scholarly journals The construction of Bacillus thuringiensis strains expressing novel entomocidal δ-endotoxin combinations

1990 ◽  
Vol 270 (1) ◽  
pp. 133-136 ◽  
Author(s):  
N Crickmore ◽  
C Nicholls ◽  
D J Earp ◽  
T C Hodgman ◽  
D J Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Parent Strain ◽  
Pieris Brassicae ◽  
Toxin Gene ◽  
Delta Endotoxin ◽  
Cabbage White Butterfly

Using our recently reported method of electroporation to transform Bacillus thuringiensis [Bone & Ellar (1989) FEMS Microbiol. Lett. 58, 171-178], cloned B. thuringiensis entomocidal delta-endotoxin genes have been introduced into several native B. thuringiensis strains. In many cases the resulting transformants expressed both their native toxins and the cloned toxin, producing strains with broader toxicity spectra. The introduction of the var. tenebrionis toxin gene into B. thuringiensis var. israelensis resulted in a strain with activity against Pieris brassicae (cabbage white butterfly), an activity which neither parent strain possesses. We discuss further the possibility of synergism and also the problems associated with introducing cloned DNA by this method.

scholarly journals Loss of Catabolite Repression Function of HPr, the Phosphocarrier Protein of the Bacterial Phosphotransferase System, Affects Expression of the cry4A Toxin Gene in Bacillus thuringiensis subsp. israelensis

2002 ◽  
Vol 184 (19) ◽  
pp. 5410-5417 ◽  
Author(s):  
Sharik R. Khan ◽  
Nirupama Banerjee-Bhatnagar
Keyword(s):  
Bacillus Thuringiensis ◽  
Mutant Strain ◽  
Catabolite Repression ◽  
Parent Strain ◽  
Inhibitory Effect ◽  
Toxin Gene ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Gene Transcripts ◽  
Bacterial Phosphotransferase System

ABSTRACT HPr, the phosphocarrier protein of the bacterial phosphotransferase system, mediates catabolite repression of a number of operons in gram-positive bacteria. In order to participate in the regulatory process, HPr is activated by phosphorylation of a conserved serine-46 residue. To study the potential role of HPr in the regulation of Cry4A protoxin synthesis in Bacillus thuringiensis subsp. israelensis, we produced a catabolite repression-negative mutant by replacing the wild-type copy of the ptsH gene with a mutated copy in which the conserved serine residue of HPr was replaced with an alanine. HPr isolated from the mutant strain was not phosphorylated at Ser-45 by HPr kinase, but phosphorylation at His-14 was found to occur normally. The enzyme I and HPr kinase activities of the mutant were not affected. Analysis of the B. thuringiensis subsp. israelensis mutant harboring ptsH-S45A in the chromosome showed that cry4A expression was derepressed from the inhibitory effect of glucose. The mutant strain produced both cry4A and σ35 gene transcripts 4 h ahead of the parent strain, but there was no effect on σ28 synthesis. In wild-type B. thuringiensis subsp. israelensis cells, cry4A mRNA was observed from 12 h onwards, while in the mutant it appeared at 8 h and was produced for a longer period. The total amount of cry4A transcripts produced by the mutant was higher than by the parent strain. There was a 60 to 70% reduction in the sporulation efficiency of the mutant B. thuringiensis subsp. israelensis strain compared to the wild-type strain.

scholarly journals Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal δ-endotoxin

1987 ◽  
Vol 248 (1) ◽  
pp. 197-201 ◽  
Author(s):  
M Z Haider ◽  
D J Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Membrane Proteins ◽  
Insect Cell ◽  
Receptor Interaction ◽  
Dual Specificity ◽  
Delta Endotoxin ◽  
Cell Membrane Proteins ◽  
Toxin Receptor ◽  
Osmotic Lysis ◽  
Initial Interaction

The mechanism of action and receptor binding of a dual-specificity Bacillus thuringiensis var. aizawai ICl delta-endotoxin was studied using insect cell culture. The native protoxin was labelled with 125I, proteolytically activated and the affinity of the resulting preparations for insect cell-membrane proteins was studied by blotting. The active preparations obtained by various treatments had characteristic specificity associated with unique polypeptides, and showed affinity for different membrane proteins. The lepidopteran-specific preparation (trypsin-treated protoxin containing 58 and 55 kDa polypeptides) bound to two membrane proteins in the lepidopteran cells but none in the dipteran cells. The dipteran-specific preparation (protoxin treated sequentially with trypsin and Aedes aegypti gut proteases, containing a 53 kDa polypeptide) bound to a 90 kDa membrane protein in the dipteran (A. aegypti) cells but bound to none in the lepidopteran cells or Drosophila melanogaster cells. The toxicity of trypsin-activated delta-endotoxin was completely inhibited by preincubation with D-glucose, suggesting a role for this carbohydrate in toxin-receptor interaction. The toxicity was also decreased by osmotic protectants to an extent proportional to their viscometric radius. These results support a proposal that initial interaction of toxin with a unique receptor determines the specificity of the toxin, following which cell death occurs by a mechanism of colloid osmotic lysis.

scholarly journals Effects of Bacillus thuringiensis δ-Endotoxin on Insect and Mammalian Cells In Vitro

1980 ◽  
Vol 15 (2) ◽  
pp. 133-139 ◽  
Author(s):  
Junko NISHITSUTSUJI-UWO ◽  
Yasuhisa ENDO ◽  
Michio HIMENO
Keyword(s):  
Bacillus Thuringiensis ◽  
Mammalian Cells ◽  
Delta Endotoxin

scholarly journals Plasmids and insecticidal activity of delta-endotoxin crystals from Bacillus thuringiensis var. israelensis.

