Bacterial (Bacillus thuringiensis subsp. thuringiensis and B. thuringiensis subsp. kurstaki) Entomocidal Preparations Decrease Chlorophyll Accumulation in Larch Needles

Chlorophyll a plus b content and absorption spectra of the homogenates from the cotyledonary leaves of 30-day-old seedlings of two larch species, Larix gmelinii (Rupr.) Rupr. and L. sibirica Ldb. were studied. The seedlings were grown on Perlite containing aqueous solutions of entomocidal biopreparations isolated from Bacillus thuringiensis subsp. thuringiensis (bitoxybacillin) and B. thuringiensis subsp. kurstaki (lepidocide) at various final concentrations (2, 6, and 12 g/l). Changes in the form of chlorophyll absorption spectra induced by biopreparations were established. A marked inhibition of pigment accumulation in the needles dependent on the biopreparation concentration was noted. At a low concentration (2 g/l), the biopreparations virtually did not affect the chlorophyll content; an increase in their concentrations resulted in a decrease in chlorophyll content in leaves by 20% (at 6 g/l) and 40% (at 12 g/l). It is concluded that bitoxybacillin and lepidocide inhibited the chlorophyll accumulation in larch needles to a similar extent.     

Transduction in Bacillus thuringiensis.

Bacteriophage CP-51, originally reported as a generalized transducing phage for Bacillus cereus and B. anthracis, has been shown to carry out generalized transduction in several strains of B. thuringiensis. A newly isolated phage, CP-54, which has a broader host range than CP-51, also mediates generalized transduction in B. thuringiensis. CP-51 and CP-54 are similar in size and morphology and are related serologically, but they are not identical. CP-54 is more cold labile than CP-51, and, as with CP-51, its stability both at 0 and 15 degrees C is enhanced by the presence of 0.02 M Mg2+. Some examples of cotransduction of linked markers in B. thuringiensis are presented, demonstrating the feasibility of chromosomal mapping in this organism. The rare occurrence of cross-transduction among strains of B. thuringiensis is probably a reflection of nonhomology rather than restriction, since phage itself did not appear to be restricted when grown on a particular host and assayed with other hosts as indicator.     

苏云金芽孢杆菌的一个新亚种(Btsubsp.sinensis)

198 9年自云南昆明市石林的红棕壤中分离到数株苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎ ｓｉｓ,Ｂｔ)菌株[1] ,对其中的一株ＹＫ30 0 4进行了生物学特性、杀虫特性研究及分类鉴定。1　材料与方法1.1　供鉴定的Ｂｔ菌株由云南昆明市石林的红棕壤中分离的苏云金芽孢杆菌ＹＫ30 0 4菌株。1.2　标准Ｂｔ菌株血清型Ｈ1 Ｈ4 1、Ｈ4 4 Ｈ55及Ｈ57 Ｈ69标准Ｂｔ菌株由法国巴斯德研究院ＤｒＬｅｃａｄｅｔ提供 ,其余为本实验室保存。1.3　生物测定用昆虫小菜蛾 (Ｐｌｕｔｅｌｌａｘｙｌｏｓｔｅｌｌａ) 3龄幼虫 ;斜纹夜盗蛾 (Ｐｒ…     

Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus.

DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.     

Molecular modeling and characterization of the B. thuringiensis and B. thuringiensis LDC-9 cytolytic proteins

The Cyt toxins are able to lyse a wide range of cell types in vitro, unlike the Cry delta-endotoxins. It exerts its activity by the formation of pores within target cell membranes. The structural information available for Cyt2Aa (PDB id: 1CBY) consists of a single domain in which two outer layers of alpha-helix wrap around a mixed beta-sheet. Beta-barrel was suggested as a possible structure of the pores. Hence, this study seeks to investigate the structural properties of other Cytolytic proteins by predicting the three-dimensional (3D) model using Cyt2Aa as template. The predicted models are expected to be significantly more accurate as all the Cyt proteins showed significant similarity with the template (PDB id: 1CBY). The refined homology models revealed similar secondary structures (alpha-helices and beta-sheets) and tertiary features as Cyt2Aa. The variation in the loop regions of the tertiary structure accounts for the differential toxicity.     

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 micrograms vs 83 micrograms, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC50 of about 600 spores/ml.     

