Development of a recombinant strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> HD-73 that produces the endochitinase ChiA74

Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine–Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.     

`Cloning of <Emphasis Type="Italic">vip1/vip2</Emphasis> genes and expression of Vip1Ca/Vip2Ac proteins in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Forty-one Bacillus thuringiensis (Bt) standard reference strains and 118 Bt local isolates were screened for vip1/vip2 genes by PCR amplification, with only three strains (HD201, HD109 and HD12) producing the desired bands. Southern blot showed that vip1/vip2 genes were located on a 10 Kb EcoRV fragment of their total DNAs. Furthermore, the vip1Ca/vip2Ac genes were cloned from a partial genomic library of HD201. Sequence homologous analysis revealed that vip2Ac gene was highly conserved and encoded a protein possibly having ADP-ribosyltransferase activity, and that vip1Ca gene was of low homology, especially at its 3-terminus. Western blot showed that Vip1Ca and Vip2Ac proteins could be detected from middle logarithmic phase to the stationary phase in Bt HD201. However, bioassays of HD201 supernatants exhibited no activity against Culex quinquefasciatus, Spodoptera exigua, S. litura, Helicoverpa amigera and Tenebrio molitor larvae. Whether Vip1Ca and Vip2Ac proteins have any toxicity to other susceptible targets still needs to be investigated.     

Characterization of cultured insect cells selected by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> crystal toxin

Summary Selection for resistance against Bacillus thuringiensis (Bt) Cry1Ac10 in the Trichoplusia ni (Hübner) cell line BTI-TN-5B1-4 (TnH5) was tested, and the development of resistance in the selected cells was like a S-form curve. Monitoring at the Cry1Ac10 50th challenge, the resistance ratio was 1, 294-fold as many as that of initial cells. But the resistance to Cry1Ac10 declined gradually when the selection was relaxed. The resistance declined rapidly at the low level of resistance and slowly at the high level of resistance. This resistant cell had high resistance to all the tested solubilized trypsin-treated mixture of crystal multitoxins of B. thuringiensis subsp. aizawai GC-91, an engineering bacterium of Bt, B. thuringiensis subsp. aizawai HD-133 and B. thuringiensis subsp. kurstaki HD-1, and low cross-resistance (19.7-fold) to activated Cry1C. Both N-acetyl-d-galactosamine (GalNAc) and tunicamycin did not inhibit the toxicity of Cry1Ac10 to the susceptible TnH5 cells. Comparison of the total proteins of the selected resistant cells with that of the nonselected susceptible cells by two-dimensional electrophoresis analysis showed that were obvious differences among the 11 protein expression. These results strongly suggest that there exists an unknown mechanism of resistance in the cell line that was different from the reported mechanisms in insects.     

The theoretical 3D structure of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry5Ba

Cry5Ba is a δ-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.     

Strategy for amplification and sequencing of insecticidal <Emphasis Type="Italic">cry1A</Emphasis> genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A feasible and fully described strategy, with a detailed list of primers, for amplifying, cloning and sequencing known and potentially novel cry1A genes harboured by a Bacillus thuringiensis strain was successfully established. Based on the analysis of conserved regions of the cry1A genes, the 1AF and 1UR oligonucleotide primers were designed to amplify the whole open reading frame of these genes. The PCR products obtained revealed the successful amplification of cry1A genes from 13 B. thuringiensis strains. These bacteria were previously known to harbour at least one cry1A gene. An Argentinean B. thuringiensis isolate INTA Mo1-12 was randomly chosen for cloning and sequencing of cry1A genes by using a primer set developed in this study. Both nucleotide and amino acid sequences similarity analysis revealed that cry1Aa and cry1Ac from B. thuringiensis INTA Mo1-12 are new natural variants, showing several differences with the other known cry1A subclasses. These genes were named by the B. thuringiensis Pesticidal Crystal Protein Nomenclature Committee as cry1Aa15 and cry1Ac21 respectively.     

