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PubMed2003

Molecular typing by pulsed-field gel electrophoresis of Bacillus thuringiensis from root voles.

The root voles intestinal strains of Bacillus thuringiensis, were characterised by pulsed-field gel electrophoresis (PFGE). For 14 isolates, three pulsotypes were found, with the use of SmaI or NotI as restriction enzymes. Strains in each pulsotypes presented identical DNA patterns, indicating that the population structure of B. thuringiensis from root voles is clonal. The similarities in banding patterns were estimated at 56% and 33% for SmaI and NotI digests, respectively. The strains under study differed significantly in the size of their entire genome, which varied between 2.4 and 4.2 Mb. No significant differences were detected among the isolates subjected to biochemical properties determined by API tests. Present study showed that genomic diversity is a common feature of B. thuringiensis originating from one ecological niche. PFGE appears to be a useful technique for use in studies on the spread of B. thuringiensis in the environment.

Animals↗
PubMed1999

[The effects of reactive oxidants on Bacillus thuringiensis parasporal crystals].

The effects of hydrogen peroxides(H2O2) and hydroxyl radicals(.OH) on the purified parasporal crystals from Bacillus thuringiensis var. Kurstaki HD-1 were studied by SDS-PAGE and bioassay. The results showed that to some extent H2O2 and .OH produced a direct and deleterious effect. Hydroxyl radicals from the system Fe(II)-EDTA-H2O2 indicated more serious damage than hydrogen peroxides. There was a 44.5% loss in activity against 12-h-old larvae of Heliothis armigera when parasporal crystals were treated with 0.3% H2O2 only. However, 85.2% activity lost when parasporal crystals were treated with 0.3% H2O2 in the presence of iron(II) and EDTA.

Animals↗
PubMed2006

Expression of Vitreoscilla hemoglobin in Bacillus thuringiensis improve the cell density and insecticidal crystal proteins yield.

The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was reduced from 18 to 12%. The maximum viable cell counts of the VHb+ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb- strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb+ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb- strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition.

Bacillus thuringiensis↗
PubMed1987

The glycoprotein toxin of Bacillus thuringiensis subsp. israelensis indicates a lectinlike receptor in the larval mosquito gut.

The mosquito-active protein crystals produced by Bacillus thuringiensis subsp. israelensis contain covalently attached aminosugars which are critical for their larvicidal activity. The 50% lethal concentrations toward Aedes aegypti larvae were increased up to 10-fold by mild periodate treatment, up to 40-fold by forming the protein crystals in the presence of tunicamycin, and up to 7-fold by the presence during the mosquito bioassays of N-acetylglucosamine or its trimer, triacetylchitotriose. Periodate-treated crystals and crystals formed in the presence of tunicamycin had greatly reduced binding capacities for wheat germ agglutinin, an N-acetylglucosamine-specific lectin. These results suggest that the B. thuringiensis subsp. israelensis glycoprotein toxin binds to a lectinlike receptor in the larval mosquito gut. Furthermore, the distinct lectin-binding patterns exhibited by diptera-active versus lepidoptera-active B. thuringiensis crystals suggest that host specificity for the microbial insecticides is determined, in part, by the carbohydrate portion of their glycoprotein crystals.

Aedes↗
PubMed1984

Inverted repeat sequences flank a Bacillus thuringiensis crystal protein gene.

Two sets of inverted repeat DNA sequences, IR2150 and IR1750, were discovered flanking the crystal protein gene on the 75-kilobase plasmid of Bacillus thuringiensis subsp. kurstaki HD73. A restriction map of ca. 40 kilobases around the crystal protein gene was constructed, and the positions of the copies of IR2150 and IR1750 were determined. Three copies of IR2150 were found flanking the crystal protein gene in an inverted orientation, and one partial and three intact copies of IR1750 were found in both inverted and direct orientations around the gene. Hybridization experiments with fragments from within IR2150 and IR1750 demonstrated the presence of multiple copies of these sequences on the chromosome of B. thuringiensis subsp. kurstaki HD73 and also revealed a strong correlation between the presence of these sequences and the presence of the crystal protein gene on plasmids from 14 strains of B. thuringiensis.

