Evaluating the Insecticidal Genes and Their Expressed Products in Bacillus thuringiensis Strains by Combining PCR with Mass Spectrometry

By a combination of PCR and mass spectrometry, a total of five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cry1Ia) were detected in genomic DNA from the wild-type Bacillus thuringiensis strain 4.0718, and three protoxins (Cry1Aa, Cry1Ac, and Cry2Aa) were identified in the strain's parasporal crystals. These results indicated that this complementary method may be useful in evaluating B. thuringiensis strains at both the gene and protein levels.     

Cloning and Characterization of Two Novel Crystal Protein Genes, <Emphasis Type="Italic">cry54Aa1</Emphasis> and <Emphasis Type="Italic">cry30Fa1</Emphasis>, from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain BtMC28

Bacillus thuringiensis strain BtMC28 was isolated from the soil sample in China. Two novel crystal protein genes were found by using the PCR-RFLP method. Moreover, the full-length sequences of two novel genes were obtained by a single oligonucleotide nested (SON)-PCR upstream and downstream strategy. Sequence analysis revealed that one gene encoded a polypeptide of 673 amino acid residues with a molecular mass of 76.3 kDa, 38% identical to Cry10Aa, and the other encoded a polypeptide of 687 amino acid residues with a molecular mass of 77.1 kDa, 74% identical to Cry30Aa. These two novel crystal protein genes were designated as cry54Aa1 and cry30Fa1 by Bt Insecticidal Crystal Proteins Nomenclature Committee, respectively. The Cry54Aa1 and Cry30Fa1 proteins retained five conserved regions commonly found in the existing Cry proteins. Cry54Aa1 protein exhibited insecticidal activities against Laphygma exigua (Lepidoptera), Helicoverpa armigera (Lepidoptera), and Aedes aegypti (Diptera) when its encoding gene was expressed in an Escherichia coli host strain. The authors, Furong Tan and Jun Zhu contributed equally to this work.     

Characterisation of the cry54Ab1 operon of Bacillus thuringiensis subsp. sichuansis strain MC28

A novel cry gene operon harbouring two open reading frames (ORFs) was discovered in a large plasmid of Bacillus thuringiensis (Bt) subsp. sichuansis MC28 (pMC189). The first ORF encoded 743 amino acids. It had a deduced molecular mass of 84.5 kDa and 73.2% identity with Cry54Aa1 protein as indicated by amino acid homology. This cry gene was designated as cry54Ab1 by the Bt Toxin Nomenclature Committee. The second ORF encoded 503 amino acids and had a deduced molecular mass of 58.2 kDa. It is here called as orf2. The whole cry54Ab1 operon, cry54Ab1 and orf2 were digested with BamHI/SalI and inserted into a shuttle vector, pSTK. The recombinant plasmids were transferred into an acrystalliferous mutant Bt HD73^?. Parasporal inclusions were observed only under expression of the whole cry54Ab1 operon. The bioassay experiments showed that expressed products of the cry54Ab1 and the whole cry54Ab1 operon were all toxic to Culex quinquefasciatus (Diptera).     

Assignment of δ-Endotoxin Genes of the Four Lepidoptera-Specific Bacillus thuringiensis Strains that Produce Spherical Parasporal Inclusions

Unique strains of Bacillus thuringiensis, that belong to the four H serogroups (serovar sumiyoshiensis, serovar fukuokaensis, serovar darmstadiensis, and serovar japonensis) and produce spherical parasporal inclusions specifically toxic to lepidopteran larvae, were examined for comparative analysis of the genes encoding δ-endotoxin proteins. Gene analysis revealed that there is no difference between the four strains in nucleotide sequences of the 1,937-bp DNA segment covering the four conserved regions and a partial sequence of the block 5 region. Surprisingly, the nucleotide sequence of the four strains showed a 100% homology with that of the corresponding region of the cry9D gene encoding a δ-endotoxin protein, which had been reported to be active on the scarabaeid coleopterans. Alignment analysis revealed that the N-terminal half (16–660) amino acid sequence of the four proteins shared relatively high homologies (27.7–35.8%) with those of the Cry9Ba, Cry9Ca, and Cry1Ba proteins, while lower homologies with those of the Cry3Aa, Cry8Ca, and Cry1Aa proteins. The results show that the cry9D gene is retained in multiple heterogeneous H serovars of Lepidoptera-specific B. thuringiensis populations naturally occurring in soil environments of Japan. Received: 4 May 1998 / Accepted: 21 July 1998     

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.     

