Cloning and Characterization of a Cytolytic and Mosquito Larvicidal δ-Endotoxin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">darmstadiensis</Emphasis>

The cytolytic δ-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 mM Na₂CO₃, pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 μg/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC₅₀ 0.5–1.0 μg/ml). Received: 27 March 2002 / Accepted: 30 April 2002     

Isolation and Characterization of a Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">morrisoni</Emphasis> PG-14 Encoding δ-Endotoxin Cry1Ac

Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes. B. thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis subsp. morrisoni (H8a8b). The plasmid and protein profiles of B. thuringiensis 656-3 were similar to those of its reference strain, subsp. morrisoni PG-14. However, PCR analysis using cry gene primers showed that B. thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B. thuringiensis 656-3 was a unique strain with respect to gene type. In addition, B. thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L. mali and C. fuscipes.     

Germinant Generation from δ-endotoxin of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain 1.1

The novel finding of this study is that the δ-endotoxin present in the spore coat of Bacillus thuringiensis strain 1.1 (Bt1.1), plays a central role in spore germination by generation of germinant via its β-glucosidase activity and is based on the following: (i) the crystals of Bt1.1 consist of the 140 kDa δ-endotoxin which exhibits β-glucosidase enzymatic activity. Besides crystals, δ-endotoxin is also located in the spore coat and at this site displays β-glucosidase activity, resulting in glucose production; (ii) glucose is an efficient germinant of both Bt1.1 and acrystalliferous Bt4.1 strain; (iii) substrates of β-glucosidase can activate the germination of Bt1.1 spores, but not those of the acrystalliferous Bt4.1 sister strain that do not contain the 140 kDa δ-endotoxin; (iv) Reduction or enhancement of enzymatic activity of δ-endotoxin, results in retardation or acceleration of germination and outgrowth, respectively. Bt1.1 cells secrete a 60 kDa polypeptide which displays β-glucosidase activity as indicated by zymogram analysis and which is immunologically related to the 140 kDa δ-endotoxin.     

Interaction of Two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> δ-Endotoxins with the Digestive System of <Emphasis Type="Italic">Lygus hesperus</Emphasis>

The active-toxin form of Cry1Ac (65 kDa) or Cry2Ab was fed to a non-susceptible insect, Lygus hesperus, in an artificial diet. Biochemical and immunocytochemical methods were used to determine the distribution of ingested toxin. The toxins did not elicit a feeding deterrent response. Cry1Ac and Cry2Ab were ingested; small amounts were absorbed into the hemolymph as holoproteins, but most was excreted. SDS-PAGE analysis of Cry1Ac and Cry2Ab incubations with salivary gland homogenate showed a small decrease in the molecular weight of the active toxins. Proteolytic processing of the toxins also occurred in vivo, within the digestive system of L. hesperus. Excreted Cry1Ac and Cry2Ab retained activity toward lepidopteran larvae. Immunocytochemical in vivo localization studies showed negligible association of Cry1Ac with L. hesperus tissues. In contrast, strong extracellular association of Cry2Ab was observed with L. hesperus midgut brush border microvilli and basement membrane, as well as with cellular outlines within the hemolymph and fat body.     

Biochemical and molecular characterization of δ-endotoxins in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The δ-endotoxins (δ-ETX) of four native strains (RT7, RT19, RT25, and RT25), and one reference strain (4L1) of Bacillus thuringiensis were biochemically and molecularly characterized to determine their potential toxic activity against lepidopteran larvae. Crystals of δ-ETX were purified through a two-phase system to determine their morphology, molar mass, solubility, and resistance to proteinases. Toxic activity and cry gene content were also determined. Crystals from native strains exhibited polyhedral, irregular and cuboidal shapes, while those from 4L1 were bipyramidal. Seven proteins with estimated molar mass ≈30–134 kDa were detected as the main components of the native δ-ETX. Only crystals from 4L1, RT24, and RT25 underwend complete solubilization at pH >12.0. Crystals from all strains produced trypsinresistant peptides. None of the cry genes associated with toxicity in lepidopterans (cry1, cry2, cry9) was found in the native strains; however, 4L1 strain harbors cry1 and cry2 genes. Strains RT19 and RT25 caused significant mortality against Trichoplusia ni larvae with partial solubilization at pH 10, strain 4L1 caused 100 % mortality. Toxicity of native strains may come from a novel cry gene.     

