful in emphasizing the distribution of cry genes and for prognostication of toxicity, and may contribute to the identification of novel candidate genes for bioengineered crop protection.     

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Cry15Aa

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542, in a crystal together with a 40 kDa accompanying protein, is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this study we examined the role of the C-terminal part of Cry15Aa and of the 40 kDa protein in crystal formation in recombinant B. thuringiensis. The contribution of the 40 kDa protein and of the Cry15Aa carboxy-terminal sequence for crystal formation, crystal solubilization, and insecticidal properties was assessed. No significant differences in toxicity against Cydia pomonella, before or after in vitro solubilization of crystal-spore preparations, were found. Although the 40 kDa protein significantly contributes to in vitro solubility and in vivo crystal formation of Cry15Aa, no direct evidence for involvement of the 40 kDa protein in toxicity of Cry15Aa was found.     

Genetic diversity and functional characterization of endophytic <Emphasis Type="BoldItalic">Bacillus thuringiensis</Emphasis> isolates from the North Western Indian Himalayas

A total of 15 endophytic Bacillus thuringiensis isolates were obtained from root nodules of six legumes (soybean, ricebean, gahat, frenchbean, lentil and pea). All of these isolates were characterized by the presence of one of two different types of crystalline inclusions (spherical and bipyramidal) and tolerance to a wide pH range (4–10; optimum 7.0) and NaCl concentrations up to 8%. Genetic diversity among the B. thuringiensis isolates was determined by repetitive extragenic palindromic PCR assays (rep-PCR) using the Bacillus cereus-repetitive extragenic palindromic, BOX, enterobacterial repetitive intergenic consensus sequence and (GTG)₅ primers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteogram of the B. thuringiensis isolates revealed the presence of two major polypeptides (24.4 and 131.0 kDa). Maximum crystal protein profile was observed in the B. thuringiensis isolates producing the spherical crystal, while those isolates producing the bipyramidal crystal protein showed four four major polypeptides (24.4, 33.8, 81.2 and 131.0 kDa). The purified crystal protein profile of the B. thuringiensis isolates revealed the presence of only one major protein of 130 kDa mass. Isolates VRB1 and VLG15 possessing the cry1 and cry2 family genes demonstrated 100% mortality against first-instar larvae of the Bihar hairy caterpillar (lepidopteran pest). Our study of the ecological and molecular diversity among newly identified B. thuringiensis isolates suggests that these could be useful in planning new strategies for integrated pest management in sustainable agricultural systems.     

Insecticidal Activity of a Bacterial Crystal Protein Expressed by a Recombinant Baculovirus in Insect Cells

Baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. Introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. The complete insecticidal crystal protein gene cryIA(b) of Bacillus thuringiensis subsp. aizawai 7.21 was engineered into the nuclear polyhedrosis virus of Autographa californica (AcNPV) in place of the polyhedrin gene. In infected Spodoptera frugiperda cells, the cryIA(b) gene was expressed at a high level without interference with AcNPV production. The crystal protein was found in the cytoplasm of S. frugiperda cells, mainly as large crystals with an ultrastructure similar to that of B. thuringiensis crystals. Infected-cell extracts inhibited feeding of the large cabbage white Pieris brassicae. The toxicity of the crystal protein expressed by AcNPV recombinants was comparable with that of the crystal protein expressed by a corresponding Escherichia coli recombinant.     

Identification of a δ-Endotoxin Gene Product Specifically Active against Spodoptera littoralis Bdv. among Proteolysed Fractions of the Insecticidal Crystals of Bacillus thuringiensis subsp. aizawai 7.29

At least three different insecticidal crystal protein genes were shown to be expressed in Bacillus thuringiensis subsp. aizawai 7.29, a strain that is potentially active against the cotton leafworm Spodoptera littoralis Bdv. Among crude K-60 fractions (60- to 70-kilodalton [kDa] molecules) that were products of proteolysed crystals containing the active domains of the protoxin molecules, we were able to distinguish several distinct components on the basis of their antigenic relationship and their larvicidal properties. A purified fraction designated SF2 was a 61-kDa component specifically active against Pieris brassicae L. and homologous to the B. thuringiensis subsp. berliner 1715 plasmid-encoded crystal protein. A second fraction designated SF1 was composed of 63- and 65-kDa polypeptides and was specifically active against S. littoralis. The SF1 fraction and particularly the 65-kDa component were not antigenically related to the 61-kDa component. The purified fractions were compared with the products of three different crystal protein genes we previously cloned from total DNA of B. thuringiensis subsp. aizawai, among them a new type of crystal protein gene encoding a protein that is specifically active against S. littoralis and other insects of the Noctuidae family. This approach led us to consider the 65-kDa component as a minimum active part of a δ-endotoxin that is encoded by this new gene. Products of the two other cloned genes can be correlated with the 61- and 63-kDa components, respectively. Thus, in B. thuringiensis subsp. aizawai 7.29, multiple δ-endotoxin genes of different structural types direct the synthesis of several δ-endotoxins with different host specificities which were identified as components of the insecticidal crystals.     

