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1.  Screening by polymerase chain reaction of Bacillus thuringiensis serotypes for the presence of cryV-like insecticidal protein genes and characterization of a cryV gene cloned from B. thuringiensis subsp. kurstaki. 
Polymerase chain reaction screening using cryV-specific oligonucleotides, designed to amplify the 5' half of cryV-type genes, revealed the presence of such genes in 7 of 21 Bacillus thuringiensis serotypes examined. Restriction analysis and hybridization studies indicated that these putative genes fall into at least three subclasses. The nucleotide sequence of the cryV-type gene cloned from B. thuringiensis subsp. kurstaki DSIR732 revealed an open reading frame coding for a protein of 719 amino acids, and lysates of Escherichia coli cells expressing the 81.2-kDa CryV732 protein were toxic to Epiphyas postvittana (Lepidoptera: Tortricidae).
PMCID: PMC182139  PMID: 8517758
2.  Biochemical and molecular characterization of the insecticidal fragment of CryV. 
Two C-terminal deletion constructs were made to study the effect of such deletions on the biological activity of the CryV protein of Bacillus thuringiensis subsp. kurstaki. The results of feeding on neonatal larvae of Ostrinia nubilalis (European corn borer [ECB]) indicated that the 50% lethal dose of the full-length CryV protein was 3.34 micrograms/g of diet (95% fiducial limits, 2.53 to 4.32 micrograms/g of diet). Removal of 71 amino acids (aa) from the C terminus had little effect on toxicity, whereas deletion of 184 aa abolished the insecticidal activity of the CryV protein completely. Truncations of the full-length CryV protein were also generated with trypsin and the midgut protease of ECB. The proteolytically treated products were characterized by determining their N-terminal amino acid sequences. The CryV protein was found to be cleaved by both proteases through a two-step process. Initially an intermediary form was generated which contained aa 45 of full-length CryV as its N-terminal end. The C-terminal end of this peptide was not experimentally determined. However, analysis of the deduced amino acid sequence of CryV indicated that the C-terminal end of the intermediary form is likely either aa 655 or 659. Further N-terminal processing of the intermediary form resulted in a protease-resistant core form. The core included aa 156 to aa 655 or 659. While the intermediary form retained 100% of the ECB larval toxicity, the core form exhibited only approximately 22% of the toxicity of the full-length protein.
PMCID: PMC168576  PMID: 9212427
3.  Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase. 
Journal of Bacteriology  1996;178(7):2141-2144.
A CryV-type protein (CGCryV) has been isolated from supernatant fluids of Bacillus thuringiensis AB88 cultures. Previous reports have suggested the cryptic nature of the cryV-type genes on the basis of the absence of CryV-type proteins in parasporal crystals. The CryV-type protein reported here is expressed early in stationary phase, and evidence indicates that it is an exported protein. Analysis of the deduced protein sequence from this gene reveals the presence of an N-terminal domain that likely acts as a signal peptide. The CGCryV protein is the first reported case of a delta-endotoxin being a secreted protein, which may influence the biological relevance of these proteins.
PMCID: PMC177917  PMID: 8606196
4.  Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. 
Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains.
PMCID: PMC167904  PMID: 8919799
5.  Characterization of cry Genes in a Mexican Bacillus thuringiensis Strain Collection 
Applied and Environmental Microbiology  1998;64(12):4965-4972.
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.
PMCID: PMC90950  PMID: 9835590
6.  Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai. 
Journal of Bacteriology  1991;173(13):3966-3976.
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.
