Parasporal Inclusions Research Articles

Expression and Characterization of a Recombinant Cry 1 Ac Crystal Protein Fused with Cry11A in Bacillus thuringiensis subsp. kurstaki Cry −B

The expression of a fusion protein comprised of two Bacillus thuringiensis crystal proteins, lepidopteran toxic CrylAc and dipteran toxic CryllA, in B. thuringiensis CryB strain was examined. To construct a recombinant gene, the cryllA gene was inserted behind the Xhol site in the middle region of the crylAc gene, but the C-terminal fragment (structure fragment) of CrylAc was removed in fusion construction (pProAcN-HA). Also, the cryllA gene was singly cloned under the crylAc promoter (pProllA). The B. thuringiensis CryB strain with pProAcN-HA (ProAcN-llA/CB) produced ovoid shaped parasporal inclusions of relatively small size, while ProllA/CB produced rhomboid shaped ones. Unexpectedly, ProAcN-llA/CB produced a protein of approximately 60 kDa while ProllA/CB and ProAc/CB produced typical 72 kDa and 130 kDa proteins, respectively. However, ProAcN-llA/CB exhibited significant dual toxicity on larvae from two insect orders, 72.7% on Plutella xylostella and 65.5% on Culex pipiens. The current results suggest that expression and crystallization of a fusion protein between toxic fragment of CrylAc and Cryl 1A was incomplete in the Cry'B strain though ProAcN-11A/CB produced parasporal inclusions and had dual toxicity.

Susceptibility of Archaea to the Antibiotic Effect of the Parasporal Inclusion Proteins from Different Bacillus thuringiensis subspecies

The proteins of parasporal inclusions from three Bacillus thuringiensis subspecies (kurstaki, amagiensis, and monterrey) inhibited growth of methanogenic archaea of two species belonging to two genera, Methanobrevibacter arboriphilus and Methanosarcina barkeri. The minimal inhibitory concentrations of these proteins were 20 to 50 micrograms/ml. Lysozyme exhibited similar bactericidal effect on archaea. The perspective of comparative studies on the effect of polyfunctional proteins on bacteria and archaea is discussed.

Characterization of a mosquitocidal Bacillus thuringiensis serovar sotto strain isolated from Okinawa, Japan.

To characterize the mosquitocidal activity of parasporal inclusions of the Bacillus thuringiensis serovar sotto strain 96-OK-85-24, for comparison with two well-characterized mosquitocidal strains. The strain 96-OK-85-24 significantly differed from the existing mosquitocidal B. thuringiensis strains in: (1) lacking the larvicidal activity against Culex pipiens molestus and haemolytic activity, and (2) SDS-PAGE profiles, immunological properties and N-terminal amino acid sequences of parasporal inclusion proteins. It is clear from the results that the strain 96-OK-85-24 synthesizes a novel mosquitocidal Cry protein with a unique toxicity spectrum. This is the first report of the occurrence of a mosquitocidal B. thuringiensis strain with an unusual toxicity spectrum, lacking the activity against the culicine mosquito.

Relocating expression of vegetative insecticidal protein into mother cell of Bacillus thuringiensis

Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth. Unlike δ-endotoxins which are produced as parasporal inclusion bodies within the cell during sporulation, VIP is secreted into the culture medium. Here we report the relocation of the expression of VIP into the mother cell compartment in a manner similar to well-characterized Cry proteins. Relocation of VIP is directed to mother cell by placing its synthesis under sporulation-dependent promoters, BtI and BtII. The insertion of cry preferred transcription termination sequence at the 3 ′ region and a STAB-SD sequence at the 5 ′ region of the gene provided stability to the vip transcript and enhanced its yield. The demonstrated expression of VIP within the cells in the form of inclusion bodies would facilitate development of a suitable formulation for the application of this class of insecticidal proteins in the field.

Microbial ecology of Bacillus thuringiensis: fecal populations recovered from wildlife in Korea.

