Cloning and Characterization of a Novel Crystal Protein from a Native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Isolate Highly Active Against <Emphasis Type="Italic">Aedes aegypti</Emphasis>

We characterized a novel Bacillus thuringiensis isolate native to Argentina (FCC 41) that exhibits a mosquitocidal activity higher than the reference B. thuringiensis subsp. israelensis. This isolate shows a rounded crystal harboring two major proteins of about 70–80 kDa. Moreover, we cloned and sequenced the encoding gene of one of the crystal proteins (Cry) consisting of an open reading frame of 2061 pb that encodes a protein of 687 amino acid residues. The deduced amino acid sequence has a predicted relative molecular mass of 78 kDa and is 52% and 45% identical to those of the reported Cry24Aa and Cry24Ba sequences, respectively. The novel Cry protein was designated as Cry24Ca, which also exhibited larvicidal activity against Aedes aegypti when its encoding gene was expressed in an Escherichia coli host strain.     

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.     

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.     

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.     

Cloning and Expression of <Emphasis Type="Italic">cyt</Emphasis>2Ba7 Gene from a Soil-Isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus thuringiensis (Bt) cyt genes coding hemolytic and cytolytic toxins constitute a gene family, which are divided into two groups: cyt1 and cyt2. A novel cyt2 gene was detected from a soil-isolated Bt strain T301, which was highly homologous to cyt2Ba1 and finally designated cyt2Ba7. Until now, Cyt2Ba has not been expressed alone in Bt or other hosts. In this study, the cyt2Ba7 gene was cloned into the vector pQE30 and expressed as a fusion protein with 6×Histidine residues in Escherichia coli. Unlike cyt1A, cyt2Ba7 was freely expressed and formed cytoplasmic inclusions without the need for a “helper” protein. The 6×His-tagged Cyt2Ba7 was purified in one step by Ni-NTA affinity chromatography, examined cytolytic activity on Sf9 cells, and developed as an antigen to obtain the antiserum against Cyt2Ba by subcutaneous injection into rabbits. This gene was also cloned into the Bt–E. coli shuttle vector pHT3101 and expressed in Bt strain 4Q7. Immunoblotting analysis revealed that the antiserum was remarkably selective and specific to Cyt2Ba. Received: 21 December 2001 / Accepted: 28 January 2002     

Sequence Analysis and Comparative Bioinformatics Study of Camelysin Gene (<Emphasis Type="Italic">calY</Emphasis>) Isolated from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus strains have been widely used for the production of fibrinolytic enzymes having role in the treatment of cardiovascular disorders. Purification and overproduction of such enzymes has increased their usage in medical fields including metalloproteinases with the ability to degrade extracellular matrix (ECM). Camelysin, a neutral metalloproteinase has been isolated from different species of bacteria like Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis with fibrinolytic, collagenolytic and actin degradation activity. This project successfully demonstrated the presence of 734-bp coding DNA sequence (CDS) encoding a 20.72331 kDa camelysin gene in local strain of Bacillus thuringiensis containing a signal peptide with cleavage site between residues 19 and 20. The sequence was submitted to GenBank (KT023597) and the sequence showed high homology with the camelysin protein of closely related Bacillus species. The alignment of related proteins through ClustalW displayed difference of four amino acids (“Q” replaced by “P” at position 169 and at position 182–184, “NQE” replaced by “HLK”) in the isolated protein. Comparison including structural and functional analysis of camelysin sequences isolated from different Bacillus species was carried out using different bioinformatics tools and software. The information would help in better understanding the properties of camelysin protein and its role in pathogenicity and clinical treatments.     

