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 δ-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.     

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.     

Screening of Bacillus thuringiensis serotypes by polymerase chain reaction (PCR) for insecticidal crystal genes toxic against coffee berry borer

Using PCR,257 isolates of Bacillus thuringiensis(Bt) were screened for cry-type genes. Of 257 isolates/strains, 60 isolates were identified as cry7/8, 10 isolates as cry3 and 36 isolates as cry 1I. One specific strain of B. thuringiensis (sumiyoshiensis; T03B 001) was investigated for the presence of cry7 and cry8 genes. Genes Cry7 and cry8 were first detected in this strain using family primers prior to analysis by exclusion polymerase chain reaction (E-PCR) using specific type primers. E-PCR conducted with the above said primers led to the identification by agarose gel electrophoresis of a remaining 1.5 Kb family band indicating a potentially novel gene. This PCR product, (1.5 Kb), was purified from the gel and cloned in pGEM-T Easy vector. Twenty recombinant colonies bearing 1.5 Kb insert were identified and three randomly selected representatives of the group, clones 7, 8 and 10, were sequenced and compared to all cry7 and cry8 sequences available from Gene Bank. Alignments with available DNA and protein sequences showed that all these clones contained a gene related to cry8Aa1. Analysis using protein sequence alignment showed that the sequence from clone 7 differed from the closest relative, known under the new nomenclature as cry 8Aa1, by 44%. The crystal proteins from B. thuringiensis sumiyoshiensis (T03B 001) was toxic to coffee berry borer larvae.     

Identification and molecular characterization of novel cry1-type toxin genes from Bacillus thuringiensis K1 isolated in Korea

To clone novel cry1-type genes from the Bacillus thuringiensis K1 isolate, about 2.4-kb-long PCR fragments were amplified with two primer sets of ATG1-F/N400-R and 1BeATG1-F/N400-R. Using PCR-RFLP, three novel cry1-type genes, cry1-1, cry1-7, and cry1-44, were obtained from B. thuringiensis K1 and the complete coding sequences of these novel genes were analyzed. The Cry1-1, Cry1-7, and Cry1-44 proteins showed maximum similarities of about 78.0%, 99.7%, and 91.0% with the Cry1Ha1, Cry1Be1, and Cry1Ac2 proteins, respectively. These novel cry1-type genes were expressed using a baculovirus expression vector system and their insecticidal activities were investigated. Whereas all three novel genes were toxic to Plutella xylostella larvae, only Cry1-1 showed insecticidal activity against Spodoptera exigua larvae.     

Purification of an active fragment of Cry1Ie toxin from Bacillus thuringiensis

The cry1I genes from Bacillus thuringiensis are a class of special genes with unique characteristics; they are silent in B. thuringiensis strains but can be over-expressed in Escherichia coli, resulting in a Cry1I-type protein with a molecular mass of approximately 81kDa. Cry1I-type protein is toxic to Lepidoptera larvae. A truncated Cry1Ie protein, IE648, which corresponds to the first 648 amino acids from the N-terminus of Cry1Ie, was purified from E. coli using Ni-NTA affinity isolation, Q-Sepharose Fast Flow chromatography, and Superdex-200 size-exclusion chromatography. It was determined using laboratory bioassays that the purified IE648 protein has good insecticidal activity. Heterologous competitive binding assays show that IE648 does not compete with Cry1Ac for binding to the brush border membrane vesicles of the Asian corn borer and does not compete with Cry1Ac at concentrations below a 500-fold excess of unlabeled Cry1Ac for binding to the peritrophic matrix of the insect. This result implies that IE648 may be a good candidate as part of a multiple-toxin strategy for the potential control of resistance in insect pests. The method of purification reported here is valuable for further research on the structure and function of IE648 and in evaluating the biosafety of this protein within transgenic plants.     