Bacillus thuringiensis↗
PubMed2000

Composition and ecological distribution of cry proteins and their genotypes of Bacillus thuringiensis isolates from warehouses in China.

The composition and distribution of insecticidal crystal proteins (Cry proteins) and their genotypes of Bacillus thuringiensis isolates from warehouses were evaluated through SDS-PAGE and PCR techniques. The results showed that the electrophoretic patterns of delta-endotoxin crystal preparations were divided into five types. The isolates containing approximately 135 kDa with a 65-kDa protein or only a approximately 135-kDa protein, which amounted to 55.74 and 35.25% of all isolates respectively, were the two major profiles of Cry protein isolated. The distribution of cry genes of B. thuringiensis from warehouses was highly variable. Cry protein genotypes detected in B. thuringiensis isolates included cry1Aa5, cry1Ab9, cry1Ac5, cry1Ba, cry1Ca1, cry1Da1, cry1Ea3, cry2, and cry3 genes, but not cry1Fa2. Among them, cry2, cry1Ac5, and cry1Ab9 genes were the most common in our B. thuringiensis isolates. Most B. thuringiensis isolates contained several cry genes in a total of 18 profiles. Among them, cry1Ac5 with cry1Ea3; cry1Aa5, cry1Ab9, cry1Ac5 with cry1Ea3; and cry1Aa5, cry1Ab9 with cry1Ac5 were the three principal profiles. The distribution of the Cry proteins and cry genes in isolates depended on geography and type of warehouses. Gene profiles may be used as markers for insecticidal activity of B. thuringiensis strains, but they did not directly reflect the toxic level of B. thuringiensis strains. The serotype of B. thuringiensis strains did not directly reflect the specific cry gene profiles in the strains, but certain relationships can be established between the serotype and cry genotype.

Bacillus thuringiensis↗
PubMed1997

Intramolecular proteolytic cleavage of Bacillus thuringiensis Cry3A delta-endotoxin may facilitate its coleopteran toxicity.

The Cry3A delta-endotoxin protein inclusion synthesized by Bacillus thuringiensis subsp. tenebrionis is soluble in alkaline and acid buffer solutions but the toxin precipitates when returned to neutral pH conditions. The midgut pH of susceptible beetle larvae is neutral to slightly acidic, a pH environment in which the Cry3A toxin is insoluble. To investigate this paradox we studied the Cry3A toxin after various proteolytic treatments. In many cases the toxin was cleaved into polypeptides that remained associated under non-denaturing conditions. Interestingly a chymotrypsinized Cry3A product was soluble under neutral pH conditions, retained full activity against susceptible beetle larvae, and exhibited specific binding to Leptinotarsa decemlineata midgut membranes.

Animals↗
PubMed1991

Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts.

We have examined expression of several insecticidal crystal protein (ICP) genes of Bacillus thuringiensis in transgenic tobacco plants and electroporated carrot protoplasts. We determined that low levels of lepidopteran toxin cryIA(b) ICP gene expression in plants and electroporated carrot cells is due to RNA instability. We used a series of 3' deleted by cryIA(b) constructs directed by the cauliflower mosaic virus 35S promoter to demonstrate that this instability is minimally contained in the first 579 bases of the gene in both systems. This instability may result from 5'----3' as well as 3'----5' RNA metabolism. The coleopteran toxic cryIIIA gene was also examined in electroporated carrot cells, and found to be poorly expressed. A model for improvement of ICP RNA stability in plants is presented.

Bacillus thuringiensis↗
PubMed1996

Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin.

The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis.

Animals↗
PubMed2001

Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

Animals↗
PubMed1990

Subdomain organization of Bacillus thuringiensis entomocidal proteins' N-terminal domains.