Characterization of Two Novel <Emphasis Type="Italic">cry8</Emphasis> Genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain BT185

Two novel cry8-type genes, cry8Ea1 and cry8Fa1, obtained from a Holotrichia parallela–specific Bacillus thuringiensis strain, BT185, were characterized. Findings showed that cry8Ea1 and cry8Fa1 encoded polypeptides of 1164 and 1174 amino acid residues, respectively. The deduced amino acid sequences of both Cry8Ea1 and Cry8Fa1 polypeptides are the most similar to that of Cry8Ba1. Eight conserved blocks (blocks 1–8) exist in Cry8Ea1 and Cry8Fa1 polypeptides compared with known Cry proteins. Cry8Ea1 and the Cry8Fa1 toxins could form spheric crystals when they were expressed in the acrystalliferous mutant strain HD73⁻. The spores and crystals from the recombinant strain containing cry8Ea1 were toxic to Holotrichia parallela, with an LC₅₀ of 0.0875 × 10⁸ colony-forming units (CFU)/g. However, Cry8Fa1 expressed in the recombinant strain was not toxic to H. parallela, Anomala corpulenta, or H. oblita.     

A <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Isolate Possessing a Spore-Associated Filament

We report on a novel bacterium, isolated during a screen for environmental isolates of Bacillus thuringiensis, that possesses a novel filamentous structure. Nucleotide sequence from the isolates 16S rRNA gene places the bacterium unambiguously within the Bacillus thuringiensis/Bacillus cereus group. Phase-contrast and electron microscopy indicate the presence of both a parasporal body and a long filament which are retained after sporulation. The filament is shown to consistently arise from the end of the exosporium and next to the parasporal body. Upon spore germination, the parasporal body/filament complex is retained on the cell wall of the resulting bacterium.Received: 4 March 2003 / Accepted: 18 April 2003     

Chitosanase activity in<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The ability to produce extracellular chitosanase (EC 3.2.1.132) was found by plate assays in 18 (23%) out of 77 crystalliferous strains of Bacillus thuringiensis. The best chitosanase producer was selected after the growth chosen in a liquid medium with colloidal chitosan as carbon source. Enzyme production was optimized (a 4-d incubation at 32 degrees C with shaking in a medium of pH 6.5 with 4% colloidal chitosan) and the enzyme was partially characterized. This is the first report on the chitosanase of B. thuringiensis.     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence observation of parasporal inclusion formation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed into the crystal-negative strain, HD-73 cry⁻, and the resulting strain was named HD-73⁻(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73⁻(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.     

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.     

The combined use of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Nesidiocoris tenuis</Emphasis> against the tomato borer <Emphasis Type="Italic">Tuta absoluta</Emphasis>

Since Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) was first detected at the end of 2006 in the Mediterranean Basin, several endemic natural enemies have been reported to prey on this exotic pest. The predator Nesidiocoris tenuis Reuter (Hemiptera: Miridae) can regulate T. absoluta populations, because it is able to prey efficiently on T. absoluta eggs. Furthermore, previous studies have demonstrated that first-instar larvae of T. absoluta are highly susceptible to Bacillus thuringiensis (Bt) treatments. In this work, we tested the combination of both approaches under greenhouse conditions. B. thuringiensis formulations were sprayed weekly for two months, three months or throughout the growing cycle, and in all cases, one N. tenuis per plant was also released. Control plants were completely destroyed by the infestation levels reached by T. absoluta. In contrast, all treatments based on B. thuringiensis treatments and releases of N. tenuis reduced leaf damage by more than 97% when compared to the untreated control, with no significant differences among them. Furthermore, yield in the control plants was significantly reduced when compared with all Bt–N. tenuis treatments. Our results demonstrate that when B. thuringiensis treatments are applied immediately after the initial detection of T. absoluta on plants, they do not interfere with N. tenuis establishment in the crop because T. absoluta eggs are available. According to our data, treatments with B. thuringiensis later in the growing season would no longer be necessary because mirids alone would control the pest.     