Assessment of cry1 gene contents of Bacillus thuringiensis strains by use of DNA microarrays

A single Bacillus thuringiensis strain can harbor numerous different insecticidal crystal protein (cry) genes from 46 known classes or primary ranks. The cry1 primary rank is the best known and contains the highest number of cry genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (cryArray) to test the feasibility of using microarrays to identify the cry gene content of B. thuringiensis strains. Specific 50-mer oligonucleotide probes representing the cry1 primary and tertiary ranks were designed based on multiple cry gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the cryArray, several well-characterized B. thuringiensis strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primary- but not tertiary-ranked cry1I probes indicated the presence of a novel cry1I gene. Amplification and partial sequencing of the cry1I gene in strains IB360 and IB429 revealed the presence of a cry1Ia gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new B. thuringiensis isolates presenting interesting insecticidal activity.     

Rapid cloning, identification, and application of one novel crystal protein gene cry30Fa1 from Bacillus thuringiensis

In this study, a fast and efficient strategy has been developed for identifying and isolating novel cry genes from Bacillus thuringiensis by combining the PCR-restriction fragment length polymorphism and the single-oligonucleotide nested-PCR method. Using this method, one novel holotype cry gene, cry30Fa1 , encoding a polypeptide of 687 amino acid residues with a molecular mass of 77.1 kDa, 74% identical to Cry30Aa1, was cloned from the B. thuringiensis strain BtMC28. Furthermore, the cry30Fa1 gene was successfully expressed in Escherichia coli BL21 (DE3). The Cry30Fa1 proteins, isolated from the cultures of recombinant E. coli , had remarkable insecticidal effects against Plutella xylostella and Aedes aegypti with LC50 at 6.477 and 15.359 μg mL⁻¹, respectively. Our results strongly suggest that this strategy is highly efficient and advantageous in terms of rapid cloning of holotype cry genes that have minimal identity to known genes. The cloning of the cry30Fa1 gene would be useful in the resources of the insecticidal crystal genes and may serve as an alternative choice of an insecticide for potential problems associated with insect resistance.     

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.     

Contribution of the 65-kilodalton protein encoded by the cloned gene cry19A to the mosquitocidal activity of Bacillus thuringiensis subsp. jegathesan.

Two new crystal protein genes, cry19A and orf2, isolated from Bacillus thuringiensis subsp. jegathesan were cloned and characterized. The cry19A gene encodes a 74.7-kDa protein, and the orf2 gene encodes a 60-kDa protein. Cry19A contains the five conserved blocks present in most B. thuringiensis delta-endotoxins. The ORF2 amino acid sequence is similar to that of the carboxy terminus of Cry4 proteins. The cry 19A gene was expressed independently or in combination with orf2 in a crystal-negative B. thuringiensis host. The proteins accumulated as inclusions. Purified inclusions containing either Cry19A alone or Cry19A and ORF2 together were toxic to Anopheles stephensi and Culex pipiens mosquito larvae. They were more toxic to C. pipiens than to A. stephensi. However, inclusions containing Cry19A and ORF2 together were more toxic than inclusions of Cry19A alone but less toxic than the wild-type inclusions of B. thuringiensis subsp. jegathesan.     

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.     

Cloning, characterization and expression of a new cry1Ab gene from Bacillus thuringiensisWB9

A new cry 1Ab-type gene, cry 1Ab17, was cloned from Bacillus thuringiensis WB9 by PCR. Nucleotide sequence indicated that the open reading frames (ORFs) consists of 3471 bases and encodes a protein of 1156 residues with a calculated molecular weight of 130.5 kDa and an pI value of 5.04. Homology comparison revealed that the deduced amino acid sequence of Cry1Ab17 had 95.4% to 99.7% identity with those of the known Cry1Ab proteins. The Cry1Ab17 was one residue longer than the known Cry1Ab (except for Cry1Ab2). Domain I (Tyr(33) to Arg(253)), II (Arg(265) to Phe(462)), III (Asn(464) to Thr(610)) of the Cry1Ab17 were 96.8%, 68.2% and 100% identical to the corresponding domains of Cry1Aa. Additionally, the cry 1Ab17 gene was expressed in Escherichia coli BL21 under the control of T7 promoter and the Cry1Ab17 isolated from the culture medium was toxic to 3rd instar Plutella xylostella larvae.     