Characterisation and expression of a novel holotype crystal protein gene, <Emphasis Type="Italic">cry56Aa1</Emphasis>, from <Emphasis Type="Italic">Bacillus</Emphasis> <Emphasis Type="Italic">thuringiensis</Emphasis> strain Ywc2-8

Bacillus thuringiensis isolate Ywc2-8, from soil in Sichuan Basin in western China, contains a spherical crystal harbouring two insecticidal crystal proteins with masses of 70 kDa and 130 kDa. A novel cry-type gene, encoding a 664 amino acid protein with 34% homology to cry29Aa1, was found and cloned from this strain. This gene belongs to a novel holotype cry and is designated as cry56Aa1. It was expressed in E. coli. Insecticidal activity assays showed that recombinant Cry56Aa1 was toxic to both Dipteran (Aedes aegypti) and Lepidopteran (Plutella xylostella and Helicoverpa armigera) pests. Cloning of this gene may help to overcome the increasing resistance of pests to currently used insecticides.     

Design and construction of a synthetic <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry4Aa gene: Hyperexpression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is, therefore, of great interest for developing a bioinsecticide to control mosquitoes. However, the expression of Cry4Aa in Escherichia coli is relatively low, which is a major disadvantage in its development as a bioinsecticide. In this study, to establish an effective production system, a 1,914-bp modified gene (cry4Aa-S1) encoding Cry4Aa was designed and synthesized in accordance with the G + C content and codon preference of E. coli genes without altering the encoded amino acid sequence. The cry4Aa-S1 gene allowed a significant improvement in expression level, over five-fold, compared to that of the original cry4Aa gene. The product of the cry4Aa-S1 gene showed the same level of insecticidal activity against Culex pipiens larvae as that from cry4Aa. This suggested that unfavorable codon usage was one of the reasons for poor expression of cry4Aa in E. coli, and, therefore, changing the cry4Aa codons to accord with the codon usage in E. coli led to efficient production of Cry4Aa. Efficient production of Cry4Aa in E. coli can be a powerful measure to prepare a sufficient amount of Cry4Aa protein for both basic analytical and applied researches.     

A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Aδ-endotoxin as two separate proteins in Bacillus thuringiensis strain L366

A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060 ) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad . cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Aδ-endotoxin. The cry5Ad sequence includes homology blocks 1–5, which are present in most B. thuringiensis δ-endotoxins. The usual C-terminal region of a Cry5Aδ-endotoxin (including homology blocks 6–8) is encoded by orf2-5Ad . Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli , after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae ( Haemonchus contortus ), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.     

A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Adelta-endotoxin as two separate proteins in Bacillus thuringiensis strain L366.

A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad. cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Adelta-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisdelta-endotoxins. The usual C-terminal region of a Cry5Adelta-endotoxin (including homology blocks 6-8) is encoded by orf2-5Ad. Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli, after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae (Haemonchus contortus), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.     

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.     

Molecular Cloning of a New Crystal Protein Gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean Sericultural Farms

A new cry1Ab-type gene encoding the 130 kDa protein of Bacillus thuringiensis NT0423 bipyramidal crystals was cloned, sequenced, and expressed in a crystal-negative B. thuringiensis host. Hybridization experiments revealed that the crystal protein gene is located on a 44 MDa plasmid of B. thuringiensis NT0423. A strong positive signal detected on the 6.6 kb HindIII fragment from B. thuringiensis NT0423 plasmid DNA was cloned and sequenced. The cry1Ab-type gene, designated cry1Af1, consisted of open reading frame of 3453 bp, encoding a protein of 1151 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 130,215 Da. With both Bt I and Br II promoter sequences were found, the B. thuringiensis NT0423 crystal protein gene promoter closely aligned with those of cry1A-type crystal protein gene. When compared with known sequences of other Cry and Cyt proteins, the Cry1Af1 protein showed maximum 93% sequence identity to Cry1Ab protein of B. thuringiensis subsp. kurstaki. The expressed Cry1Af1 protein in a crystal-negative B. thuringiensis host appears to have strong insecticidal activity against lepidopteran larvae (Plutella xylostella). Crystals containing Cry1Af1 were about six times more toxic than the wild-type crystals of B. thuringiensis NT0423. Received: 20 February 2001 / Accepted: 17 April 2001     

Assessment of protoxin composition of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by use of polyacrylamide gel block and mass spectrometry

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zujiao Fu and Yunjun Sun contributed equally to this work.     