A new Tunisian strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis><Emphasis Type="Italic">kurstaki</Emphasis> having high insecticidal activity and δ-endotoxin yield

BLB1 is a new Bacillus thuringiensis kurstaki strain, isolated from a Tunisian soil sample. Assay of toxicity of BLB1 crystal proteins resulted in an LC50 of 70.32 ng of toxin per mg of flour against third instar Ephestia kuehniella with confidence limits of (31.6–109.04 ng). This LC50 is less than that of the commercial strains HD1 used as a reference. The characterization of this strain by scanning transmission electron microscopy, analysis of its cry genes content by PCR-sequencing, and analysis of its δ-endotoxin patterns demonstrate that it belongs to the same subgroup than HD1, but ruled out the involvement of cry gene content or protoxin activation in the hypertoxicity of this strain. Taking into account the δ-endotoxin/spore ratio for each strain, and by allowing the estimation of the production level per spore, it might be concluded that BLB1 production is the highest, when compared with that of HD1. On the basis of its toxicity, BLB1 could be considered as a strain of great interest and would allow the production of quantities of bioinsecticides at low cost.     

Activity of wild-type and hybrid<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> δ-endotoxins against<Emphasis Type="Italic"> Agrotis ipsilon</Emphasis>

Twelve Cry1 and two Cry9 delta-endotoxins from Bacillus thuringiensis were tested for their activity against black cutworm ( Agrotis ipsilon). A. ipsilon was not susceptible to many toxins, but three toxins had significant activity. Cry9Ca was the most toxic, followed by Cry1Aa and Cry1Fb. Hybrids between these three active proteins were made by in vivo recombination and analyzed for activity against A. ipsilon. Analysis of hybrids between Cry1Aa and Cry1Fb indicated that domain I of Cry1Aa protein was involved in its higher activity.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

Insoluble but enzymatically active <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.     

Bioinformatics Characterization of Potential New Beta-Glucuronidase from <Emphasis Type="Italic">Streptococcus</Emphasis> <Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.     

Evidence of the Involvement of E358, A498 and C571 of a New Cry1Ac δ-endotoxin of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in its High Insecticidal Activity Against <Emphasis Type="Italic">Ephestia kuehniella</Emphasis>

A new cry1Ac-type gene was cloned from Bacillus thuringiensis strain BLB1, sequenced and expressed. The deduced amino acid sequence of the polypeptide has a predicted molecular mass of 132.186 kDa. The amino acid sequence alignment of BLB1 Cry1Ac with those of the published ones showed that this is a new delta-endotoxin. When compared with Cry1Ac of Bacillus thuringiensis strain HD1, it was found that BLB1 Cry1Ac harbours three mutations: V358E localized in domain II and V498A and Y571C localized in domain III. When the BLB1 Cry1Ac toxin was expressed in an acrystalliferous strain of B. thuringiensis (HD1CryB), bipyramidal crystals were produced. The spore–crystal mixture of this recombinant strain was at least two-fold more active against larvae of the lepidopteran Ephestia kuehniella than that of the recombinant strain expressing Cry1Ac of HD1. The study of the structural effect of these mutations suggested that they may stabilize key regions involved in the binding of the domains II and III to insect receptors.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

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

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

New family of pectinase genes <Emphasis Type="Italic">PGU1</Emphasis>b–<Emphasis Type="Italic">PGU3b</Emphasis> of the pectinolytic yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Behavioral Response of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae) to Cotton with and Without Expression of the CrylAc δ-Endotoxin Protein of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Berliner

Behavioral responses of larvae and adults of cotton bollworm, Helicoverpa armigera to cotton with (GK-12) and without (SI-3) expression of the CrylAc -endotoxin protein of Bacillus thuringiensis (Bt) Berliner were observed during 2001 and 2002. Our results showed that 8.3% individuals fed with flowers and bolls of GK-12 could develop from neonate to pupa; however, pupal weight decreased by 48.6% and duration of development was delayed by 7.6 days compared with those fed with flowers–bolls of SI-3. Deterrence index (DI) of larvae decreased in later instars, which indicated that the Bt toxin decreased with age. Feeding frequency of 4th-instar larvae on GK-12 leaves decreased by 38.8%, but movement frequency increased by 37.1%. Larvae moved at least one plant away by the age of 10 days in both pure and mixed plantings of SI-3 and GK-12 in the field. Adults preferred to lay eggs on SI-3. The total number of eggs deposited on SI-3 plants in 3 days were about 232 and 95% greater than that on GK-12 plants at bud–flower stage and flower–boll stage, respectively. Based on the behavior of larva and adults in response to the transgenic cotton, the potential effect of refuge strategy in resistance management of H. armigera is discussed.