Correspondence of high levels of beta-exotoxin I and the presence of cry1B in Bacillus thuringiensis

Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 micro g of beta-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 micro g/ml. However, these amounts of beta-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of beta-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of beta-exotoxin I.     

RFLP analysis of cry1 and cry2 genes of Bacillus thuringiensis isolates from India

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.     

Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains.

An extended PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to lepidopteran, coleopteran, and dipteran pests (Ben-Dov et al., Appl. Environ. Microbiol. 63:4883-4890, 1997). To optimize identification of all reported cry genes, this methodology needs a complete PCR set of primers. In the study reported here, a set of universal (Un9) and specific primers for multiplex rapid screening for all four known genes from the cry9 group was designed. PCR analyses were performed for cry9 genes on 16 standard strains and 215 field isolates of B. thuringiensis. Among the standard strains, only B. thuringiensis subsp. aizawai HD-133, which harbors cry1 and cry2 genes, was positive with Un9 but negative to all four specific primers for cry9 genes. DNA of 22 field-collected isolates was also found to be positive with Un9. These isolates were classified into three cry9 profiles using specific primers; all of them harbor cry1 and cry2. This newly designed set of primers complements the existing PCR methodology for most currently known cry genes.     

Cloning and expression of the cry1Ea4 gene of Bacillus thuringiensis and the comparative toxicity of its gene product

A new cry gene (cry1Ea4) was cloned and sequenced from a Bacillus thuringiensis isolate native to Mexico (LBIT-147). The gene coded for a 133kDa protoxin which had greater than 99% homology with the holotype Cry1Ea1, as only four mismatches were found between the two amino acid sequences. When the Cry1Ea4 toxin was expressed in a crystal-negative strain of B. thuringiensis, bipyramidal crystals were produced. Purified crystals from this recombinant strain and from the holotype (Cry1Ea1) were bioassayed against first instar larvae of the tobacco hornworm. Statistically different mean LC₅₀ values indicated that Cry1Ea4 was more toxic than its holotype. This increase in toxicity may be attributed to the three amino acids which differ from the holotype sequence in the toxic fragment.     

Isolation and characterization of strain of Bacillus thuringiensis subsp. kenyae containing two novel cry1-type toxin genes

To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cry1-5 and cry1-12, in addition to the cry1Ja1 gene. The deduced amino acid sequences of cry1-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and Cry1Ja crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.     

A novel Bacillus thuringiensis strain LLB6, isolated from bryophytes, and its new cry2Ac-type gene

AIMS: To isolate and characterize the novel Bacillus thuringiensis strains from bryophytes collected from Wuyi Mountain, Fujian Province of China, and identify new B. thuringiensis strains and toxins active against mosquitoes. METHODS AND RESULTS: Twelve novel B. thuringiensis strains were isolated from 76 bryophyte samples. According to the results of this preliminary screening, LLB6 was the most toxic to Aedes albopictus. Then phase-contrast as well as scanning electron microscopy, bioassays, cloning, sequencing and expression were performed to characterize the novel isolate LLB6 and its new gene cry2Ac5. CONCLUSIONS: Bacillus thuringiensis occurred naturally on bryophytes. LLB6 isolated from Physcomitrium japonicum was toxic to A. albopictus. A new cry2Ac5 gene of LLB6 was detected, cloned and expressed successfully. Bioassays on A. albopictus showed that the expressed Cry2Ac5 was also toxic to the third instar larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of B. thuringiensis strains isolated from bryophytes. It represents a specific source of new B. thuringiensis strains and is of great importance for the knowledge of the ecology of B. thuringiensis. Novel LLB6 harboring the new gene cry2Ac5 and its expressed Cry2Ac5 protein revealed activity against A. albopictus and became a new member of B. thuringiensis toxins.     

Cloning and expression of two crystal protein genes, cry30Ba1 and cry44Aa1, obtained from a highly mosquitocidal strain, Bacillus thuringiensis subsp. entomocidus INA288

Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.