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.     

Analysis of non-active engineered Bacillus thuringiensis crystal proteins

Abstract Crystal proteins of Bacillus thuringiensis are known for their insecticidal specificity. This specificity is, to a large extent, determined by the interaction of the proteins with high-affinity binding sites on the epithelial membrane of the midgut of sensitive insects. In particular, domain II of the three domains of the toxic moiety has been implicated in specificity. To determine which sequences of the protein are involved in binding, loops of domain II which terminate in the molecular apex of CryIA(b) were replaced by the corresponding regions of CryIE, a protein with different binding characteristics and insect specificity. In contrast to expression of the wild-type genes, expression of the mutant alleles in Escherichia coli resulted in the formation of biologically inactive, insoluble aggregates. Although these aggregates could be solubilized in vitro using urea, in contrast to the wild-type CryIA(b), the mutant proteins did not correctly refold as is shown by their increased protease sensitivity and lack of biological activity. The results indicate that engineering CryI proteins, based on the CryIIIA structure, is likely to prove difficult, particularly since the conformation of CryIIIA and CryI proteins might differ in domain II.     

On the formation of crystal proteins during sporulation in Bacillus thuringiensis var. thuringiensis

The sporulation potential of Bacillus subtilis as a function of position in the cell cycle was determined by transferring cells from growth medium to sporulation medium at various times during growth. Growth was induced by incubating heat-activated spores in rich medium or by diluting stationary phase vegetative cultures with fresh growth medium. The results supported earlier observations that sporulation potential is cell cycle dependent. The rise in sporulation potential was studied by exposing cultures to the inhibitors of cell wall and protein synthesis, vancomycin and chloramphenicol. The delay in the appearance of the peak of sporulation potential caused by these inhibitors compared with the reported lack of effect of nalidixic acid, indicates that the appearance of sporulation potential requires synthesis of a macromolecular component other than deoxyribonucleic acid. The effect of nalidixic acid in preventing the decline of the sporulation potential was compared with the effect of high temperature on a mutant temperature sensitive for the initiation of DNA replication. It was found that prevention of chromosome completion with nalidixic acid maintained a high sporulation potential, whereas prevention of chromosome re-initiation in the temperature sensitive mutant did not affect the decline in sporulation potential as the cells enter stationary phase.Abbreviations NAL Nalidixic acid - HPUra 6-(p-hydroxyphenylazo)-uracil - VAN Vancomycin - CAM Chloramphenicol - BHI Brain heart infusion broth - c.f.u. Colony forming units     

Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.     

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.     

Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins

[This corrects the article on p. 1821 in vol. 59.].     

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.     

Bacillus thuringiensis Vip3 mutant proteins: Insecticidal activity and trypsin sensitivity

Vip3 is a novel insect toxin isolated from Bacillus thuringiensis (Bt), and could be used as an alternative toxin for Bt δ-endotoxins for transgenic insect control. Vip3 mutants with deletion, addition or mutations at the very end of the C-terminus were generated. The deletion and addition of a few amino acid residues at the C-terminus totally abolished the insecticidal activity. The mutation of the last two residues from IK to LG also resulted in the total loss of its insecticidal activity; however, the mutation from IK to LR increased its activity substantially against beet armyworm. Interestingly, all the inactive mutants were found to be highly sensitive to trypsin digestion, while the active mutant generated a trypsin-resistant polypeptide of 62-kDa upon trypsin digestion. However, this 62-kDa polypeptide expressed in E. coli from the 5' end truncated vip3 gene was biologically inactive and sensitive to trypsin digestion. Thus, the N-terminal part of the protein is required to form the 62-kDa trypsin resistant core. This study suggested that the 62-kDa peptide core could be a hallmark of active insecticidal Vip3 proteins.     

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins.

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.     