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

The dual-activity insecticidal protein,Cry2Aa,does not enhance the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis

Cry2Aa, one of the major insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki HD1, is known to be active against both lepidopteran and dipteran larvae. In order to determine whether Cry2Aa could enhance or synergize the mosquitocidal activity of B. thuringiensis subsp. israelensis, we constructed a plasmid vector that harbored the cry2Aa operon and transformed crystalliferous and acrystalliferous strains of this bacterium. The wild-type B. thuringiensis subsp. israelensis, a recombinant B. thuringiensis subsp. israelensis producing Cry2A along with its native major mosquitocidal proteins, and a recombinant B. thuringiensis subsp. israelensis producing Cry2Aa alone were tested against three major mosquito species — Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Our results demonstrated that Cry2Aa does not synergize or enhance the mosquitocidal activity of B. thuringiensis subsp. israelensis against these important vectors of disease.     

Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests

A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding a coleopteran-specific insecticidal protein was constructed and introduced into wild Bacillus thuringiensis subsp. aizawai G03, which contained cry1Aa, cry1Ac, cry1Ca, and cry2Ab genes and was highly toxic to lepidopteran insect pests. The genetically engineered strain were named G033A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry3Aa7 gene was expressed normally and produced a 67 kDa protein in G033A, and the flat rectangular crystals of Cry3Aa7 toxin protein was observed under scanning electron microscope. The recombinant plasmid was maintained in bacteria cultured for 180 generations in culture media containing no antibiotics. Synthesis of the Cry3Aa7 toxin conferred high and broad toxicity to the recombinant strain G033A against coleopteran order, elm leaf beetle (Pyrrhalta aenescens) (LC₅₀ 0.35 mg/ml), for which the parental strain G03 was not toxic. Both the parental strain G03 and recombinant strain G033A showed strong insecticidal activity to lepidopteran pests, beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella), and cotton bollworm (Helicoverpa amigera), respectively. The lethal concentration 50% (LC₅₀) of G033A against S. exigua, P. xylostella, and H. amigera was 4.26, 0.86, and 1.76 μg/ml, respectively.     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.     

Isolation and Characterization of a Bacillus thuringiensis ssp. kurstaki Strain Toxic to Spodoptera exigua and Culex pipiens

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species. Received: 19 January 2001 / Accepted: 21 February 2001     

An engineered Bacillus thuringiensis strain with insecticidal activity against Scarabaeidae (Anomala corpulenta) and Chrysomelidae (Leptinotarsa decemlineata and Colaphellus bowringi)

A genetically-engineered Bacillus thuringiensis (Bt) strain, 3A-HBF, with a broad insecticidal spectrum was constructed by introducing the recombinant plasmid pSTK-3A containing cry3Aa7 into the wild-type Bt strain HBF-1 containing the cry8Ca2 gene. The Cry3Aa7 protein produced by strain 3A-HBF was verified by SDS-PAGE and Western blotting. Flat rectangular crystals of Cry3Aa7 protein were observed besides spherical crystals (Cry8Ca2). The plasmid pSTK-3A was stable when strain 3A-HBF was grown in medium without antibiotics. The growth rate of 3A-HBF was not significantly different from that of the recipient strain, HBF-1. Strain 3A-HBF showed toxicity against two families of pests, Scarabaeidae and Chrysomelidae pests, which are susceptible to Cry8Ca (Anomala corpulenta) and Cry3Aa (Leptinotarsa decemlineata and Colaphellus bowringi). The 50% lethal concentrations of 3A-HBF against A. corpulenta, L. decemlineata and C. bowringi were 0.730 × 10⁸ c.f.u./g dry soil, 1.74 μg/ml and 1.15 μg/ml, respectively.     

Cloning of cry2Aa and cry2Ab genes from new isolates of Bacillus thuringiensis and their expression in recombinant Bacillus thuringiensis and Escherichia coli strains

Bacillus thuringiensis (Bt) is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of Bt are promising candidates for management of resistance development in insects due to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. Two insecticidal crystal protein genes of Bt, viz. cry2Aa and cry2Ab were cloned from new isolates of Bt, 22-4 and 22-11, respectively. Expression of both the genes was studied in an acrystalliferous strain of Bt (4Q7) by fusing the cry2Aa and cry2Ab genes downstream of cry2Aa promoter and orf1 + orf2 sequences. Western blot analysis revealed a low level expression of the cloned cry2Aa and cry2Ab genes in the recombinant Bt strains. High-level expression of cry2Aa and cry2Ab genes was achieved in the recombinant E. coli by cloning the cry2A genes under the control of the T7 promoter.