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1.
Only part of the protoxin gene of Bacillus thuringiensis subsp. berliner 1715 is necessary for insecticidal activity. 总被引:1,自引:0,他引:1
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Escherichia coli strains harboring deletion mutations of the insecticidal protoxin gene of Bacillus thuringiensis subsp. berliner 1715 were constructed. Although these strains did not produce intact protoxin, cell extracts from one of the mutants were extremely toxic to tobacco hornworm (Manduca sexta) larvae, indicating that only a part of the protoxin gene is required for insecticidal activity. 相似文献
2.
The association of 20 kb heterologous DNA fragments with the parasporal crystals from native and recombinant Bacillus thuringiensis strains was analyzed, respectively. The cry2Aa10 gene cloned in plasmid pHC39 was transformed into B. thuringiensis subsp. kurstaki strains CryˉB and HD73, producing recombinant strains CryˉB(pHC39) and HD73(pHC39). SDS-PAGE and scanning electron microscopy
analyses demonstrated that the recombinant CryˉB(pHC39) produced cuboidal crystals of Cry2Aa10 protoxin, while recombinant
HD73(pHC39) produced both bipyramidal crystals of Cry1Ac1 protoxin and cuboidal crystals of Cry2Aa10 protoxin. Bioassay results
proved that recombinant HD73(pHC39) showed higher insecticidal activity to Helicoverpa armigera than CryˉB(pHC39). It was found that 20 kb DNA fragments were present in bipyramidal and cuboidal crystals from both native
and recombinant strains, and the 20 kb heterologous DNAs contained chromosome-specific and resident large plasmid-borne DNA
fragments, suggesting the 20 kb heterologous DNA fragment embodied in crystals came randomly from the bacterial chromosomal
and plasmid genome. This was the first investigation devoted exclusively on the origin of 20 kb DNA fragments in the parasporal
crystals of B. thuringiensis. The data provides a basis for further investigation of the origin of 20 kb DNAs in the crystals and the interaction of DNA
and protoxins. 相似文献
3.
Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki. 总被引:7,自引:6,他引:1
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Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable. 相似文献
4.
Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable. 相似文献
5.
Generation and Analysis of Soybean Plastid Transformants Expressing <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1Ab Protoxin 总被引:4,自引:0,他引:4
Dufourmantel N Tissot G Goutorbe F Garçon F Muhr C Jansens S Pelissier B Peltier G Dubald M 《Plant molecular biology》2005,58(5):659-668
We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia gemmatalis). 相似文献
6.
[目的]将特异性杀虫毒蛋白基因Bt cry3A转入桑粒肩天牛(Apriona germari Hope,Ag)幼虫肠道常驻内生菌中,构建能在天牛幼虫肠道中定殖并表达特异性杀虫基因Bt cry3A的工程菌.[方法]以传统方法和16S rDNA分子生物学分析等方法分离、鉴定Ag幼虫肠道优势的常驻内生菌,从中筛选出适合转化的候选菌株.利用电转化技术将含有对鞘翅目昆虫具专一性毒力Bt cry3A基因的Escherichia coli-Bacillus thuringiensis穿梭表达质粒pHT305a和pHT7911分别转入Ag幼虫肠道常驻内生菌短短芽孢杆菌(Brevibacillus brevis Ag12,Ag12)和苏云金芽孢杆菌(Bacillus thuringiensis Ag13,Ag13)中.[结果]从Ag幼虫肠道共分离获得18个不同种的可培养细菌菌株,并从中选取菌株Ag12和Ag13作为出发菌株转入Bt cry3A基因.经质粒稳定性试验、转化子生长特性测试、伴胞晶体电镜检测、毒蛋白SDS-PAGE分析、工程菌定殖性分析以及生物毒力测试,结果显示cry3A基因已经成功转入Ag幼虫的常驻内生菌短短芽孢杆菌和苏云金芽孢杆菌中,并且工程菌Ag12-305a、Ag13-305a、Ag 12-7911和Ag13-7911都能在天牛幼虫肠道内稳定生长、繁殖并表达分子量约65kDa的伴孢晶体杀虫蛋白Cry3A.[结论]Bt cry3A基因已成功转入桑粒肩天牛幼虫肠道优势常驻内生菌中,获得了四株能在桑粒肩天牛幼虫肠道内定殖,并能表达目的杀虫基因Btcry3A的转基因工程菌. 相似文献
7.
8.
The insecticidal protoxin from Bacillus thuringiensis has been shown to be a major component of the spore coat. We have developed a novel surface display system using B. thuringiensis spores in which the N-terminal portion of the protoxin is replaced with a heterologous protein. The expression vector with a sporulation-specific promoter was successfully used to display green fluorescent protein and a single-chain antibody (scFv) gene that encodes anti-4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (anti-phOx) antibody. The spores that carry the anti-phOx antibody can bind to phOx specifically. 相似文献
9.