PMCID: PMC208042  PMID: 2061280
7.  A Change in a Single Midgut Receptor in the Diamondback Moth (Plutella xylostella) Is Only in Part Responsible for Field Resistance to Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai 
A population (SERD3) of the diamondback moth (Plutella xylostella L.) with field-evolved resistance to Bacillus thuringiensis subsp. kurstaki HD-1 (Dipel) and B. thuringiensis subsp. aizawai (Florbac) was collected. Laboratory-based selection of two subpopulations of SERD3 with B. thuringiensis subsp. kurstaki (Btk-Sel) or B. thuringiensis subsp. aizawai (Bta-Sel) increased resistance to the selecting agent with little apparent cross-resistance. This result suggested the presence of independent resistance mechanisms. Reversal of resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai was observed in the unselected SERD3 subpopulation. Binding to midgut brush border membrane vesicles was examined for insecticidal crystal proteins specific to B. thuringiensis subsp. kurstaki (Cry1Ac), B. thuringiensis subsp. aizawai (Cry1Ca), or both (Cry1Aa and Cry1Ab). In the unselected SERD3 subpopulation (ca. 50- and 30-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai), specific binding of Cry1Aa, Cry1Ac, and Cry1Ca was similar to that for a susceptible population (ROTH), but binding of Cry1Ab was minimal. The Btk-Sel (ca. 600-and 60-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) and Bta-Sel (ca. 80-and 300-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) subpopulations also showed reduced binding to Cry1Ab. Binding of Cry1Ca was not affected in the Bta-Sel subpopulation. The results suggest that reduced binding of Cry1Ab can partly explain resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai. However, the binding of Cry1Aa, Cry1Ac, and Cry1Ca and the lack of cross-resistance between the Btk-Sel and Bta-Sel subpopulations also suggest that additional resistance mechanisms are present.
PMCID: PMC1389152  PMID: 16535597
8.  Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins. 
Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.
PMCID: PMC182717  PMID: 2014985
9.  Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins. 
We compared responses to six insecticidal crystal proteins from Bacillus thuringiensis by a Cry1A-resistant strain (NO-QA) and a susceptible strain (LAB-P) of the diamondback moth, Plutella xylostella. The resistant strain showed > 100-fold cross-resistance to Cry1J and to H04, a hybrid with domains I and II of Cry1Ab and domain III or Cry1C. Cross-resistance was sixfold to Cry1Bb and threefold to Cry1D. The potency of Cry1I did not differ significantly between the resistant and susceptible strains. Cry2B did not kill resistant or susceptible larvae. By combining these new data with previously published results, we classified responses to 14 insecticidal crystal proteins by strains NO-QA and LAB-P. NO-QA showed high levels of resistance to Cry1Aa, Cry1Ab, and Cry1Ac and high levels of cross-resistance to Cry1F, Cry1J, and H04. Cross-resistance was low or nil to Cry1Ba, Cry1Bb, Cry1C, Cry1D, Cry1I, and Cry2A. Cry1E and Cry2B showed little or no toxicity to susceptible or resistant larvae. In dendrograms based on levels of amino acid sequence similarity among proteins, Cry1F and Cry1J clustered together with Cry1A proteins for domain II, but not for domain I or III. High levels of cross-resistance to Cry1Ab-Cry1C hybrid H04 show that although Cry1C is toxic to NO-QA, domain III or Cry1C is not sufficient to restore toxicity when it is combined with domains I and II of Cry1Ab. Thus, diamondback moth strain NO-QA cross-resistance extends beyond the Cry1A family of proteins to at least two other families that exhibit high levels of amino sequence similarity with Cry1A in domain II (Cry1F and Cry1J) and to a protein that is identical to Cry1Ab in domain II (H04). The results of this study imply that resistance to Cry1A alters interactions between the insect and domain II.