N-Terminal domain (65 kD) of delta-endotoxin produced by Bacillus thuringiensis ssp. alesti, as shown by limited proteolysis, consists of two subdomains of molecular mass 30 and 33 kD that correspond, respectively, to conservative and variable regions of the delta-endotoxin primary structure. Furthermore, proteolysis of these subdomains leads to their conversion into at least two fragments of molecular mass 10 kD stable to proteinase action. Such a pattern of molecular organization appears to be common for several structurally related delta-endotoxins that belong to the kurstaki group. Entomicidal protein produced by ssp. israelensis (70 kD), which differs strongly from alesti and other kurstaki group delta-endotoxins, retains a similar type of molecular organization and consists of two subdomains with molecular mass of approximately 35 kD. Apparently, the characteristic pattern of the delta-endotoxins' molecular structure reflects separation of functions (e.g., host recognition and toxicity per se) between domains and subdomains of these proteins.

Amino Acid Sequence↗
PubMed1998

Direct evidence for rapid degradation of Bacillus thuringiensis toxin mRNA as a cause of poor expression in plants.

It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial. Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms. To resolve this controversy, we compared the expression of an A/U-rich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5' and 3' flanking sequences. Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays. In contrast, mRNA half-life measurements demonstrated directly that the wild-type transcript was markedly less stable than that encoded by the synthetic gene. Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum). These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants. Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.

Bacillus thuringiensis↗
PubMed2000

Characterization of Bacillus thuringiensis ser. balearica (Serotype H48) and ser. navarrensis (serotype H50): two novel serovars isolated in Spain.

The novel strains of Bacillus thuringiensis PM9 and NA69, isolated from soil samples in Spain, were classified and characterized in terms of their crystal proteins, plasmid profile, cry genes content, and their toxicological properties against several species of Lepidoptera, Coleoptera, and Diptera. Both strains share morphological and biochemical characteristics with previously described B. thuringiensis strains, although their unique H antigens identify them as two new serotypes. Two new serovar names, B. thuringiensis serovar balearica (H serotype 48) and B. thuringiensis serovar navarrensis (H serotype 50) are proposed for the type strains PM9 and NA69, respectively.

Animals↗
PubMed2005

Association analysis between serotype, cry gene content, and toxicity to Helicoverpa armigera larvae among Bacillus thuringiensis isolates native to Spain.

Serotyping, cry gene content, and toxicity to Helicoverpa armigera were determined for 178 isolates of Bacillus thuringiensis native to Spain. A total of 13 different cry1 and cry2 genes were detected when isolates were screened by PCR analysis. Results showed that cry2 and cry1Ia were the most frequent cry genes in the collection (74 and 57%, respectively); whereas cry1D, cry1Aa, cry1Ab, and cry1C were only moderately abundant (49, 48, 47, and 36%, respectively). The most uncommon cry genes were cry1Ac, cry1E, cry1B, cry1Ib, cry1Ad, cry1F, and cry1G, with frequencies of 24, 14, 13, 8, 5, 5, and 1%, respectively. The distribution of some cry genes was somewhat associated with particular serovars. For example, genes cry1C and cry1D were especially frequent in the serovar aizawai, while cry1B was very frequent in the serovar thuringiensis. Bioassays against H. armigera larvae showed a wide variation in the insecticidal potency, even among strains sharing the same set of cry genes and within the same serotype.

Animals↗
PubMed2001

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity.

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were futile, but it was degraded by proteases with broad specificity indicating the presence of a peptide. Carbohydrate was detected by labeling with digoxigenin hydrazide following periodate oxidation. Mild alkaline hydrolysis destroyed toxin and antibody binding, suggesting O-linked glycans are involved in the activity. GC/MS composition analysis showed that the predominant sugars were galactose, glucose, and N-acetyl galactosamine with lesser amounts of N-acetyl glucosamine, glucuronic acid, xylose, and fucose. The carbohydrate moiety accounted for 73% of its total mass. Amino acid analysis showed a high content of aspartic/asparagine, threonine, and serine residues in the protein moiety. The purified glycoconjugate was not visualized using Coomassie or silver staining procedures, but stained "blue" using the cationic dye Stains-all. BTR-270 was labeled with biotin and used as a diagnostic probe for screening and identifying toxins that bind to the receptor. Toxin-binding kinetics obtained using a biosensor demonstrated that the receptor binds Cry1Aa and Cry1Ab toxins with high affinity, and displays a weaker affinity for Cry1Ac, in correlation with the toxicity of these toxins towards gypsy moth. Arch.

