Expression and characterization of a recombinant Cry1Ac crystal protein with enhanced green fluorescent protein in acrystalliferous Bacillus thuringiensis

AIMS: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B. thuringiensis Cry(-)B strain was examined. METHODS AND RESULTS: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter. The EGFP gene was cloned into pProMu and named pProMu-EGFP. The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm. The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody. The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB. However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae. CONCLUSIONS: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B. thuringiensis crystal protein and a foreign protein were validated.     

苏云金芽胞杆菌cry1Ac与烟草几丁质酶tchiB双价基因克隆表达及其杀虫增效作用研究

将苏云金芽胞杆菌(Bacillus thuringiensis,Bt)4.0718菌株质粒上的cry1Ac基因和烟草几丁质酶tchiB基因 (去掉信号肽或去信号肽再加肠激酶位点)构建了重组基因。经过双酶切和亚克隆,将带有cry1Ac基因上游启动序列和下游终止序列的重组基因片段克隆至穿梭载体pHT315,分别构建重组质粒pHUAccB6、pHUAccB7,在大肠杆菌中扩增后,将两个重组质粒分别电转化苏云金芽胞杆菌无晶体突变株XBU001中,获得重组菌株HAccB6和HAccB7。经液体双相胞晶分离提取离心后,将晶体和上清液分别进行SDS-PAGE分析,双价基因重组与cry1Ac基因在无晶体突变株中表达量相比较,几丁质酶活性提高5.2倍,双价重组蛋白表达量显著提高,主要产生130kDa蛋白条带。经定量分析：双价重组目的晶体蛋白占总蛋白量的61.38%;Cry1Ac蛋白占总蛋白量的42%。发酵上清液经60％硫酸铵沉淀,显示出一条分子量为18kDa新蛋白条带。经原子力显微镜和电子显微镜观察,表达后的重组蛋白呈菱形或椭原形晶体,其规格约为1.5×3.0μm;经生测分析,重组菌株HAccB6和 HAccB7毒力相近,与HAc菌株比较毒力提高4.5倍,对棉铃虫（Helicourpa armigora）具有高效杀虫活性,其3d LC₅₀值分别为9.1μg/mL和11.34μg/mL。研究结果表明,烟草几丁质酶与cry1Ac双价基因重组表达产物具有杀虫增效作用。     

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC₅₀ = 0.84 mg l^?1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.     

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.     

Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp. jegathesan encoding a highly mosquitocidal protein.

A gene, designated cry11B, encoding a 81,293-Da crystal protein of Bacillus thuringiensis subsp. jegathesan was cloned by using a gene-specific oligonucleotide probe. The sequence of the Cry11B protein, as deduced from the sequence of the cry11B gene, contains large regions of similarity with the Cry11A toxin (previously CryIVD) from B. thuringiensis subsp. israelensis. The Cry11B protein was immunologically related to both Cry11A and Cry4A proteins. The cry11B gene was expressed in a nontoxic strain of B. thuringiensis, in which Cry11B was produced in large amounts during sporulation and accumulated as inclusions. Purified Cry11B inclusions were highly toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. The activity of Cry11B toxin was higher than that of Cry11A and similar to that of the native crystals from B. thuringiensis subsp. jegathesan, which contain at least seven polypeptides.     

Expression and characterization of a recombinant Cry1Ac crystal protein fused with an insect-specific neurotoxin ω-ACTX-Hv1a in Bacillus thuringiensis

In order to assess possible enhancement of biopesticide activity, the fusion gene of crystal protein gene cry1Ac with the insect-specific neurotoxin ω-ACTX-Hv1a gene and egfp was expressed in Bacillus thuringiensis acrystalliferous strain Cry-B under the control of the native gene expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell and the GFP signal could be clearly observed. A 166 kDa full-length fusion protein was identified by immunoblot analysis. Virulence of the fusion inclusions was at least fivefold higher toward larvae of Spodoptera exigua. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in B. thuringiensis by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity.     