Effects of constant light on circadian rhythmicity in mice lacking functional <Emphasis Type="Italic">cry</Emphasis> genes: dissimilar from <Emphasis Type="Italic">per</Emphasis> mutants

Mutations in each of the genes mPer1, mPer2, mCry1 and mCry2 separately cause deviations from the wild type circadian system. Differences between these mutant strains have inspired the hypothesis that the duality of circadian genes (two mPer and two mCry genes involved) is related to the existence of two components in the circadian oscillator (Daan et al., J Biol Rhythms 16:105–116, 2001). We tested the predictions from this theory that the circadian period (τ) lengthens under constant illumination (LL) in mCry1 and mPer1 mutant mice, while it shortens in mCry2 and mPer2 mutants. mCry1 ^−/− and mCry2 ^−/− knockout mice both consistently increased τ with increasing light intensity, as did wild type mice. With increasing illumination, rhythmicity is reduced in mCry1, mCry2 and mPer1, but not in mPer2 deficient mice. Results for mPer mutant mice are in agreement with data reported on these strains earlier by Steinlechner et al. (J Biol Rhythms 17:202–209, 2002), and also with the predictions from the model. The increase in cycle length of the circadian system by light in the mCry2 deficient mice violates the predictions. The model is thereby rejected: the mCry genes do not play a differential role, although the opposite responses of mPer mutants to light remain consistent with a functional Evening–Morning differentiation.     

Screening of <Emphasis Type="Italic">cry2</Emphasis> genes in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolates from Argentina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identification of cry2 genes from Bacillus thuringiensis (Bt) was established. Strains from different sources of Argentina were analyzed to study the distribution of cry2 genes. The results showed that cry2Aa/cry2Ab profile was the most abundant irrespective of source and represented 56 of 59 Bt isolates (94.9%). Three different cry2 profiles were found in this collection, one of which was novel.     

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.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

The chemotactic characteristics of the S and R dissociants of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The chemotactic responses of Bacillus thuringiensis subsp. dendrolimus (strain 49) and thuringiensis (strain 2002) and their morphological dissociants were studied by using some natural and artificial substances as effectors. The 12-h-old wild-type cells (S variants) of both strains were found to be motile and similar in their chemotactic responses, whereas the chemotactic responses of the R variants were different.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 87–91.Original Russian Text Copyright © 2005 by Lebenko, Sekerina, Chemerilova.     

Cry2A toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> expressed in insect cells are toxic to two lepidopteran insects

The cry2Aa and cry2Ab genes from a Brazilian Bacillus thuringiensis strain were introduced into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in order to evaluate the heterologous proteins expression in insect cells and their toxicity to different insects. The recombinant viruses (vAcCry2Aa and vSynCry2Ab) were amplified in Trichoplusia ni (BTI-Tn5B1-4) cells and used to infect Spodoptera frugiperda larvae. Total extracts from S. frugiperda infected with the recombinant viruses were analysed by SDS-PAGE, which detected the presence of polypeptides around 65 kDa. Cuboid-shaped protein crystals were observed in insect extracts by light and scanning electron microscopy. Bioassays, using the heterologous proteins showed toxicity against second instar A. gemmatalis larvae (Cry2Aa) with a LC₅₀ of 1.03 μg/ml and second instar S. frugiperda larvae (Cry2Ab) with a LC₅₀ of 3.45 μg/ml. No toxic activity was detected for Aedes aegypti and Culex quinquenfaciatus.     

Barley-based medium for the cost-effective production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Summary Studies conducted for the multiplication of Bacillus thuringiensis (Bt) using barley Hordeum vulgare as the carbon source led to the development of a protocol for the cost-effective, mass production of Bt. The production employs the simple shake flask method and can be easily adopted with a production potential of 1.5 kg Bt per day approximately at an overall production cost of Rs. 360 kg^-1 (8 US dollars). The protocol is suitable for promoting localized production of Bt at the village/district level. The product when tested as 0.1% (w/v) spray against the castor semilooper, Achoea janata proved highly effective, causing immediate feeding cessation of the larvae followed by 85% and 100% mortality by 48 and 72 h after treatment, respectively.     

<Emphasis Type="Italic">Prays oleae</Emphasis> Midgut Putative Receptor of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Vegetative Insecticidal Protein Vip3LB Differs from that of Cry1Ac Toxin

Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins.     

