Cloning of a New Bacillus thuringiensis cry1I-Type CrystalProtein Gene

A new cry1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cry1Ib, and Cry1Ic proteins, respectively. The upstream sequence of the cry1Id1 structural gene was not functional as promoter in B. subtilis. The Cry1Id1 protein, purified from recombinant E. coli cells, had a toxicity comparable to that of Cry1Ia against Plutella xylostella, but it was significantly less active than Cry1Ia against Bombyx mori. Cry1Id1 was not active against the coleopteran insect, Agelastica coerulea. Received: 19 January 2000 / Accepted: 22 February 2000     

New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

Characterization of cry Genes in a Mexican Bacillus thuringiensis Strain Collection

Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1, cry3, cry5, cry7, cry8, cry9, cry11, cry12, cry13, cry14, cry21, and cyt genes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containing cry1 genes were the most abundant in our collection (49.5%). Thirty-three different cry1-type profiles were identified. B. thuringiensis strains harboring cry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes. cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carrying cry5, cry12, cry13, cry14, or cry21 genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.     

Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments

This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.     

Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.     

Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China

Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.     

Characterization of a cry2A Gene Cloned from an Isolate of Bacillus thuringiensis serovar sotto

A cry2A-type gene, designated as cry2(SKW), was cloned from Bacillus thuringiensis serovar sotto SKW01-10.2-06, and some unique features of the gene were revealed. The cry2(SKW) gene encoded a polypeptide of 635 residues with a predicted molecular mass of 71,137 Da. Cry2(SKW) had 95.4% identity with Cry2Aa in amino acid sequence and was two residues longer than Cry2Aa. Two open reading frames (ORFs), designated as orf1 and orf2, were present upstream of the cry2(SKW) and showed high homology with the corresponding ORFs in the cry2Aa operon. The Orf2 from SKW01-10.2-06 contained a region of repeated sequences. However, unlike Cry2Aa, Cry2(SKW) formed the cuboidal crystalline inclusions when the cry2(SKW) gene was expressed in an acrystalliferous B. thuringiensis strain in the absence of the upstream ORFs. Furthermore, Cry2(SKW) was less toxic to a lepidopteran species, Bombyx mori, than Cry2Aa in spite of high homology between the two proteins. Received: 17 July 1996 / Accepted: 5 December 1996     

Cloning and Characterization of Two Novel Genes, cry24B and s1orf2, from a Mosquitocidal Strain of Bacillus thuringiensis serovar sotto

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.     

Characterization of the cry1Ac17 Gene from an Indigenous Strain of Bacillus thuringiensis subsp. kenyae

An indigenously isolated strain of Bacillus thuringiensis subsp. kenyae exhibited toxicity against lepidopteran as well as dipteran insects. The lepidopteran active cry1Ac protoxin gene coding sequence of 3.5 kb from this strain was cloned into vector pET28a(+). However, it could not be expressed in commonly used Escherichia coli expression hosts, BL21(DE3) and BL21(DE3)pLysS. This gene is classified as cry1Ac17 in the B. thuringiensis toxic nomenclature database. The coding sequence of this gene revealed that it contains about 3% codons, which are not efficiently translated by these expression hosts. Hence, this gene was expressed in a modified expression host, Epicurian coli BL21-Codonplus (DE3)-RIL. The expression of gene yielded a 130-kDa Cry1Ac17 protein. The protein was purified and its toxicity was tested against economically important insect pests, viz., Helicoverpa armigera and Spodoptera litura. LC₅₀ values obtained against these insects were 0.1 ng/cm³ and 1231 ng/cm², respectively. The higher toxicity of Cry1Ac17 protein, compared to other Cry1Ac proteins, toward these pests demonstrates the potential of this isolate as an important candidate in the integrated resistance management program in India.     

苏云金芽孢杆菌B-Pr-88菌株中cry2Ab4基因的表达和杀虫活性研究

以我室自行分离的对鳞翅目夜蛾科害虫具有高毒力的Bt菌株B-Pr-88为材料,用PCR-RFLP方法从其质粒DNA文库中筛选到含cry2Ab基因的一个阳性克隆pZF858,序列测定发现,该片段含有cry2Ab全长基因,开放读码框为1902bps,编码由633个氨基酸组成的70.7kD蛋白,氨基酸同源性与已公布的cry2Ab基因同源性均为99.8%,经Bt基因国际命名委员会正式命名为cry2Ab4。根据cry2Ab4基因开放阅读框架(ORF)两端序列,设计合成一对特异引物L2ab5和L2ab3,PCR扩增获得cry2Ab4完整ORF,与大肠杆菌表达载体pET-21b连接,构建了重组表达质粒pET-2Ab4,质粒导入大肠杆菌BL21(DE3),IPTG诱导后,SDS-PAGE电泳证实该基因表达了60kD的蛋白,生物测定表明,Cry2Ab4对棉铃虫和大豆食心虫具有高毒力,同时对小菜蛾和二化螟有一定的杀虫活性,而对亚洲玉米螟和甜菜夜蛾没有杀虫活性。     

Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

On the basis of the known cry gene sequences of Bacillus thuringiensis, three sets of primers were designed from four conserved blocks found in the delta-endotoxin-coding region. The primer pairs designed amplify the regions between blocks 1 and 5, 2 and 5, and 1 and 4. In silico analyses indicated that 100% of the known three-domain cry gene sequences can be amplified by these sets of primers. To test their ability to amplify known and unknown cry gene sequences, 27 strains from the CINVESTAV (LBIT series) collection showing atypical crystal morphology were selected. Their DNA was used as the template with the new primer system, and after a systematic amplification and sequencing of the amplicons, each strain showed one or more cry-related sequences, totaling 54 different sequences harbored by the 27 strains. Seven sequences were selected on the basis of their low level of identity to the known cry sequences, and once cloning and sequencing of the complete open reading frames were done, three new cry-type genes (primary ranks) were identified and the toxins that they encode were designated Cry57Aa1, Cry58Aa1, and Cry59Aa1 by the B. thuringiensis Toxin Nomenclature Committee. The rest of the seven sequences were classified Cry8Ka2, Cry8-like, Cry20Ba1, and Cry1Ma1 by the committee. The crystal morphology of the selected strains and analysis of the new Cry protein sequences showed interesting peculiarities.Bacillus thuringiensis has been safely used for the control of insect pests within the orders Lepidoptera, Coleoptera, and Diptera for the last 50 years (19). It is an aerobic, Gram-positive bacterium whose insecticidal activity is based on the presence of parasporal crystalline inclusions formed during the sporulation process. These parasporal bodies or crystals are assembled by the so-called Cry proteins, expressed by the cry genes (21).B. thuringiensis shows great variability, as has been demonstrated by the huge number of strains isolated around the world (19), by the number of serotypes known to date (a total of 84) (20), and by the great number of different cry gene sequences accumulated so far (a total of 492) (8), as well as by the number of molecular characterization tools that have been developed, such as sequencing of the flagellin gene and of the gyrB and aroE genes, the band patterns from repetitive extragenic palindromic-PCR analyses, and the plasmid patterns, among others (17, 18, 25, 27), all indicating the great variability within this species.In spite of this variability, some similarity has been found in the three-domain Cry proteins, evidenced by the presence of three conserved domains in the tertiary structure of the active toxin (delta-endotoxin), even from Cry toxins showing low levels of identity (i.e., the Cry1-type, Cry3-type, and Cry4-type toxins): one domain with a bundle of α helices and two domains of β sheets (3). This uniformity is, at least in part, a reflection of the five conserved blocks in the gene structure, which are present in almost all the cry genes (10).The great number of sequences known to date is mostly a result of the strong interest in finding novel Cry proteins, with the foci being on three main purposes: (i) the search for a new range of activities, (ii) the search for higher levels of toxicity, and (iii) the search for alternative toxins in case of resistance development. The search for novel Cry toxins has followed different strategies, such as (i) the development of DNA libraries and their expression in acrystalliferous mutants of B. thuringiensis (24); (ii) hybridization with degenerate oligonucleotides, partial sequences, or complete cry genes used as probes (5, 12); and (iii) PCR amplification of putative novel cry genes by using multiple primers (14) or a combination of general primers, followed either by restriction analysis (26) or by a second amplification using a mixture of general and specific primers (13).These strategies have shown certain levels of efficiency in detecting new cry genes, but they also have some limitations. For example, the screening of a large library is time-consuming and requires a high level serendipity, and the use of Southern analysis with either degenerate or specific probes is limited to finding only sequences related to known cry genes. The amplification of putatively new cry genes by PCR has been widely used in a series of different strategies. However, most of them are limited to finding genes with sequences similar to those used in the primer design. Previous attempts to design universal primers used only nine cry types, which significantly limits the search for actual new genes. Besides, the new sequences described were limited to the amplicons and no new cry genes were reported (6).This report describes the design of a system of three sets of primers, based on conserved regions of the cry family, potentially able to amplify all the known three-domain cry genes. The use of this strategy with B. thuringiensis strains showing atypical crystal morphology allowed the identification of three new cry types and four more new cry holotypes.     