Insecticidal activity of Bacillus thuringiensis cells]

The influence of sixteen different nutrient media on the entomopathogenic activity of three Bacillus thuringiensis strains was studied. The medium composition based on potato, yeast extract, and molasses was optimized. B. thuringiensis No 1 grown on the media No 7 and 9 displayed the highest entomopathogenic activity (94.3 and 90.6%, respectively).     

Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins

Biotinylation of Bacillus thuringiensis insecticidal crystal proteins (ICPs) was evaluated for its potential use in an alternative ICP screening method and in the characterization of ICP receptors. In vivo biological activity of CryIA(b), as inferred from bioassays with Manduca sexta and Ostrinia nubilalis and from histopathological effects on O. nubilalis midgut cells induced by force feeding, was not affected by biotinylation at moderate biotinylation ratios. A competitive radioreceptor assay showed that there was only a minor reduction in binding affinity of biotin-labeled CryIA(b) for M. sexta brush border membrane vesicles. On midgut tissue sections, the binding pattern along the midgut epithelium and the staining intensity of biotinylated ICPs detected with streptavidin-enzyme conjugate were virtually identical to the binding pattern and staining intensity of native CryIA(b) detected with antibodies. The specificity of biotinylated ICP binding to larval midgut tissue was demonstrated by performing homologous competition experiments. The relationship between different ICP receptor types in Plutella xylostella, as inferred from radioligand binding studies, was confirmed by the results of heterologous competition experiments performed with biotinylated and native ICPs.     

苏云金芽孢杆菌杀虫晶体蛋白超量表达的机制

杀虫晶体蛋白是苏云金芽孢杆菌主要杀虫成分,进一步提高杀虫晶体蛋白的表达量是苏云金芽杆菌高效工程菌构建的主要途径。本文讨论了ｃｒｙ基因启动子活性、ｍＲＮＡ稳定性、不同ｃｒｙ基因间的协同表达发及伴了孢晶体的形成等几个方面在转录水平或转录后水平上对杀虫晶体蛋白表达的影响。     

杀蚊苏云金芽孢杆菌及其晶体蛋白研究进展

自从发现苏云金芽孢杆菌Bacillusthuringiensis(Bt)具有杀蚊活性以来,目前已发现多种Bt亚种或血清型对蚊虫具有杀虫活性,同时也发现了一些新的杀蚊晶体蛋白。在对杀蚊晶体蛋白的分子结构进行研究的基础上,对其的作用机理有了一定的了解。近年来利用DNA重组技术显著提高杀蚊晶体蛋白的合成和将不同菌种的杀蚊晶体蛋白进行联合表达,为有效控制蚊虫危害展示广阔前景。     

Biochemistry and genetics of insect resistance to Bacillus thuringiensis insecticidal crystal proteins

Abstract Current knowledge of biochemical mechanisms of insect resistance to Bacillus thuringiensis is reviewed. Available information on resistance inheritance and on patterns of cross-resistance is included. Modification of the binding sites for B. thuringiensis insecticidal crystal proteins has been found in different populations of three insect species. This resistance mechanism seems to be inherited as a single recessive or partially recessive major gene, and the resistance levels reached are high. Altered proteolytic processing of B. thuringiensis crystal proteins has been suggested to be involved in one case of resistance. From the available data it seems that binding site modification is the most significant resistance mechanism under field conditions.     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

Activated insecticidal crystal proteins from Bacillus thuringiensis serovars killed adult house flies

Insecticidal crystal proteins (ICP) from Bacillus thuringiensis serovar kurstaki HD-1 and HD-73 were activated by immobilized trypsin or chymotrypsin. The activated toxins (10 μ g or more) as well as unactivated ICP killed adult house flies but not larvae. Bacillus thuringiensis strain son diego did not kill house flies. In this experimental system, the average life span of the adult house fly was 8 days and the activated toxins reduced it to 2 days. The unactivated insecticidal crystal protein also reduced it to 4 days at the same concentration as the activated toxin.