PMCID: PMC168069  PMID: 8702276
10.  Determination and Distribution of cry-Type Genes of Bacillus thuringiensis Isolates from Taiwan 
Using PCR with a set of specific oligonucleotide primers to detect cryI-type genes, we were able to screen the cry-type genes of 225 Bacillus thuringiensis soil isolates from Taiwan without much cost in time or labor. Some combinations of cry genes (the cry-type profile) in a single isolate were unique. We identified five distinct profiles of crystal genes from the B. thuringiensis soil isolates from Taiwan. The cry genes included cryIA(a), cryIA(b), cryIA(c), cryIC, cryID, and cryIV. Interestingly, 501 B. thuringiensis isolates (93.5% of the total number that we identified) were isolated from areas at high altitudes. The profiles of cry-type genes were distinct in all isolation areas. The distribution of cry-type genes of our isolates therefore depended on geography. Using PCR footprinting to detect cryIC-type genes, we identified two distinct cryIC footprints from some of our isolates, indicating that these isolates may contain novel cryIC-type genes. B. thuringiensis isolates containing cryIA(a)-, cryIA(b)-, and cryIA(c)-type genes exhibited much greater activity against Plutella xylostella than did other isolates, indicating that multiple cry-type genes may be used as markers for the prediction of insecticidal activities.
PMCID: PMC201665  PMID: 16349324
11.  Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae. 
A novel cryIC-type gene was isolated from a strain of Bacillus thuringiensis subsp. galleriae. A new polymerase chain reaction (PCR) technique with a set of several oligonucleotide primer pairs specific to the cryIC gene was used to screen a number of B. thuringiensis strains. PCR amplified several DNA fragments ranging from 100 bp to 1 kb for B. thuringiensis strains containing a cryIC gene. PCR fragments amplified from the Bacillus thuringiensis subsp. galleriae HD29 DNA differed from the fragments amplified from other cryIC-containing strains, indicating strain HD29 contained a novel cryIC-type gene. To isolate crystal genes homologous to cryIC, an HD29 gene library was probed with a 984-bp fragment of the amino-terminal coding region of the cryIC gene cloned from Bacillus thuringiensis subsp. aizawai HD229. A putative toxin gene was isolated from a phage that hybridized strongly to the cryIC probe. Translation of the putative toxin DNA sequence revealed an open reading frame of 1,176 amino acids whose predicted molecular mass was 132.8 kDa. Comparisons of the toxin gene sequence with sequences of other cry genes indicated that this gene is a subclass of cryIC. We propose to designate this gene cryIC(b). In Escherichia coli, the cryIC(b) gene produced a protein of approximately 130 kDa toxic to Spodoptera exigua and Trichoplusia ni.
PMCID: PMC202250  PMID: 8476286
12.  An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1-Type Genes 
Applied and Environmental Microbiology  2013;79(21):6706-6711.
The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.
PMCID: PMC3811518  PMID: 23995930
13.  Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes. 
A two-step procedure was used to place a cryIC crystal protein gene from Bacillus thuringiensis subsp. aizawai into the chromosomes of two B. thuringiensis subsp. kurstaki strains containing multiple crystal protein genes. The B. thuringiensis aizawai cryIC gene, which encodes an insecticidal protein highly specific to Spodoptera exigua (beet armyworm), has not been found in any B. thuringiensis subsp. kurstaki strains. The cryIC gene was cloned into an integration vector which contained a B. thuringiensis chromosomal fragment encoding a phosphatidylinositol-specific phospholipase C, allowing the B. thuringiensis subsp. aizawai cryIC to be targeted to the homologous region of the B. thuringiensis subsp. kurstaki chromosome. First, to minimize the possibility of homologous recombination between cryIC and the resident crystal protein genes, B. thuringiensis subsp. kurstaki HD73, which contained only one crystal gene, was chosen as a recipient and transformed by electroporation. Second, a generalized transducing bacteriophage, CP-51, was used to transfer the integrated cryIC gene from HD73 to two other B. thuringiensis subsp. kurstaki stains. The integrated cryIC gene was expressed at a significant level in all three host strains, and the expression of cryIC did not appear to reduce the expression of the endogenous crystal protein genes. Because of the newly acquired ability to produce the CryIC protein, the recombinant strains showed a higher level of activity against S. exigua than did the parent strains. This two-step procedure should therefore be generally useful for the introduction of an additional crystal protein gene into B. thuringiensis strains which have multiple crystal protein genes and which show a low level of transformation efficiency.
