Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action

The spore-forming bacterium Bacillus thuringiensis produces intracellular inclusions comprised of protoxins active on several orders of insects. These highly effective and specific toxins have great potential in agriculture and for the control of disease-related insect vectors. Inclusions ingested by larvae are solubilized and converted to active toxins in the midgut. There are two major classes, the cytolytic toxins and the delta-endotoxins. The former are produced by B. thuringiensis subspecies active on Diptera. The latter, which will be the focus of this review, are more prevalent and active on at least three orders of insects. They have a three-domain structure with extensive functional interactions among the domains. The initial reversible binding to receptors on larval midgut cells is largely dependent upon domains II and III. Subsequent steps involve toxin insertion into the membrane and aggregation, leading to the formation of gated, cation-selective channels. The channels are comprised of certain amphipathic helices in domain I, but the three processes of insertion, aggregation and the formation of functional channels are probably dependent upon all three domains. Lethality is believed to be due to destruction of the transmembrane potential, with the subsequent osmotic lysis of cells lining the midgut. In this review, the mode of action of these delta-endotoxins will be discussed with emphasis on unique features.     

昆虫中肠液性质对苏云金芽孢杆菌伴孢晶体毒力的影响

综述了昆虫中肠液性质对苏云金芽孢杆菌Bacillus thuringiensis伴孢晶体毒力的影响。中肠液的酸碱度和蛋白酶是影响伴孢晶体溶解与原毒素活化的两大因素。中肠液的酸碱度不仅影响到伴孢晶体的溶解速度,还影响到各种蛋白酶的活性表现;而蛋白酶则直接参与了原毒素的活化,其组成与活性影响着原毒素的活化速度和杀虫专一性。因中肠液蛋白水解能力过高而导致原毒素的过度降解是某些昆虫对苏云金芽孢杆菌低度敏感的主要原因,而中肠液对原毒素活化能力的降低则与昆虫抗性的形成有关。此外,中肠液的沉淀作用及其它生理生化特性也影响着原毒素毒力的正常发挥。     

Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: A critical review

Bacillus thuringiensis (Bt) Cry toxins constitute the active ingredient in the most widely used biological insecticides and insect-resistant transgenic crops. A clear understanding of their mode of action is necessary for improving these products and ensuring their continued use. Accordingly, a long history of intensive research has established that their toxic effect is due primarily to their ability to form pores in the plasma membrane of the midgut epithelial cells of susceptible insects. In recent years, a rather elaborate model involving the sequential binding of the toxins to different membrane receptors has been developed to describe the events leading to membrane insertion and pore formation. However, it was also proposed recently that, in contradiction with this mechanism, Bt toxins function by activating certain intracellular signaling pathways which lead to the necrotic death of their target cells without the need for pore formation. Because work in this field has largely focused, for several years, on the elaboration and promotion of these two models, the present revue examines in detail the experimental evidence on which they are based. It is concluded that the presently available information still supports the notion that Bt Cry toxins act by forming pores, but most events leading to their formation, following binding of the activated toxins to their receptors, remain relatively poorly understood.     

Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae

Bacillus thuringiensis is a Gram‐positive aerobic bacterium that produces insecticidal crystalline inclusions during sporulation phases of the mother cell. The virulence factor, known as parasporal crystals, is composed of Cry and Cyt toxins. Most Cry toxins display a common 3‐domain topology. Cry toxins exert intoxication through toxin activation, receptor binding and pore formation in a suitable larval gut environment. The mosquitocidal toxins of Bt subsp. israelensis (Bti) were found to be highly active against mosquito larvae and are widely used for vector control. Bt subsp. jegathesan is another strain which possesses high potency against broad range of mosquito larvae. The present review summarizes characterized receptors for Cry toxins in mosquito larvae, and will also discuss the diversity and effects of 3‐D mosquitocidal Cry toxin and the ongoing research for Cry toxin mechanisms generated from investigations of lepidopteran and dipteran larvae.     

Cryptic endotoxic nature of Bacillus thuringiensis Cry1Ab insecticidal crystal protein

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.     

Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis

Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.     

Differential effects of pH on the pore-forming properties of Bacillus thuringiensis insecticidal crystal toxins

The effect of pH on the pore-forming ability of two Bacillus thuringiensis toxins, Cry1Ac and Cry1C, was examined with midgut brush border membrane vesicles isolated from the tobacco hornworm, Manduca sexta, and a light-scattering assay. In the presence of Cry1Ac, membrane permeability remained high over the entire pH range tested (6.5 to 10.5) for KCl and tetramethylammonium chloride, but was much lower at pH 6.5 than at higher pHs for potassium gluconate, sucrose, and raffinose. On the other hand, the Cry1C-induced permeability to all substrates tested was much higher at pH 6.5, 7.5, and 8.5 than at pH 9.5 and 10.5. These results indicate that the pores formed by Cry1Ac are significantly smaller at pH 6.5 than under alkaline conditions, whereas the pore-forming ability of Cry1C decreases sharply above pH 8.5. The reduced activity of Cry1C at high pH correlates well with the fact that its toxicity for M. sexta is considerably weaker than that of Cry1Aa, Cry1Ab, and Cry1Ac. However, Cry1E, despite having a toxicity comparable to that of Cry1C, formed channels as efficiently as the Cry1A toxins at pH 10.5. These results strongly suggest that although pH can influence toxin activity, additional factors also modulate toxin potency in the insect midgut.     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.     