A total of 34 fecal samples, collected from 14 species of wild mammals in Korea, were examined for the occurrence of Bacillus thuringiensis. The organism was detected in 18 (53%) samples. Among the three food-habit groups, herbivorous animals yielded the highest frequency (69%) of samples positive for B. thuringiensis, followed by omnivorous animals (50%). Of the six fecal samples from carnivorous animals, only one sample contained B. thurin giensis. Among 527 isolates belonging to the Bacillus cereus - B. thuringiensis group, 43 (8%) were assigned to B. thurin giensis on the basis of the formation of parasporal inclusions. Of the 43 isolates, 13 were serologically allocated to the nine H-antigenic serotypes: H3ad (serovar sumiyoshiensis), H15 (dakota), H17/27 (tohokuensis/ mexicanensis), H19 (tochigiensis), H21 (colmeri), H29 (amagiensis), H31/49 (toguchini/muju), H42 (jinghongiensis), and H44 (higo). Other isolates were untestable or untypable by the 55 reference H antisera available. Insecticidal activity was associated with 23% of the fecal populations: three isolates killed larvae of the silkworm, Bombyx mori (Lepidoptera), and seven exhibited larvicidal activity against the mosquito, Aedes aegypti (Diptera). There was no larvicidal activity against the three lepidopterous insects: Plutella xylostella, Spodoptera exigua, and Spodoptera litura. The overall results suggest that wild animals in Korea are in contact with naturally occurring B. thuringiensis at high frequencies through the daily food intake of plants.

Cloning and study of the expression of a novel cry1Ia-type gene from Bacillus thuringiensis subsp. kurstaki

Cloning and expression of a new cry1Ia-type gene of Bacillus thuringiensis. PCR amplification, using gene cry1I-specific primers revealed the presence of such a gene in the strain BNS3 of Bacillus thuringiensis subsp. kurstaki. The cloning and sequencing from BNS3 of the cry1Ia-type gene, called crybns3-3, showed an open reading frame of 2160-bp, encoding a protein of 719 amino acid residues. Both nucleotide and amino acid sequences similarity analysis revealed that the crybns3-3 is a new cry1Ia-type gene, presenting several differences from the cry1Ia-type genes. The study of the expression of crybns3-3 by Northern blot and RT-PCR showed that it was transcribed. The expression of crybns3-3 under the control of BtI and BtII promoters revealed that Crybns3-3 would co-crystallize with the endogenous delta-endotoxins. crybns3-3 is a novel cry1Ia gene isolated from B. thuringiensis subsp. kurstaki strain BNS3. The characteristics of crybns3-3 indicate that it is a new cry1Ia-type gene. Amino acid residue substitutions presented in Crybns3-3 could be exploited for both toxicity and specificity studies. Crybns3-3 would interact and co-crystallize at least partially with the endogenous delta-endotoxins of BNS3, and then participate in the formation of the parasporal crystal inclusions.

Characterization of flagellar antigens and insecticidal activities of Bacillus thuringiensis populations in animal feces.

In total, 287 Bacillus thuringiensis isolates, recovered from feces of 28 zoo-maintained animal species, were examined for flagellar (H) antigenicity and insecticidal activity. Serologically, 209 isolates (72.8%) were allocated to the 8 H serogroups, 4 were untypable, and 74 were untestable. Among the 8 H serotypes detected, H3abc (serovar kurstaki) predominated at a high frequency of 88.0%, followed by H6 (serovar entomocidus) with a frequency of 7.7%. Insecticidal activity was associated with 67.2% of the fecal populations: 188 isolates were toxic to both Bombyx mori (Lepidoptera: Bombycidae) and Aedes aegypti (Diptera: Culicidae), 2 isolates were specific for B. mori, and 3 isolates were toxic to A. aegypti only. Of the isolates with dual toxicity, 97.9% belonged to the serovar kurstaki, producing bipyramidal parasporal inclusions. All of the H7 (serovar aizawai) isolates were toxic to both insects.

Mannose-specific lectin activity of parasporal proteins from a lepidoptera-specific Bacillus thuringiensis strain.