Bt4.0718 cry1Ac基因的克隆与表达分析

在基因库中比对14种cry1Ac基因序列,发现了同源性很高的上游启动子区域和下游终止子区域。根据这一同源序列设计引物,从B t4.0718中扩增出包含双启动子和终止子的4.2 kb片段,用PCR-RFLP检测确定其中含有cry1Ac基因。然后将此片段克隆到穿梭载体pHT304中,转化大肠杆菌DH5α和B t无晶体突变株XZM-101。同时,利用原子力显微镜观察发现重组菌株BXZM34能够产生菱形晶体。     

苏云金芽胞杆菌杀虫晶体蛋白基因cry1Ab16的克隆及表达

通过对已知cry1类基因以及已发表的cry1Ab的序列进行分析,分别设计了引物P1、P2、P3和P4,首次从无晶体的芽胞杆菌AC11中扩增到一个苏云金芽胞杆菌杀虫晶体蛋白（Insecticidal crystal protein, ICP）cry1Ab类基因。测序结果显示该基因与已知的cry1Ab1基因有8个核苷酸不同,编码的蛋白有7个氨基酸差异。此基因已登录GenBank,并命名为新亚型基因cry1Ab16 (Ac. NO. AF375608)。Southern杂交结果进一步证实该基因存在于菌体的质粒上。将cry1Ab16基因克隆到Escherichia coli表达载体pQE30上并转化E. coli M15。Western印迹分析表明,E. coli M15表达了130 kD的Cry1Ab16蛋白,但此蛋白不稳定,大部分降解成65 kD的蛋白。将表达Cry1Ab16 蛋白的大肠杆菌用涂布法对三龄小菜蛾（Plutella xylostella）毒力测定,其LC50为258.3mg/L;对其他夜蛾科害虫的生长发育也有明显的抑制作用。     

Cloning of Two New cry Genes from Bacillus thuringiensis subsp. wuhanensis Strain

With PCR products as probes, we have cloned two new cry-type genes from Bacillus thuringiensis subsp. wuhanensis. The deduced amino acid sequence of the first clone is 77.3% identical to Cry1Ga1. The deduced protein sequence of the second clone is 69.8–78.7% identical to that of Cry1B group. The nomenclature assignment of these two clones is, therefore, named Cry1Gb1 and Cry1Bd1, respectively. The Cry1Bd1 is toxic to Plutella xylostella larvae, and the Cry1Gb1 is toxic to Pieris rapae larvae. Received: 2 August 1999 / Accepted: 18 October 1999     

Cloning, Characterization, and Expression of a New cry1Ab Gene from DOR Bt-1, an Indigenous Isolate of Bacillus thuringiensis

A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper (Achaea janata L.) cadavers from castor bean (Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as cry1Ab26 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(+) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.     

Genetic Diversity of cry Gene Sequences of Bacillus thuringiensis Strains Analyzed by Denaturing Gradient Gel Electrophoresis

PCR has been widely used to identify cry-type genes, to determine their distribution, to detect new such genes and to predict insecticidal activities. We describe here a molecular approach to analyze the genetic diversity of B. thuringiensis cry-like genes based on denaturing gradient gel electrophoresis (DGGE). This analysis demonstrated that different B. thuringiensis isolates can be distinguished according to its PCR-DGGE profile of cry-like genes. Identification of the resolvable DNA fragments was easy to accomplish by DNA sequencing, which was confirmed in this work. Importantly, the strategy allowed the identification of unknown B. thuringiensis cry-like sequences present in a single strain that remained cryptic after PCR analysis using degenerate primers. The method developed in this work contributes to the availability of molecular techniques for both B. thuringiensis strains and cry-like genes identification and discovery.     

Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae

Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with ¹²⁵I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution.     

苏云金芽胞杆菌cry2Ab、cry9Ea基因的表达及活性分析

旨在为农业害虫防治提供更多的苏云金芽胞杆菌基因资源,从中国吉林市龙潭山土壤样品中分离得到野生菌株命名为Bt LTS-7,扫描电镜显示该菌株产生晶体形状为双锥体和正方体,聚丙烯酰胺凝胶电泳显示该菌株产生130 kD和71 kD的晶体蛋白,通过PCR鉴定出该菌株中含有cry2Ab和cry9Ea基因,并成功克隆到了这两个新基因,并被Bt国际命名委员会正式命名为cry2Ab28和cry9Ea9,将两个基因分别在大肠杆菌Rosetta( DE3)中表达,并进一步对其表达蛋白进行杀虫活性测定.结果显示,Cry2Ab28蛋白对棉铃虫(Helicoverpa armigera)初孵幼虫具有杀虫活性,LC50为32.45 μg/mL.Cry9Ea9蛋白对小菜蛾初孵幼虫(Plutella xylostella)具有较高杀虫活性,LC50为0.77μg/mL.     

Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp. kurstaki

A 4 kb BamHI-HindIII fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp. kurstaki strain BNS3, was cloned. The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues. Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes. The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.