苏云金芽孢杆菌及其杀虫晶体蛋白 作用机制的研究进展

综合叙述了苏云金芽胞杆菌Bacillus thuringiensis和杀虫晶体蛋白的作用机制及在不同水平上解释这些机制的一些流行模型和有关亚分子结构的作用。     

Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

Summary Previous studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm). Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane. In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system. We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances. These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations. These properties appeared to be pH dependent. At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed. Among the channels recorded at pH 6.0, a 25–35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses. We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.The assistance of A. Mazza and G.A.R. Mealing is gratefully acknowledged. The trypsin-activated, HPLC-purified CryIC toxin isolated from B. thuringiensis var. entomocidus crystal was a kind gift from M. Pusztai, Institute for Biological Sciences, NRC, Ottawa.     

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.     

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。     

增强Bt Cry毒素杀虫作用的重要途径:增效因子的利用及晶体蛋白的遗传改良

Bt Cry毒素广泛应用于害虫防治,但存在杀虫谱窄、活性低、靶标害虫易产生抗性等缺点.为了弥补这些不足,采取适当措施增强Cry毒素的杀虫作用十分必要.本文围绕Cry毒素的作用机理,论述了利用丝氨酸蛋白酶抑制剂、几丁质酶、增效蛋白、钙粘蛋白片段和Cyt毒素等增效因子提高Cry毒素的杀虫活性,延缓昆虫抗性的研究进展;探讨了利用基因定点突变、蛋白融合和杂交以及晶体蛋白末端小片段的去除等分子技术手段对毒素蛋白进行遗传改良,改善Cry毒素的杀虫性能,扩大其杀虫范围.     

苏云金芽孢杆菌的筛选和初步鉴定

采集四川温江昆虫孳生地的土壤样本94份,利用醋酸钠-抗生素法分离、筛选,共获得苏云金芽孢杆菌9株。镜检可观察到大菱形、小菱形、方形、圆形等四种主要形态的伴胞晶体;采用cryⅠ、cryⅡ、cryⅢ、cryⅤ基因的通用对9株Bt分离菌进行的PCR检测结果表明：9株菌全部含cryⅠ和cryⅤ基因,2株菌含cryⅡ基因,5株菌含cryⅢ基因,而且各菌株均包含2—3个基因型。利用这些菌株对菜青虫进行了室内和室外生物毒力测定,达到了较好的杀虫效果。     

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.     

Mechanism of action of Bacillus thuringiensis insecticidal delta-endotoxin: interaction with phospholipid vesicles

Bacillus thuringiensis (Bt) crystal delta-endotoxin from three subspecies and the product of a cloned crystal protein gene were activated in vitro and their interaction with phospholipid liposomes studied. Despite their diverse spectrum of activity, all these toxins were found to cause a rapid increase in the light scattering of liposome suspensions, which reflects a morphological change in the lipid bilayer. When liposomes loaded with radioactive markers were incubated with B. thuringiensis aizawai IC1 toxin, a relatively rapid release of more than 70% of the trapped markers occurred after an initial lag. Activated Bta IC1 and B. thuringiensis israelensis toxins were shown to bind to phospholipid vesicles. Two of the five conserved domains (D1-D5) detectable in the sequence of a range of Bt toxins are predicted to be highly hydrophobic. It is suggested that these, together with an additional conserved hydrophobic region showing structural homology and two predicted amphiphilic helices, play a major part in the interaction of these toxins with target membranes.     

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.     

Identification of an insecticidal crystal protein from Bacillus thuringiensis DSIR517 with significant sequence differences from previously described toxins.

The nucleotide (nt) sequence of a DNA segment containing the majority of a gene cloned from Bacillus thuringiensis DSIR517 encoding a 130 kDa insecticidal crystal protein has been determined. Sequence analysis reveals an open reading frame (ORF) of 3453 nt. The ATG initiation codon, which is preceded by a potential ribosome-binding site sequence, was confirmed by N-terminal amino acid sequencing. The ORF extends beyond the 3' terminus of the cloned fragment; however, the high degree of homology between the deduced amino acid sequence of this ORF and other Cry proteins suggests the clone lacks only five C-terminal amino acids. Making this assumption, the ORF of 3468 nt encodes a protein of 1156 amino acids with an estimated molecular mass of 129700 Da. Analysis of the deduced amino acid sequence reveals a number of features characteristic of Cry proteins. Alignment of the Cry 517 protein sequence with other Cry proteins suggests it is most closely related to the cryIA-E genes but sufficiently different to form a new cryI gene subclass.