Display of avian influenza virus nucleoprotein on <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> cell surface using CTC as a fusion partner

The S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on the cell surface of B. thuringiensis. By fusing np with the anchoring motif of ctc, four recombinant plasmids were constructed. They harbored fusion gene ctc-np, csa-ctc-np (csa representing csaAB operon, very important in anchoring S-layer protein on cell surface), ctc-npp (npp representing the part fragment of np), and csa-ctc-npp, respectively. Five recombinant strains were obtained by transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The vegetative cells of five strains were used as agglutinogens for slide agglutination assays. The assays showed recombinant NP proteins successfully displayed on the cell surface of five strains. After immunization of chickens with spores by oral route, all five strains elicited a humoral response to NP and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that one of five strains, CN (bearing csa-ctc-npp), exhibited the highest immunogenicity among five strains, which suggested that the best way of constructing ctc fusion gene was the csa-ctc-npp. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine with B. thuringiensis surface display system.     

The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans

Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis–C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen–host.     

Small molecule inhibitors of bacterial transcription complex formation

Knoevenagel condensation was employed to generate a set of molecules potentially capable of inhibiting the RNA polymerase-σ⁷⁰/σ^A interaction in bacteria. Synthesis was achieved via reactions between a variety of indole-7-carbaldehydes and rhodanine, N-allylrhodanine, barbituric acid or thiobarbituric acid. A library of structurally diverse compounds was examined by enzyme-linked immunosorbent assay (ELISA) to assess the inhibition of the targeted protein–protein interaction. Inhibition of bacterial growth was also evaluated using Bacillus subtilis and Escherichia coli cultures. The structure–activity relationship studies demonstrated the significance of particular structural features of the synthesized molecules for RNA polymerase-σ⁷⁰/σ^A interaction inhibition and antibacterial activity. Docking was investigated as an in silico method for the further development of the compounds.     

Studies on the regulation of 1-aminocyclopropane-1-carboxylate synthase in tomato using monoclonal antibodies

A partially purified preparation of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) from tomato (Lycopersicon esculentum (Mill.) fruit tissue was used to generate monoclonal antibodies (MAb) specific for the two different MAbs yielded a 50-kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An enzyme-linked immunosorbent assay (ELISA) capable of detecting <1 ng of antigen was developed. The ELISA system was used to demonstrate that two of the MAbs recognized different epitopes on the ACC-synthase protein. Wound-induced increases in ACC-synthase activity in tomato fruit tissue were correlated with changes in ELISA-detectable protein. In-vivo labeling of wounded tissue with [³⁵S]methionine followed by extraction and immunopurification in the presence of various protease inhibitors yielded one major radioactive band of 50 kDa molecular mass. Pulse labeling with [³⁵S]methionine at various times after wounding indicated that the wound-induced increase in ACC-synthase activity involved de-novo synthesis of a rapidly turning over 50-kDa polypeptide.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - ELISA enzyme-linked immunosorbent assay - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Nonenzymatic Glycosylation of Lepidopteran-Active Bacillus thuringiensis Protein Crystals

We used high-pH anion-exchange chromatography with pulsed amperometric detection to quantify the monosaccharides covalently attached to Bacillus thuringiensis HD-1 (Dipel) crystals. The crystals contained 0.54% sugars, including, in decreasing order of prevalence, glucose, fucose, arabinose/rhamnose, galactose, galactosamine, glucosamine, xylose, and mannose. Three lines of evidence indicated that these sugars arose from nonenzymatic glycosylation: (i) the sugars could not be removed by N- or O-glycanases; (ii) the sugars attached were influenced both by the medium in which the bacteria had been grown and by the time at which the crystals were harvested; and (iii) the chemical identity and stoichiometry of the sugars detected did not fit any known glycoprotein models. Thus, the sugars detected were the product of fermentation conditions rather than bacterial genetics. The implications of these findings are discussed in terms of crystal chemistry, fermentation technology, and the efficacy of B. thuringiensis as a microbial insecticide.     