Characterization of a highly pathogenic<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strain isolated from common cockchafer,<Emphasis Type="Italic">Melolontha melolontha</Emphasis>

A bacterial isolate (Mm2) of Melolontha melolontha was identified and characterized. Based on various morphological, physiological, biochemical and molecular characteristics, it was identified as Bacillus thuringiensis subsp. tenebrionis. This isolate was compared to the reference strains by electron microscopy, SDS-PAGE analysis, plasmid pattern, cry gene content and insecticidal activity. Cells of the isolate harbored flat square inclusions containing a protein component of approximately equal to65 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique proteinase-resistant peptide of approximately equal to 50 kDa. Plasmid pattern showed similar bands to those of the reference strain, PCR analysis showed that the isolate has cry3 gene. Toxicity tests (against 5 coleopteran species) showed 80 % insecticidal activity against the larvae of M. melolontha. The isolate Mm2 may be valuable as biological control agent for M. melolontha and other coleopteran insects.     

Dissection of <Emphasis Type="Italic">cry</Emphasis> Gene Profiles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Isolates in Taiwan

On the basis of the newly revised nomenclature system of cry genes, the PCR amplification method has been adopted to resolve the cry gene combinations of 294 Bacillus thuringiensis isolates from five selected areas of Taiwan. Our results indicate that cry1 (especially cry1A + 1B + 1F) and cry2 were the most abundant cry genes in Taiwan. In contrast, cry3 and cry6 genes were detected only on Yang Ming Mountain, while the cry13 gene was found only on Snow Mountain. In addition, some distinctive combinations of cry genes were detected in distinct areas of Taiwan, such as cry1C, cry1D, cry1C + 1D, cry4, cry1 + 4, cry1 + 11, cry4 + 11, and cry1 + 4 + 11 in the Taipei area; cry1A + 1C + 1F in the Taichung area; cry1E and cry1A + 1B + 1I on Yang Ming Mountain; cry1 + 13, cry1 + 2 + 11, and cry1 + 2 + 13 on Snow Mountain; and cry1 + 5 and cry1 + 2 + 5 on Jade Mountain. These data clearly indicate that the distribution of cry gene combinations of B. thuringiensis isolates seems to be geographically related.     

Biological characterization of two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains toxic against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Two Bacillus thuringiensis strains isolated from diseased Spodoptera frugiperda larvae collected in the northwest of Argentina were molecularly and phenotypically characterized. Insecticidal activity against Spodoptera frugiperda larvae was also determined. Both strains were highly toxic against first instar larvae. One strain (Bacillus thuringiensis LSM) was found to be even more toxic than the reference strain Bacillus thuringiensis var. kurstaki 4D1. This strong biological effect was represented by both a higher mortality which reached 90%, and a shorter LT₅₀. Molecular characterization showed that Bacillus thuringiensis LSM carried a cry gene profile identical to that of Bacillus thuringiensis var. kurstaki 4D1. Evaluation of length polymorphism of the intergenic transcribed spacers between the 16S and 23S rDNA genes revealed an identical pattern between native strains and Bacillus thuringiensis var. kurstaki 4D1. In contrast, phenotypic characterization allowed differentiation among the isolates by means of their extracellular esterase profiles. Lytic activity that would contribute to Bacillus thuringiensis effectiveness was also studied in both strains. Analyses like those presented in the current study are essential to identify the most toxic strains and to allow the exploitation of local biodiversity for its application in biological control programmes.     

Protection from UV-B Damage of Mosquito Larvicidal Toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">israelensis</Emphasis> Expressed in <Emphasis Type="Italic">Anabaena</Emphasis> PCC 7120

A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed δ-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of B. thuringiensis subsp. israelensis as a mosquito biological control agent. Received: 20 November 2001 / Accepted: 31 December 2001     

Identification of a Novel DNA Methyltransferase Activity from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A DNA methyltransferase activity was identified in a strain of Bacillus thuringiensis that was found to protect DNA from cleavage by the restriction endonuclease HaeIII at overlapping sites. Site-directed mutagenesis was used to confirm the recognition sequence of the methyltransferase as ACGGC. RID= ID= <E5>Correspondence to:</E5> N. Crickmore; <E5>email:</E5> n.crickmore&commat;sussex.ac.uk Received: 13 September 2002 / Accepted: 7 October 2002     

Chitinase production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: Their potential in antifungal biocontrol

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na⁺, Mg²⁺, Cu²⁺, and Ca²⁺ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn²⁺, Hg²⁺, and Ag⁺. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.