Lectin activity, agglutinating sheep erythrocytes, was associated with parasporal inclusion proteins from a Lepidoptera-specific isolate of Bacillus thuringiensis serovar galleriae (H5ab). The activity was generated when parasporal inclusions were solubilized in an alkaline condition. Proteolytic processing was not required for generation of the lectin activity; the activity level was not affected by the presence/absence of the three proteases (trypsin, chymotrypsin, and proteinase K). SDS-PAGE analysis revealed that (1) alkali-solubilized parasporal inclusion proteins consisted of two major components of 130 kDa and 65 kDa, and (2) proteinase K treatment of alkali-solubilized proteins yielded a single major protein of 60 kDa. Lectin activity of our isolate was strongly inhibited by preincubation with D-mannose, but not with the six other monosaccharides: D-galactose, D-glucose, L-fucose, N-acetyl- D-glucosamine, N-acetyl- D-galactosamine, and N-acetylneuraminic acid. In contrast, D-mannose did not inhibit the in vivo larvicidal activity of the proteins against the silkworm, Bombyx mori.

Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp. kurstaki.

A 4 kb BamHI-HindIlI fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp. kurstaki strain BNS3, was cloned. The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues. Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes. The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.

Antitrichomonal strains of Bacillus thuringiensis.

Parasporal inclusion proteins from a total of 816 Bacillus thuringiensis strains isolated in Japan were examined for antitrichomonal activity against Trichomonas vaginalis. Ten strains of B. thuringiensis inhibited the growth of T. vaginalis in 48 h cultures at 37 degrees C. Moreover, the B622 and B626 strains clearly showed trichomonacidal effects against T. vaginalis. The H antigen serotypes of both strains were identified as H13/29 (pakistani/amagiensis). The parasporal inclusion protein from both strains consisted of three major polypeptides of 77, 45 and 25 kDa.

Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera LeConte.

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.

High frequency of Bacillus thuringiensis in feces of herbivorous animals maintained in a zoological garden in Japan.

A total of 71 fecal samples, collected from 56 animal species (47 mammals, four reptiles, and five avians), were examined for the occurrence of Bacillus thuringiensis. Most of the animals were residents of the Fukuoka Municipal Zoo, Fukuoka, Japan. The organism was detected in 32 (45%) samples. Among 2,955 colonies of the Bacillus cereu/B. thuringiensis group examined, 531 (18%) were assigned to B. thuringiensis on the basis of the formation of parasporal inclusions. Fecal samples from animals feeding on vegetable matter contained B. thuringiensis at high frequencies. Examples included feces from the chimpanzee, gorilla, Japanese black bear, polar bear, green iguana, and ostrich. In contrast, only a few isolates were recovered from feces of carnivorous animals, in particular, feline mammalians including the lion, tiger, leopard, and jaguar. The results suggest that a daily food intake of plant origin yields the feces containing B. thuringiensis at high levels.

Isolation and characterization of a Bacillus thuringiensis ssp. kurstaki strain toxic to Spodoptera exigua and Culex pipiens.

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species.

The receptor of Bacillus sphaericus binary toxin in Culex pipiens (Diptera: Culicidae) midgut: molecular cloning and expression

Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39–43% identities with insect maltases (α-glucosidases and α-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant α-glucosidase activity but no α-amylase activity. These results support the view that Cpm1 is an α-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains.

A 28 kDa protein of the Bacillus thuringiensis serovar shandongiensis isolate 89-T-34-22 induces a human leukemic cell-specific cytotoxicity

A 28 kDa protein that exhibits cytocidal activity specific for human leukemic T (MOLT-4) cells was purified from proteinase K-digested parasporal inclusion of a Bacillus thuringiensis serovar shandongiensis isolate. The N-terminal sequence of the protein was identical with that of the 32 kDa protein, regarded as a protoxin, of the inclusion proteins. The median effective concentration of this protein was 0.23 μg/ml against MOLT-4 cells and its specific activity was 7.9 times greater than that of the whole inclusion proteins. The 28 kDa protein induced necrosis-like cytotoxicity against MOLT-4 cells and the cytopathic effect with the passage of time was characterized by cell swelling, nuclear membrane isolation and chromatin condensation.

Specificity of lectin activity of Bacillus thuringiensis parasporal inclusion proteins.

Sheep erythrocyte-agglutinating parasporal inclusion proteins from four Bacillus thuringiensis strains (FITC-20, FITC-73, FITC-76 and IBC-1456) were examined for lectin activity against erythrocytes from four mammalian (rabbit, horse, cow and guinea pig) and an avian (chicken) species. Of the five erythrocyte species, only rabbit cells were agglutinated with the protein of the human cancer cell-killing strain IBC-1456. No haemagglutination (HA) activities were shown in other protein-erythrocyte combinations. The lectin activity of the strain IBC-1456 against rabbit cells was strongly inhibited by preincubation with D-galactose. Overall results revealed that the B. thuringiensis lectins have a preference for sheep erythrocytes.