An enzyme-linked immunosorbent assay to detect alkali-soluble spore surface antigens of strains of Nosema bombycis (Microspora: Nosematidae)

Spore surface antigens of strains of Nosema bombycis were extracted with alkaline solutions and used in an indirect enzyme-linked immunosorbent assay. Treatment of N. bombycis spores with 0.1 n potassium carbonate or potassium hydroxide solution at 27°C for 30 min was sufficient for the extraction of the antigens. Usually, 10⁸ spores of N. bombycis liberated ca. 30 μg spore surface proteins. The indirect enzyme-linked immunosorbent assay detected as little as 60 ng of spore surface proteins (ca. 2000 spore-equivalent antigen). The alkali-soluble spore surface antigens of N. bombycis contained a specific antigen and were stable under storage at −20°C for more than 1 year. The serological assay separated the Nosema isolates pathogenic to the silkworm into three groups.     

Enzyme-linked immunosorbent assay for detection of Aeromonas hydrophila serogroup O:19

An enzyme-linked immunosorbent assay has been developed for the detection of Aeromonas hydrophila serogroup O:19 isolated from epizootics in eels. The enzyme-linked immunosorbent assay specificity was confirmed after testing A. hydrophila O:19 and non-O:19 strains from different origins, as well as other Aeromonas species and other fish pathogens such as Vibrio vulnificus biotype 2, V. furnisii, V. damsela, Yersinia ruckerii and Edwardsiella tarda. The detection limits for A. hydrophila O:19 cells were around 10⁴–10⁵ cells/well. Artificially infected eels were analyzed and the immunodetection was confirmed by cultural methods. With this methodology A. hydrophila O:19 was successfully detected in infected eels and water samples. We described two subgroups within the serogroup O:19 (Guinée and Jansen system), one of them presents a 50 kDa outer membrane protein as a strong thermostable antigen which is not present in the other group.     

Symbiotic Relationship of Thiothrix spp. with an Echinoderm

Immunoassay procedures were used to investigate the symbiotic relationship of Thiothrix spp. in the intestinal cecum of the spatangoid species Echinocardium cordatum. Thiothrix spp. were identified in nodule samples from E. cordatum digestive tubes based on microscopic examination, enzyme-linked immunosorbent assay, and indirect immunofluorescence. Thiothrix spp. protein made up as much as 84% of the total protein content of the nodules. This is the first identification of Thiothrix spp. internally symbiotic with marine invertebrates.     

Bacillus thuringiensis isolates from Korean forest environments

We investigated the distribution, toxicity, morphology, and protein profiles of Bacillus thuringiensis isolates from forests in Korea to isolate naturally occurring novel B. thuringiensis. A total of 170 B. thuringiensis isolates were obtained from 832 samples producing spore and parasporal inclusion bodies. In toxicity tests for lepidopteran, dipteran, and coleopteran insects, 57.6% isolates were toxic only to Lepidoptera, 5.3% were toxic only to Diptera, and 24.1% were toxic to both Diptera and Lepidoptera. The remaining collections (13.0%) were not toxic to the tested insects. The shapes of the parasporal crystals produced in B. thuringiensis isolates were bipyramidal, spherical, ovoid, or irregular. As their toxicities varied with parasporal crystal shape, B. thuringiensis isolates possessing bipyramidal or irregular parasporal crystals were largely toxic to lepidopteran species whereas those producing spherical parasporal crystals were mainly toxic to dipteran species. B. thuringiensis toxic to both dipteran and lepidopteran insects contained 130- and 70-kDa parasporal crystals, whereas B. thuringiensis toxic to lepidopteran insects expressed 130-kDa parasporal crystals. The results suggest that forest areas in Korea are a rich source of B. thuringiensis and need to be further explored to discover novel B. thuringiensis isolates.     

A Bacillus thuringiensis S-Layer Protein Involved in Toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.     

New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518

We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes—cry55Aa1, cry6Aa2, and cry5Ba2—were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.