On the formation of crystal proteins during sporulation in Bacillus thuringiensis var. thuringiensis

The sporulation potential of Bacillus subtilis as a function of position in the cell cycle was determined by transferring cells from growth medium to sporulation medium at various times during growth. Growth was induced by incubating heat-activated spores in rich medium or by diluting stationary phase vegetative cultures with fresh growth medium. The results supported earlier observations that sporulation potential is cell cycle dependent. The rise in sporulation potential was studied by exposing cultures to the inhibitors of cell wall and protein synthesis, vancomycin and chloramphenicol. The delay in the appearance of the peak of sporulation potential caused by these inhibitors compared with the reported lack of effect of nalidixic acid, indicates that the appearance of sporulation potential requires synthesis of a macromolecular component other than deoxyribonucleic acid. The effect of nalidixic acid in preventing the decline of the sporulation potential was compared with the effect of high temperature on a mutant temperature sensitive for the initiation of DNA replication. It was found that prevention of chromosome completion with nalidixic acid maintained a high sporulation potential, whereas prevention of chromosome re-initiation in the temperature sensitive mutant did not affect the decline in sporulation potential as the cells enter stationary phase.Abbreviations NAL Nalidixic acid - HPUra 6-(p-hydroxyphenylazo)-uracil - VAN Vancomycin - CAM Chloramphenicol - BHI Brain heart infusion broth - c.f.u. Colony forming units     

Homology modeling of Cry10Aa toxin from B. thuringiensis israelensis and B. thuringiensis subsp. LDC-9

A three dimensional model was developed for Cry10Aa protein sequence of B. thuringiensis LDC-9 and B. thuringiensis israelensis that has not been solved empirically by X-ray crystallography or NMR. Homology modeling was employed for the structure prediction using Cry2Aa as template protein, a high-resolution X-ray crystallography structure. The model predicted for the B. thuringiensis LDC-9 Cry10Aa protein reveals a partial N-terminal domain only due to its partial sequence of 104 amino acids. B. thuringiensis israelensis Cry10Aa model contains three domains such as domain I, a bundle of eight alpha helices with the central relatively hydrophobic helix surrounded by amphipathic helices while domain II and III contain mostly beta-sheets. Significant structural differences within domain II in this model among all Cry protein structures indicates that it is involved in recognition and binding to cell surfaces. Comparison of B. thuringiensis israelensis predicted structure with available experimentally determined Cry structures reveals identical folds. The distribution of electrostatic potential on the surface of the molecules in the model is non-uniform and identifies one side of the alpha-helical domain as negatively charged indicating orientation of toxic molecules toward the cell membrane during the initial binding with a cell surface receptor. The collective knowledge of Cry toxin structures will lead to a more critical understanding of the structural basis for receptor binding and pore formation, as well as allowing the scope of diversity to be better appreciated. This model will serve as a starting point for the design of mutagenesis experiments aimed to improve the toxicity and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.     

Novel cloning vectors for Bacillus thuringiensis.

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.     

Spore and crystal formation inBacillus thuringiensis var.thuringiensis during growth in cystine and cysteine

The effect of the addition of different concentratons of cystine and cysteine on sporulation and parasporal crystal formation inBacillus thuringiensis var.thuringiensis was studied. The effect was well pronounced when the cystine/cysteine additions were made after the stationary phase. Heat stable spores and crystals were formed when the culture was provided with a low concentration of cystine/cysteine (0.05 per cent w/v). At a moderate concentration of cystine or cysteine (0.15%), only heat labile spores were formed without the production of the crystal. When the cystine/cysteine concentration was high (0.25%), spore and crystal formation were completely inhibited. Partial reversal of inhibition of sporulation was brought about by sodium sulphate or Zinc sulphate and lead, copper, cadmium or cobalt acetate at 0.2 mM or at 0.2% of sodium or potassium pyruvate, citrate, cisaconitate, oxalosuccinate, ∞ -keto-glutarate, succinate, fumarate, malate, or oxalacetate. Glutamate (0.2%) overcame the inhibitory effect of cystine/cysteine completely. The structural changes observed using phase contrast microscopy were dependent upon the concentration of cystine/cysteine.     

Chitinase from Bacillus thuringiensis subsp. pakistani

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.     

Safety of Bacillus thuringiensis for earthworms.

In vitro chemotaxis by invertebrate hemocytes is demonstrated by the attraction of granulocytes from the operculate snail Viviparus malleatus to heat-killed Staphylococcus aureus and to N-acetyl-d-glucosamine. A soluble constituent of the hemolymph, a bacterial agglutinin, was necessary for this positive response to both of these chemotactic agents. Agglutination studies revealed the presence of two nonhomologous agglutinins in the hemolymph of V. malleatus.