Isolation and characterization of a novel Bacillus thuringiensis strain expressing a novel crystal protein with cytocidal activity against human cancer cells

AIMS: To characterize a novel, unusual, Bacillus thuringiensis strain, to clone its Cry gene and determine the spectrum of action of the encoded Cry protein. METHODS AND RESULTS: The B. thuringiensis strain, referred to as M15, was isolated from dead two-spotted spider mites (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae). It is an autoagglutination-positive strain and is therefore non-serotypeable. A sporulated culture produces a roughly spherical parasporal inclusion body, the crystal, tightly coupled to the spore. Although the crystal appears to be composed of at least two major polypeptides of 86 and 79 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Southern hybridization indicates that the corresponding crystal protein gene is likely present in only one copy. The crystal protein gene was cloned and, based on nucleotide sequence homology with an orthologous cry31Aa1 gene, assigned the name cry31Aa2. Although initially isolated from spider mites, B. thuringiensis M15 is non-toxic to spider mites and it does not produce the wide spectrum beta-exotoxin. Assays on mammalian cells, however, reveal that Cry31Aa2, when cleaved with trypsin, is cytocidal to some human cancer cells but not to normal human cells. No cytocidal activity was induced after protease treatment of Cry31Aa2 with either chymotrypsin or proteinase K. Trypsin, chymotrypsin and proteinase K cleavage sites were determined. CONCLUSIONS: The B. thuringiensis strain M15 exhibits specific cytocidal activities against some human cancer cells. Significance and Impact of the Study: This study raises questions as to the actual role of this bacterial strain and its crystal protein in the environment. It may be possible to further develop the Cry31Aa2 protein to target specific human cancer cells.     

Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis.

A novel mosquitocidal protein gene, cry20Aa, was cloned from Bacillus thuringiensis subsp. fukuokaensis (H-3a: 3d: 3e). The gene product, Cry20Aa, was naturally truncated and had a molecular mass of 86,138 Da. The Cry20Aa protein possessed five conserved sequence blocks, as do most other insecticidal Cry toxins. However, an amino acid comparison of Cry20Aa with other mosquitocidal toxins, including Cry4A, Cry4B, Cry10A, Cry11A, and Cry11B, demonstrated that Cry20Aa was quite different from other toxins except for the conserved blocks. The N terminus of Cry20Aa was, however, homologous to the N termini of Cry4A and Cry10A. Interestingly, an inverted repeat (IR1) sequence in the open reading frame of the cry20Aa gene caused incomplete expression of Cry20Aa. When this internal IR1 sequence was altered with no change of amino acid sequence, acrystalliferous B. thuringiensis cells transformed with cry20Aa gene dramatically produced crystal inclusions. However, the intact 86-kDa Cry20Aa protein is highly labile, and it is rapidly degraded to polypeptides of 56 and 43 kDa. To increase expression of the cry20Aa gene, the p20 chaperonelike protein and the cyt1Aa promoter were utilized. While p20 did not increase Cry20Aa expression or stability, chimeric constructs in which the cry20Aa gene was under control of the cyt1Aa promoter overexpressed the Cry20Aa protein in acrystalliferous B. thuringiensis. The expressed Cry20Aa protein showed larvicidal activity against Aedes aegypti and Culex quinquefasciatus. However, the mosquitocidal activity was low, probably due to rapid proteolysis to inactive 56- and 43-kDa proteins.     

Comparative study on effect of different promoters on expression of cry1Ac in Bacillus thuringiensis chromosome

AIMS: The aim of this work was to investigate the effect of cry3A promoter on the expression of cry1Ac in Bacillus thuringiensis chromosome and stably enhance the production of different cry genes under the control of cry3A promoter. METHODS AND RESULTS: The cry1Ac gene, which is specific to Lepidopteran larvae, was integrated into the chromosome of a B. thuringiensis plasmid-free and acrystalliferous strain BMB171, under the control of cry3A promoter and cry1Ac promoter, respectively. The expression of cry1Ac genes in the chromosome of host strain was investigated. The results from sodium dodecyl sulfate-polyacrymide gel electrophoresis, crystal observation and bioassay showed that either integrated with cry3A promoter (cry3Apro-cry1Ac) or with its native promoter (cry1Acpro-cry1Ac), cry1Ac gene could efficiently and stably express in the chromosome. The production of cry3Apro-cry1Ac gene was higher than that of cry1Acpro-cry1Ac gene. CONCLUSIONS: The cry3A promoter enhanced the expression of cry1Ac gene efficiently either on the chromosome or on the plasmid in B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: So far, the comparative studies on cry3A promoter and other cry promoters were carried on B. thuringiensis plasmids. This system offers an additional method for potentially improving the efficacy of B. thuringiensis insecticidal proteins efficiently, stably and safely.     