PMCID: PMC167583  PMID: 7487039
14.  Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera 
The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD50]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD50 of HD-1 with the FFD50 expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.
PMCID: PMC183447  PMID: 16348504
15.  Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis. 
Journal of Bacteriology  1988;170(10):4732-4738.
A gene encoding a 72,357-dalton (Da) crystal protein of Bacillus thuringiensis var. israelensis was isolated from a native 75-MDa plasmid by the use of a gene-specific oligonucleotide probe. Bacillus megaterium cells harboring the cloned gene (cryD) produced significant amounts of the 72-kDa protein (CryD), and the cells were highly toxic to mosquito larvae. In contrast, cryD-containing Escherichia coli cells did not produce detectable levels of the 72-kDa CryD protein. The sequence of the CryD protein, as deduced from the sequence of the cryD gene, was found to contain regions of homology with two previously described B. thuringiensis crystal proteins: a 73-kDa coleopteran-toxic protein and a 66-kDa lepidopteran- and dipteran-toxic protein of B. thuringiensis subsp. kurstaki. A second gene encoding the B. thuringiensis subsp. israelensis 28-kDa crystal protein was located approximately 1.5 kilobases upstream from and in the opposite orientation to the cryD gene.
PMCID: PMC211515  PMID: 2902069
16.  Bacillus thuringiensis monogenic strains: screening and interactions with insecticides used against rice pests 
Brazilian Journal of Microbiology  2012;43(2):618-626.
The screening of Bacillus thuringiensis (Bt) Cry proteins with high potential to control insect pests has been the goal of numerous research groups. In this study, we evaluated six monogenic Bt strains (Bt dendrolimus HD-37, Bt kurstaki HD-1, Bt kurstaki HD-73, Bt thuringiensis 4412, Bt kurstaki NRD-12 and Bt entomocidus 60.5, which codify the cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1C, cry2A genes respectively) as potential insecticides for the most important insect pests of irrigated rice: Spodoptera frugiperda, Diatraea saccharalis, Oryzophagus oryzae, Oebalus poecilus and Tibraca limbativentris. We also analyzed their compatibility with chemical insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil), which are extensively used in rice crops. The bioassay results showed that Bt thuringiensis 4412 and Bt entomocidus 60.5 were the most toxic for the lepidopterans, with a 93% and 82% mortality rate for S. frugiperda and D. saccharalis, respectively. For O. oryzae, the Bt kurstaki NRD-12 (64%) and Bt dendrolimus HD-37 (62%) strains were the most toxic. The Bt dendrolimus HD-37 strain also caused high mortality (82%) to O. poecilus, however the strains assessed to T. limbativentris caused a maximum rate of 5%. The assays for the Bt strains interaction with insecticides revealed the compatibility of the six strains with the four insecticides tested. The results from this study showed the high potential of cry1Aa and cry1Ba genes for genetic engineering of rice plants or the strains to biopesticide formulations.
PMCID: PMC3768848  PMID: 24031872
Bacillus thuringiensis; Cry proteins; Insects
17.  Genetic and Biochemical Characterization of Field-Evolved Resistance to Bacillus thuringiensis Toxin Cry1Ac in the Diamondback Moth, Plutella xylostella 
Applied and Environmental Microbiology  2004;70(12):7010-7017.
The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.
PMCID: PMC535196  PMID: 15574894
18.  Genetic and Biochemical Approach for Characterization of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Field Population of the Diamondback Moth, Plutella xylostella 
Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F4 to F8) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F9 found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F9. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F9 to F13) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F15). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F2 progeny from a backcross of F1 progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.
PMCID: PMC92015  PMID: 10742234
19.  Insecticidal Activity of Bacillus thuringiensis Cry1Bh1 against Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and Other Lepidopteran Pests 
Applied and Environmental Microbiology  2013;79(24):7590-7597.
Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis-resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis-resistant strains of Plutella xylostella and Spodoptera frugiperda. Cry1Bh1 is active against susceptible and B. thuringiensis-resistant colonies of O. nubilalis, P. xylostella, and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda. Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.
PMCID: PMC3837808  PMID: 24077715
20.  Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species. 
Applied and Environmental Microbiology  1992;58(12):3921-3927.
Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2.
PMCID: PMC183205  PMID: 1476436
21.  Unique regulation of crystal protein production in Bacillus thuringiensis subsp. yunnanensis is mediated by the cry protein-encoding 103-megadalton plasmid. 
In sporulating cultures of Bacillus thuringiensis subsp. yunnanensis HD977, two cell types are observed: cells forming only spores and cells forming only crystals. Curing analysis suggested that the crystal proteins are plasmid encoded. Through plasmid transfer experiments, it was established that a 103-MDa plasmid is involved in the crystal production. Conjugal transfer of this plasmid to Cry- recipient cells of Bacillus thuringiensis subsp. kurstaki HD73-26 conferred the ability to produce crystals exclusively on asporogenous cells of the recipient, indicating that the 103-MDa plasmid mediates the unique regulation of Cry protein production. When the dipteran-specific cryIVB gene was introduced into wild-type (Cry+) and Cry- backgrounds of B. thuringiensis subsp. yunnanensis by phage CP51ts45-mediated transduction, similar to all other B. thuringiensis strains, irregular crystals of CryIVB protein were produced by spore-forming cells in both backgrounds. However, the synthesis of the bipyramidal inclusions of B. thuringiensis subsp. yunnanensis was still limited only to asporogenous cells of the transductant. Thus, it appears that the unique property of exclusive crystal formation in asporogenous cells of B. thuringiensis subsp. yunnanensis is associated with the crystal protein gene(s) per se or its cis acting elements. As the crystals in B. thuringiensis subsp. yunnanensis were formed only in asporogenous cells, attempts were made to find out whether crystal formation had any inhibitory effect on sporulation. It was observed that both Cry+ and Cry- strains of B. thuringiensis subsp. yunnanensis (HD977 and HD977-1, respectively) exhibited comparable sporulation efficiencies. In addition, the Cry- B. thuringiensis subsp. kurstaki host (HD73-26) and its Cry+ transconjugant (HD73-26-16), expressing the B. thuringiensis subsp. yunnanensis crystal protein, were also comparable in their sporulation efficiencies, indicating that production of the crystal proteins of B. thuringiensis subsp. yunnanensis does not affect the process of sporulation.
PMCID: PMC168575  PMID: 9212426
22.  Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. 
Journal of Bacteriology  1989;171(2):965-974.
Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryB1 and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryB1 gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryB1 is the distal gene of an operon which is comprised of three open reading frames (designated orf1, orf2, and cryB1). The proteins encoded by cryB1 and orf2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orf1 or cryB2 gene products are present in cuboidal crystals. The protein encoded by orf2 has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orf1 and orf2 are not toxic to either test insect.
PMCID: PMC209689  PMID: 2914879
23.  A Holistic Approach for Determining the Entomopathogenic Potential of Bacillus thuringiensis Strains 
Applied and Environmental Microbiology  1998;64(12):4782-4788.
The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed. Cloning and sequencing of the cry1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B. thuringiensis isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B. thuringiensis strains may be higher than previously thought.
PMCID: PMC90922  PMID: 9835562
24.  Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp. jegathesan encoding a highly mosquitocidal protein. 
Applied and Environmental Microbiology  1995;61(12):4230-4235.
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.
PMCID: PMC167734  PMID: 8534090
25.  Differential Role of Manduca sexta Aminopeptidase-N and Alkaline Phosphatase in the Mode of Action of Cry1Aa, Cry1Ab, and Cry1Ac Toxins from Bacillus thuringiensis 
Applied and Environmental Microbiology  2013;79(15):4543-4550.
Aminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in Escherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP- or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp. kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.
PMCID: PMC3719532  PMID: 23686267

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