1985 ◽  
Vol 49 (3) ◽  
pp. 573-580 ◽  
Author(s):  
Michio HIMENO ◽  
Masao IKEDA ◽  
Kikuo SEN ◽  
Naoto KOYAMA ◽  
Tohru KOMANO ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Insecticidal Activity ◽  
Delta Endotoxin

Improvement of delta-endotoxin production from local Bacillus thuringiensis Se13 using Taguchi’s orthogonal array methodology

2018 ◽  
Vol 43 (6) ◽  
pp. 662-670 ◽  
Author(s):  
Ardahan Eski ◽  
Zihni Demirbağ ◽  
İsmail Demir
Keyword(s):  
Bacillus Thuringiensis ◽  
Incubation Period ◽  
Orthogonal Array ◽  
Incubation Temperature ◽  
Taguchi Methods ◽  
Optimum Level ◽  
Optimum Conditions ◽  
Delta Endotoxin ◽  
Initial Ph ◽  
Four Levels

Abstract Objective The insecticidal activity of Bacillus thuringiensis directly depends on the yield of delta-endotoxins. In this study, various nutritional and cultural parameters influencing delta-endotoxin synthesis by a local isolate of B. thuringiensis Se13 were investigated using Taguchi methods. Methods In the first experiment, four factors, incubation period, incubation temperature, initial pH and medium, each at four levels, were selected and an orthogonal array layout of L16 was carried out. In the second experiment, Taguchi’s orthogonal array method of L27 was used to evaluate the effects of the different concentration of medium components. Taguchi’s signal–noise ratio and variance analysis were applied to determine the effect of the factors. After each experiment, verification studies were carried out using determined optimum conditions. Results The optimum conditions for incubation period, incubation temperature, initial pH, and medium determined as 72 h, 30°C, pH 9, and M4 medium, respectively. In the second experiment, soybean flour (5%), glucose (5%), KH2PO4 (0.3%), K2HPO4 (0.1%), MgSO4 (0.4%) were determined as the optimum conditions. The delta-endotoxin yield was elevated to 1559.25 μg mL−1 when the factors were adjusted to optimum level. Conclusion Optimization using the Taguchi method appeared to be a good choice for the overproduction of delta-endotoxin.

The initial stages in the action of an insecticidal delta-endotoxin of Bacillus thuringiensis var. israelensis on the epithelial cells of the malpighian tubules of the insect, Rhodnius prolixus

1988 ◽  
Vol 90 (1) ◽  
pp. 131-144
Author(s):  
S.H. Maddrell ◽  
N.J. Lane ◽  
J.B. Harrison ◽  
J.A. Overton ◽  
R.B. Moreton
Keyword(s):  
Bacillus Thuringiensis ◽  
Intercellular Junctions ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Ultrastructural Changes ◽  
Cytoplasmic Vacuolization ◽  
Large Numbers ◽  
Delta Endotoxin ◽  
High Concentrations ◽  
Very High

The effects of the 27 X 10(3) Mr insecticidal delta-endotoxin from Bacillus thuringiensis var. israelensis have been studied using, as a model system, isolated insect Malpighian tubules. At all concentrations of the toxin higher than 1 microgram ml-1 (4 X 10(−8) moll-1) applied to the outer surface of the tubules, fluid secretion failed within about 30 min. Except at very high concentrations, where failure always takes at least 30 s, there was an inverse relationship between the concentration of toxin and the time of failure of toxin-treated tubules. During exposure to toxin, the tubules were initially unaffected for a relatively long period and then rapid failure occurred. If the tubules were removed into toxin-free saline just before failure would have occurred, fluid secretion remained normal for at least 2 h, but on return to the origin toxin-containing saline failure was almost immediate. The toxin was found not to bind to the basement membrane. Ultrastructural changes became evident as tubule failure occurred. These initially involved modifications to the basal side of the cells, but later also to the luminal microvilli. Intercellular junctions became disassociated and cytoplasmic vacuolization occurred. The population of intramembranous particles in the basal membranes became reduced with time. Our findings suggest the following hypothesis for the initial stages in the interaction of the toxin with the tubules. Toxin molecules attach to the accessible cell membranes progressively and irreversibly. They do not readily associate by diffusing laterally in the membrane, so that toxic effects develop only when sufficiently large numbers of them attach close together. The molecules may then associate in some way as a complex, perhaps forming a pore in the membrane. Relatively few such pores lead rapidly to cell failure and death.

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