Cloning and characterization of a novel gene cry9Ec1 encoding lepidopteran-specific parasporal inclusion protein from a Bacillus thuringiensis serovar galleriae strain

A novel delta-endotoxin gene from a lepidopteran-specific Bacillus thuringiensis serovar galleriae strain was cloned, and the full sequence of the cry gene was determined. The cloned 6.5-kb DNA fragment included the full sequence of the cry gene and three open reading frames located upstream of the cry gene. The gene, designated cry9Ec1, encodes a polypeptide of 1154 amino acid residues with a predicted molecular weight of 130 237. The deduced amino acid sequence of the Cry9Ec1 protein had the highest homology (77.7%) with the Cry9Ea1 protein when compared with existing Cry proteins. The expression, in an acrystalliferous B. thuringiensis strain, of the cry9Ec1 gene was high when controlled by the cyt1A2 promoter, leading to the formation of large spherical inclusions. The purified crystals from the recombinant strain were toxic when tested against two lepidopteran species, Bombyx mori and Plutella xylostella. However, the Cry9Ec1 protein gave no toxicity against Spodoptera litura, Spodoptera exigua, Plodia interpunctella, Helicoverpa zea, and Culex pipiens molestus.     

苏云金杆菌辅助蛋白P19对杀虫晶体蛋白Cry11Aa表达的影响

苏云金杆菌以色列亚种的p19基因、cry11Aa基因和p20基因位于同一操纵子上,据推测辅助蛋白P19可能与Cry11Aa蛋白的晶体化相关。本研究利用穿梭载体pHT3101构建了两个重组质粒pHcy1和pHcy3,两质粒均携带cry11Aa基因,但后者完全缺失了cry11Aa基因上游的p19基因。将重组质粒电激转化至苏云金杆菌无晶体突变株4Q7中进行蛋白表达,SDS-PAGE结果表明在4Q7(pHcy1)和4Q7(pHcy3)中均能检测到正常表达的Cry11Aa蛋白,但单位体积培养液的Cry11Aa蛋白在辅助蛋白P19存在时的表达量明显高于其单独表达的表达量;透射电镜观察显示两菌株中的Cry11Aa蛋白形成了大小相近、形状相似的双梯形晶体;另外,生物测定结果表明重组菌株4Q7(pHcy1)和4Q7(pHcy3)对三龄致倦库蚊的杀虫活性没有显著性差异。该现象说明辅助蛋白P19的缺失对Cry11Aa蛋白的晶体形成和杀蚊活性没有影响,但P19作为分子伴侣在一定程度上帮助提高了Cry11Aa蛋白的表达水平。     

苏云金芽孢杆菌的cry2A芽孢期启动子和分子伴侣ORF1-ORF2对Cry11Aa蛋白表达的影响

[目的]分析苏云金芽孢杆菌的cry2A型芽孢期启动子对晶体蛋白Cry11Aa的协调作用和分子伴侣ORF1-ORF2对Cry11Aa表达的促进功能.[方法]3个包括cry11Aa编码区的重组质粒pHcy1、pHcy2和pHcy4被构建并电激转化到苏云金芽孢杆菌晶体缺陷株4Q7中,其中pHcy1质粒携带cry11Aa基因自身启动子和分子伴侣p19基因,pHcy2携带cry2A型芽孢期启动子和分子伴侣orf1-orf2基因,pHcy4质粒在pHcy1的上游插入了cry2A型芽孢期启动子和分子伴侣orf1-orf2基因.SDS-PAGE分析了Cry11Aa蛋白在各重组苏云金菌株中的表达情况,并通过生物测定确定了其对蚊虫的生物活性.[结果]SDS-PAGE结果表明,Cry11Aa蛋白在4Q7(pHcy1)和4QT(pHcy4)均获得了表达,在4Q7(pHcy2)中未检测到Cry11Aa蛋白,推测晶体蛋白Cry11A不能利用cry2A型启动子进行表达调控;Cry11Aa蛋白在等体积4Q7(pHcy4)培养液中的表达量是4Q7(pHcy1)菌株的1.25倍,暗示着分子伴侣ORF1-ORF2在某种程度上能提高Cry11Aa的蛋白表达量.4Q7(pHcy1)和4Q7(pHcy4)形成的Cry11Aa蛋白晶体的形状和大小相似,两者对致倦库蚊的生物活性没有明显差异,LC50s分别为59.33 ng/mL和66.21 ng/mL,.[结论]推测晶体蛋白Cry11A能否成功表达与其使用启动子的类型和两者的协调配合有关.分子伴侣ORF1-ORF2虽然在某种程度上能提高Cry11Aa的蛋白表达量,但对提高Cry11Aa蛋白的杀蚊毒力没有显著性帮助.     