Two delta-endotoxin genes, cry9Da and a novel related gene, commonly occurring in Lepidoptera-specific Bacillus thuringiensis Japanese isolates that produce spherical parasporal inclusions.

Six Lepidoptera-specific Bacillus thuringiensis isolates, which belong to the four H serovars (sotto, fukuokaensis, canadensis, and galleriae) and produce spherical parasporal inclusions, were examined for assignment of the classes of the delta-endotoxin genes. Gene analysis was conducted by PCR technique with primers designed to probe the genes cry9Ca and cry9Da. The data revealed that the delta-endotoxin of a serovar canadensis isolate is encoded by the gene cry9Da, while those of the five other strains are encoded by an undescribed delta-endotoxin gene. DNA fragments from five strains had an identical 1917-bp nucleotide sequence, covering the four conserved regions and a partial sequence of the block 5 region. The deduced amino acid sequence exhibited a 70.6% homology to that of the corresponding region of the Cry9Ea delta-endotoxin protein which is active on the order Lepidoptera, and a 63.1% homology to the Cry9Ca protein highly toxic to the noctuid lepidopterans. The results showed that Japanese isolates of B. thuringiensis producing spherical parasporal inclusions with Lepidoptera-specific activity are categorized into two groups: one produces the class Cry9Da protein and the other a novel delta-endotoxin allied to the class Cry9. It also appeared that heterogeneous multiple H serovars are involved in each group.

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A computer-based image processing technique was adapted for rapid screening of Bacillus thuringiensis. Sporulated colonies of the B. thuringiensis/B. cereus group were observed under a phase-contrast microscope and the images were recorded by a CCD camera. The data were converted into binary images after reducing the background noises and thickening the edge lines of spores and inclusions. Parasporal inclusions were then distinguished from spores by a difference in curvatures and sizes. The system automatically differentiated in five seconds between B. thuringiensis and B. cereus by the ratio of spores and parasporal inclusions.

Bacillus thuringiensis and its use in agriculture

Bacillus thuringiensis (Bt) is an ubiquitous Gram positive, spore forming bacterium that forms parasporal crystal inclusions. These proteinaceous inclusions are called crystal (Cry) proteins or delta- endotoxins, which are toxic to insects. Hence preparations of Bt are being used as bioinsecticides for the past four decades in commercial agriculture and forest management for the control of certain insect species belonging to the orders of Lepidoptera, Diptera and Coleoptera. Bt is a major source for transfer of genes into plants to impart insect resistance. Since 1996 millions of hectares are grown with insect resistant transgenic plants containing Bt genes, in different parts of the world. Commercial release of transgenic Bt cotton in India is expected soon.

Morphology of a spectrum of parasporal endotoxin crystals from cultures of Bacillus thuringiensis ssp. kurstaki isolate A3-4

Morphology of the parasporal δ-endotoxin crystals of Bacillus thuringiensis subsp. kurstaki isolate A3-4, a native (Taiwan) strain was studied by scanning (SEM) and transmission (TEM) electron microscopy. The typical bipyramidal crystals and an unusual parasporal inclusion with an embedded body were observed. The parasporal δ-endotoxin crystal with an embedded body, which was in different size and shape was well demonstrated in thin sections by transmission electron microscopy. The sporulated cells had multiple individually separated inclusions up to four crystals in one cell, which was unique to this isolate, and has not been reported before. The δ-endotoxin crystals included in the cell or released from the cell after batch fermentation or fed-batch fermentation did not show any altered morphological characteristics. However, judging from thin-sections of TEM, cells and the included parasporal crystals from fed-batch fermentation appeared larger than those from batch fermentation. It was observed that release of spore and parasporal crystals from the bacterial cell produced from batch cultures was earlier than that of fed-batch cultures. Preliminary bioassay results showed that the isolate cultures from both types of culture were equally effective against Plutellaxylostella larvae. Based on the morphological observations, this strain may have a multiple insecticidal activities toward different insect species.