The delta-endotoxin of Bacillus thuringiensis subspecies kurstaki is an intracellular crystalline proteinaceous inclusion which, upon ingestion, is toxic to lepidopteran insects. Upon dissolution at pH > 9 it yields a protein subunit called protoxin. Under appropriate conditions, protoxin is hydrolyzed to a toxin molecule, which is responsible for killing the insect. It is known that this toxic activity decreases considerably above pH 10. In this study, circular dichroism spectroscopy has been used to examine the secondary structures of the protoxin and toxin molecules at different pH values to determine if there are detectable conformational changes associated with their pH-dependent functional properties. At pH 10, where toxic activity is approximately maximal, both the protoxin and toxin molecules were found to assume a conformation that is on an average approx. 26% alpha-helix and approx. 45% beta-structure. As the pH was increased above 10, where the insecticidal activity decreases, the magnitude of the CD spectrum at 222 nm decreased for protoxin and the calculated alpha-helix contents of both protoxin and toxin molecules decreased. The net secondary structure did not change significantly at pH values below 10. Significant conformational differences are observed between the secondary structure of the protoxin and toxin molecules at different pH values. The pH-dependent changes in secondary structure of the protoxin and toxin can be correlated with the effects of pH on the insecticidal activity of these proteins. 相似文献
10.
We studied mechanisms of resistance to Bacillus thuringiensis insecticidal crystal protein Cry1C in the diamondback moth, Plutella xylostella (L.). Binding assays with midgut brush border membrane vesicles prepared from whole larvae showed no significant difference between resistant and susceptible strains in binding of radioactively-labeled Cry1C. These results indicate that reduced binding of Cry1C to midgut membrane target sites did not cause resistance to Cry1C. Thus, the mechanism of resistance to Cry1C differs from that observed in several previously reported cases of resistance to Cry1A toxins in diamondback moth. We tested Cry1C toxin and Cry1C crystalline protoxin against resistant and susceptible larvae using leaf disk bioassays. After adjusting for the size difference between Cry1C toxin and protoxin, we found that with resistant larvae, toxin was significantly more toxic than protoxin. In contrast, with susceptible larvae, no significant difference in toxicity occurred between Cry1C toxin and protoxin. The resistance ratios for Cry1C were 19 for toxin and 48 for protoxin. These results suggest that reduced conversion of Cry1C protoxin to toxin is a minor mechanism of resistance to Cry1C. Because neither reduced binding nor reduced conversion of protoxin to toxin appear to be major mechanisms, one or more other mechanisms are important in diamondback moth resistance to Cry1C. 相似文献
11.
Vazquez-Padron RI de la Riva G Agüero G Silva Y Pham SM Soberón M Bravo A Aïtouche A 《FEBS letters》2004,570(1-3):30-36
Cry1Ab is one of the most studied insecticidal proteins produced by Bacillus thuringiensis during sporulation. Structurally, this protoxin has been divided in two domains: the N-terminal toxin core and the C-terminal portion. Although many studies have addressed the biochemical characteristics of the active toxin that corresponds to the N-terminal portion, there are just few reports studying the importance of the C-terminal part of the protoxin. Herein, we show that Cry1Ab protoxin has a unique natural cryptic endotoxic property that is evident when their halves are expressed individually. This toxic effect of the separate protoxin domains was found against its original host B. thuringiensis, as well as to two other bacteria, Escherichia coli and Agrobacterium tumefaciens. Interestingly, either the fusion of the C-terminal portion with the insecticidal domain-III or the whole N-terminal region reduced or neutralized such a toxic effect, while a non-Cry1A peptide such as maltose binding protein did not neutralize the toxic effect. Furthermore, the C-terminal domain, in addition to being essential for crystal formation and solubility, plays a crucial role in neutralizing the toxicity caused by a separate expression of the insecticidal domain much like a dot/anti-dot system. 相似文献
12.
T G Benoit G R Wilson D L Bull A I Aronson 《Applied and environmental microbiology》1990,56(8):2282-2286
Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores. 相似文献
13.
Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores. 相似文献
14.
Cheng Du Wing C. Chan Timothy W. McKeithan Kenneth W. Nickerson 《Applied microbiology》2005,71(6):3337-3341
The insecticidal protoxin from Bacillus thuringiensis has been shown to be a major component of the spore coat. We have developed a novel surface display system using B. thuringiensis spores in which the N-terminal portion of the protoxin is replaced with a heterologous protein. The expression vector with a sporulation-specific promoter was successfully used to display green fluorescent protein and a single-chain antibody (scFv) gene that encodes anti-4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (anti-phOx) antibody. The spores that carry the anti-phOx antibody can bind to phOx specifically. 相似文献
15.
Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis. 总被引:20,自引:5,他引:15
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The parasporal crystalline protoxin of Bacillus thuringiensis contains a single glycoprotein subunit that has a molecular weight of approximately 1.2 X 10(5). The carbohydrate consists of glucose (3.8%) and mannose (1.8%). At alkaline pH, the proendotoxin is apparently solubilized and activated by an autolytic mechanism involving an inherent sulfhydryl protease that renders the protoxin insecticidal. Activation generates protons, degraded polypeptides, sulfhydryl group reactivity, proteolytic activity, and insect toxicity. Chemical modification of the sulfhydryl groups inhibits the proteolytic and insecticidal activities, suggesting that cysteine residues may be present in the active site of the protein. 相似文献
16.
Selection and characterization of Bacillus thuringiensis strains toxic against pyralid stored‐product pests
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H. Azzouz J. Kebaili‐Ghribi F. Daoud N. Abdelmalak K. Ennouri N. Belguith‐Ben Hassan S. Tounsi S. Rouis 《Journal of Applied Entomology》2015,139(9):690-700
In this study, we present the selection and the characterization of Bacillus thuringiensis strains toxic against pyralid pests of stored products. Among 201 new B. thuringiensis strains isolated from different countries, two strains (BLB249 and BLB384) showed greater toxicity than the commercialized strain B. thuringiensis kurstaki HD1 against Ephestia kuehniella larvae. Morphological, molecular and biochemical investigations revealed that these strains were similar to HD1 but presented different cry gene content. Additional bioassays revealed that only strain BLB249 displayed higher toxicity than HD1 against Plodia interpunctella larvae. The study of Cry protoxin activation by midgut proteases of E. kuehniella and P. interpunctella larvae supported that higher toxicity of BLB249 and BLB384 strains compared to HD1 was not due to differential protoxin activation. Moreover, the toxic strains produced δ‐endotoxins and spores in similar amounts to HD1. Interestingly, the δ‐endotoxin production and the yield of BUPM26 strain were 32.87% and 35.12% greater than that of HD1. The potent insecticidal activities of BLB249 and BLB384 strains and the high level of δ‐endotoxin production by BUPM26 strain make them excellent candidates for use against E. kuehniella and P. interpunctella larvae. 相似文献
17.
The 16 cysteine residues of reduced protoxin from Bacillus thuringiensis subsp. kurstaki HD-73 can be quantitatively reacted with: (a) iodoacetic acid, to give carboxymethyl protoxin; (b) iodoacetamide, giving carbaminomethyl protoxin and (c) N-(beta-iodoethyl)trifluoroacetamide to give aminoethyl protoxin. The carboxymethyl derivative was found to be significantly more soluble at neutral pH values where both the native protoxin and the carbaminomethyl derivative exhibit low solubilities. At the alkaline pH values (pH 9.5-10.5) normally used to solubilize the crystal protein, the native protein was slightly more soluble than either the carboxymethyl or the carbaminomethyl derivatives. The aminoethyl derivative had an extremely low solubility at all pH values. Succinic anhydride reacted with only 35% of the lysine residues in both the carboxymethyl and the carbaminomethyl protoxin derivatives. Nonetheless, these succinylated protoxins exhibited significantly increased solubilities at neutral pH values. All the derivatives were found to retain full insecticidal activity toward spruce budworm (Choristeneura fufimerana) larvae. It is concluded that all the cysteine residues and modified lysine residues are on the surface of the protein and that derivatization does not alter the conformation of the solubilized protoxin. 相似文献
18.
Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests. 相似文献
19.
Eric J. Sturman Timothy J. Rydel Meiying Zheng Jeffrey W. Seale Sonya Franklin 《Protein science : a publication of the Protein Society》2014,23(11):1491-1497
For almost half a century, the structure of the full‐length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt‐derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice‐based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability. 相似文献
20.
Rose Monnerat Luke Masson Roland Brousseau Marianne Pusztai-Carey Dominique Bordat Roger Frutos 《Current microbiology》1999,39(3):159-162
Whole-crystal preparations from strains HD-1 and HD-133, activated Cry1Ab and Cry1C toxins as well as Cry1Aa, Cry1Ac, Cry1D,
and Cry2Aa protoxins were tested for toxicity to 2nd-instar larvae of the diamondback moth, Plutella xylostella. Mortality data recorded after 2 and 5 days provided different results that were related to differential rates of solubilization,
activation, and degradation of insecticidal crystal proteins. The two most active proteins are Cry1Ab and Cry1C, which are
both present in HD-133. The Cry1Ab protoxin is activated within 2 days, whereas activation of the Cry1C protoxin occurs between
2 and 5 days. HD-133 is more active than HD-1 immediately after infection and remains toxic over 5 days owing to the sequential
activation of its crystal components. Solubility properties of crystals and rates of activation of protoxins influence the
overall toxicity of HD-1 and HD-133 to the diamondback moth.
Received: 30 March 1999 / Accepted: 3 May 1999 相似文献