Synergistic interaction between sublethal doses of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Campoletis chlorideae</Emphasis> in managing <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) is an important solitary larval endoparasitoid of the tomato fruit borer Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in India. The interaction between Bacillus thuringiensis subspecies kurstaki (Btk) HD-1 and C. chlorideae was studied under laboratory condition to explore their compatibility in managing H. armigera. The results had indicated that the growth and development of H. armigera was affected in a dose-dependent manner upon feeding on sublethal doses of Btk HD-1-treated diets. There were no larval survivors in lethal doses of Btk HD-1 (LC₇₀ and LC₉₀). The growth and survival of the parasitoid were normal when the host larvae were fed with sublethal doses or subjected to short time exposure to lethal doses of Btk HD-1. However, the parasitoid offsprings developed slowly and pupal as well as adult period, adult weight and adult emergence rate were reduced significantly if the parasitoid was developing inside a severely Bt intoxicated host larvae. There were no evident differences in longevity of parasitoid adults that were fed on honey solution containing different concentrations of Btk HD-1 as compared to adults fed only on honey solution. This indicates no direct adverse effect of Btk HD-1 on C. chlorideae. Further, the gravid female parasitoid did not discriminate Btk HD-1 intoxicated and normal H. armigera larvae for oviposition. The result implies that spore crystal formulation of Btk HD-1 can be effectively used in a synergistic manner along with existing natural or prereleased population of C. chlorideae in organic farming or as components in biointensive IPM module for managing H. armigera.     

Insecticidal crystal proteins from native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>: numerical analysis and biological activity against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Fourteen strains of Bacillus thuringiensis collected from both larvae showing disease symptoms and soil samples in northwest Argentina were characterized by insecticidal activity against Spodoptera frugiperda. First instar larvae and protein profile SDS-PAGE analysis of whole cell proteins not only allowed the differentiation of native Bacillus thuringiensis but also revealed the possibility of applying protein profile analysis in classification of toxicity patterns. Cluster analysis showed that there were two main groups. Interestingly, one of them only contained the most pathogenic native strains. The biomass-bound protease activity of native pathogenic isolates and the reference strain Bt 4D1 is also reported.     

Isolation and heterologous expression of PHA synthesising genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> R1

The polyhydroxyalkanoate biosynthesis gene locus from Bacillus thuringiensis R1 was isolated, cloned and analyzed at the molecular level. We found that a ∼5 kb SacI–ClaI digested fragment of genomic DNA from B. thuringiensis R1 encoding the PHA synthesising genes, conferred PHA producing ability to E. coli. The fragment was sequenced and found to be of 4787 bp with five open reading frames. Sequence alignment with closely related species of Bacillus in the existing database revealed that the ORFs correspond to phaP, phaQ, phaR, phaB and phaC genes. However, E. coli harboring phaP, phaQ, phaR, phaB and phaC locus produced very low PHA. Furthermore, complementation of the locus with phaA from Ralstonia eutropha increased the PHA production in the recombinant E. coli from 3.0% to 24% of cell dry mass. The putative promoter regions and ribosome binding sites were identified for each of the gene. Conserved domains for PHA synthase and aceto-acetyl-coA reductase were also identified. We hence conclude that the PHA operon of Bacillus thuringiensis R1 consists of phaP, phaQ, phaR, phaB, phaC and complementation of the same with phaA is accountable for its high PHA production.     

Novel roles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> to control plant diseases

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.     

Characterization of the highly variable <Emphasis Type="Italic">cry</Emphasis> gene regions of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strain ly4a3 by PCR-SSCP profiling and sequencing

Characterization of cry gene contents can help to predict the insecticidal activities of Bacillus thuringiensis isolates and in the searching of new cry genes. PCR-Single-strand conformation polymorphism (SSCP) profiling and sequencing of the highly variable cry gene regions were used to characterize cry gene content of B. thuringiensis strain ly4a3. The highly variable regions with about 1100 bp in sizes were amplified using a degenerate primer pair for cry genes, OL2(d) and OL5(r). A library of the PCR product was constructed, and all white colonies were subjected to PCR using another degenerate primer pair for cry genes, OL3(d) and OL5(r), with products about 250 bp in sizes. Two different profiles were observed based on SSCP profiling for the PCR products. The cry genes in the two corresponding colonies were sequenced and their deduced amino acids showed high identities to Cry1Ab (84.5%∼98.4%) and Cry1I (88.78%∼98.4%), respectively. This method allows the quick characterization of cry gene content of B. thuringiensis isolates and the detection of new cry genes.