The 60-kilodalton protein encoded by orf2 in the cry19A operon of Bacillus thuringiensis subsp. jegathesan functions like a C-terminal crystallization domain

The cry19A operon of Bacillus thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of Culex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC(95)] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC(95) = 8.2 μg/ml).     

Disruption of a cadherin gene associated with resistance to Cry1Ac {delta}-endotoxin of Bacillus thuringiensis in Helicoverpa armigera

A laboratory strain (GY) of Helicoverpa armigera (Hubner) was established from surviving larvae collected from transgenic cotton expressing a Bacillus thuringiensis var. kurstaki insecticidal protein (Bt cotton) in Gaoyang County, Hebei Province, People's Republic of China, in 2001. The GYBT strain was derived from the GY strain through 28 generations of selection with activated Cry1Ac delivered by diet surface contamination. When resistance to Cry1Ac in the GYBT strain increased to 564-fold after selection, we detected high levels of cross-resistance to Cry1Aa (103-fold) and Cry1Ab (>46-fold) in the GYBT strain with reference to those in the GY strain. The GYBT strain had a low level of cross-resistance to B. thuringiensis var. kurstaki formulation (Btk) (5-fold) and no cross-resistance to Cry2Aa (1.4-fold). Genetic analysis showed that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive gene. The cross-resistance pattern and inheritance mode suggest that the Cry1Ac resistance in the GYBT strain of H. armigera belongs to "mode 1," the most common type of lepidopteran resistance to B. thuringiensis toxins. A cadherin gene was cloned and sequenced from both the GY and GYBT strains. Disruption of the cadherin gene by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. Tight linkage between Cry1Ac resistance and the cadherin locus was observed in a backcross analysis. Together with previous evidence found with Heliothis virescens and Pectinophora gossypiella, our results confirmed that the cadherin gene is a preferred target for developing DNA-based monitoring of B. thuringiensis resistance in field populations of lepidopteran pests.     

大肠杆菌中利用苏云金芽胞杆菌强启动子p1Ac指导Cry1Ac蛋白表达的特性分析

对利用苏云金芽胞杆菌(Bacillus thuringiensis,Bt)强启动子——cry1Ac基因启动子p1Ac指导cry基因在大肠杆菌中的表达进行了研究。结果显示,大肠杆菌中由启动子p1Ac指导表达的Cry1Ac蛋白与苏云金芽胞杆菌来源的Cry1Ac蛋白在碱溶性、胰蛋白酶活化、杀虫活性等方面有较好的一致性,从而解决了目前商业化载体大肠杆菌表达cry基因时形成不易溶解的包涵体问题。同时,还对p1Ac指导的cry1Ac基因在大肠杆菌中表达的发酵条件进行了初步探索。     

Use of a Cry1Ac-Resistant Line of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae) to Detect Novel Insecticidal Toxin Genes in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

This paper describes a screening strategy incorporating resistant insect lines for discovery of new Bacillus thuringiensis toxins against a background of known genes that would normally mask the activity of additional genes and the application of that strategy. A line of Helicoverpa armigera with resistance to Cry1Ac (line ISOC) was used to screen Cry1Ac-expressing strains of B. thuringiensis for additional toxins with activity against H. armigera. Using this approach, a number of Cry1Ac-producing strains with significant toxicity toward Cry1Ac-resistant H. armigera were identified. When the insecticidal protein complement of one of these strains, C81, was examined in detail, a novel cry2 gene (cry2Af1) was detected.