Amino Acids↗
PubMed2005

Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac.

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.

Alleles↗
PubMed1995

Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration.

The Bacillus thuringiensis toxin-binding properties of midgut epithelial cells from two strains of Heliothis virescens were compared. One H. virescens strains (YHD2) which was selected against CryIAc toxin had over 10,000-fold resistance to CryIAc toxin relative to the susceptible strain and was cross-resistant to CryIAa and CryIAb. The second H. virescens strain (YDK) was susceptible to these toxins in the order CryIAc > CryIAb > CryIAa. Receptor-binding properties of CryIAa, CryIAb, and CryIAc toxins were compared between the susceptible and resistant strains. Saturation and competition-binding experiments were performed with brush border membrane vesicles prepared from midguts of the susceptible and resistant insects and 125I-labeled toxins. In the susceptible strain, saturable, specific, and high-affinity binding of all three toxins was observed. The relative binding-site concentration was directly correlated with toxicity (CryIAc > CryIAb > CryIAa). In the resistant strains, the binding affinities of CryIAb and CryIAc were similar to that observed with the susceptible strain and ony minor differences in binding-site concentration (Bmax) were observed. The major difference between the two strains was the total lack of binding of CryIAa toxin to the brush border membrane vesicles of the resistant strain. Heterologous competition-binding experiments and ligand blot analysis supported the hypothesis that there were multiple binding sites for the toxins. On the basis of results of the present study, we propose that alterations in binding proteins shared by all three toxins are a major factor in resistance. This suggests that not all receptors of CryIAc might be involved in toxic function.

Animals↗
PubMed1995

Interaction of the insecticidal crystal protein CryIA from Bacillus thuringiensis with amino acid transport into brush border membranes from Bombyx mori larval midgut.

The activities of three related Bacillus thuringiensis delta-endotoxins, designated CryIA(a), CryIA(b), and CryIA(c), as inhibitors of K(+)-dependent amino acid transport into membrane vesicles prepared from the anterior and posterior portions of Bombyx mori larval midgut were measured. Under experimental conditions similar to those occurring in vivo (membrane potential approximately -100 mV, inside negative; pH 7.2in/8.8out; an inwardly directed K+ gradient) CryIA(a) toxin produced a clear dose-dependent inhibitory effect on leucine uptake by both the anterior and the posterior gut membrane vesicles, giving half-maximal inhibition constants (IC50) of 2.6 +/- 0.3 and 2.1 +/- 0.2 microgram toxin/mg membrane protein, respectively. The other two delta-endotoxins were practically inactive. A dose-dependent inhibition of amino acid transport by CryIA(a) toxin was also observed on the carrier-mediated K(+)-independent component, i.e., the leucine-only form. This result strongly indicates that the activity of the K+/amino acid cotransporter is directly affected after binding of delta-endotoxin to the brush border membrane. When the extravesicular pH was lowered to pH 7.2, the interaction of CryIA(a) toxin with the brush border appeared more complex, as suggested by the Hill coefficient of the dose-response curves higher than 1. In conclusion, our data indicate that (i) CryIA(a) toxin specifically inhibited K+/leucine symport along the length of the midgut; (ii) the interaction between cotransporters and toxin is affected by the pH of the medium; and (iii) the K+/leucine cotransporter or a strictly associated protein may serve as membrane receptor for CryIA(a) delta-endotoxin in the B. mori larval midgut.

Animals↗