scholarly journals Regulation of the Packaging of Bacillus thuringiensis δ-Endotoxins into Inclusions

2001 ◽  
Vol 67 (11) ◽  
pp. 5032-5036 ◽  
Author(s):  
Lily Chang ◽  
Rian Grant ◽  
Arthur Aronson
Keyword(s):  
Bacillus Thuringiensis ◽  
Consensus Sequence ◽  
Luria Bertani ◽  
Insect Larvae ◽  
Inclusion Formation ◽  
Reducing Conditions ◽  
Mrna Ratio ◽  
Yeast Extract Medium ◽  
Relative Deficiency ◽  
Binding Sequence

ABSTRACT During sporulation, many Bacillus thuringiensis subspecies synthesize several related δ-endotoxins which are packaged into bipyramidal intracellular inclusions. These inclusions are solubilized in the alkaline, reducing conditions of the midguts of susceptible insect larvae and are converted by proteolysis to active toxins. The toxins insert into the membranes of cells lining the midgut and form cation-selective channels, which results in lethality. There are three δ-endotoxins, Cry1Ab3, Cry1Ca1, and Cry1Da1, present in the inclusions produced by a B. thuringiensis subsp.aizawai cell. While the ratio of the steady-state mRNAs for these three protoxins has been shown to differ (cry1Ab3/cry1Ca1/cry1Da1 mRNA ratio, 4:2:1), the half-lives of the cry1Da1 and cry1Ab3 mRNAs were found to be similar, indicating that there were differences in the transcription rates. The relative contents of these δ-endotoxins in purified inclusions from B. thuringiensissubsp. aizawai have been measured previously, and an even greater relative deficiency of the Cry1Da1 protoxin (ratio, 20:12:1) was found. In order to account for this deficiency, other steps which could be involved in inclusion formation, such as translation and packaging, were examined. The three cry genes have the same dual overlapping promoters, but the ribosome binding sequence for the cry1Da1 gene was not the consensus sequence. Translation was enhanced about fourfold by changing to the consensus sequence. In addition, the relative amount of Cry1Da1 protoxin in inclusions was twofold lower when cells were sporulated in Luria-Bertani (LB) medium than when cells were sporulated in a glucose-yeast extract medium. This difference was attributable to packaging since the relative amounts of Cry1Da1 antigen in cells sporulating in the two media were the same. Some factor(s) required for packaging of the Cry1Da1 protoxin in inclusions is apparently limiting in LB medium. Differences in the initial transcription rates, translation efficiencies, and packaging all contribute to the δ-endotoxin composition of an inclusion.

Technology of production of biopesticides based on Bacillus thuringiensis

2021 ◽  
Vol 9 (16) ◽  
pp. 28-38
Author(s):  
Anatolii Bezusov ◽  
◽  
Valentyna Krutiakova ◽  
Olena Myroshnichenko ◽  
Nataliia Dotsenko ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Corn Oil ◽  
Raw Materials ◽  
Culture Fluid ◽  
Scientific Work ◽  
Insect Larvae ◽  
Effective Prevention ◽  
Research Results ◽  
Wide Range ◽  
Vegetable Raw Materials

Subject of research. Biopesticides are based on live cultures of specially selected beneficial microorganisms with controlled properties. They have a pronounced phytoprotective and stimulating effect, thus providing effective prevention and protection of plants from diseases. The obtained biological product increases productivity, improves the quality and structure of the crop, does not accumulate in plants, which allows to obtain environmentally friendly agricultural products and does not harm the environment. Among the large number of bacteria, as a source of microbiological insecticide is Bacillus thuringiensis, which infects lepidopteran pests and leads to their death. Preparations based on this strain are effective when used in low concentrations of solutions. The Bacillus thuringiensis strain produces several toxins with insecticidal action, including β-exotoxin and δ-endotoxin. Toxic effect is manifested and leads to paralysis of the intestinal tract of parasites. Preparations β-exotoxin and δ-endotoxin are obtained by culturing Bacillus thuringiensis bacteria in a liquid medium. The scientific work proposes a method of industrial production using by-products of vegetable raw materials, which makes it economically feasible to use such substrates. The purpose of the study is the development of technology, formulation of nutrient medium, process parameters for the cultivation of bacteria of the genus Bacillus thuringiensis and obtaining a culture fluid containing substances of the class of biopesticides. Methods. Standard and generally accepted methods of research of bioproducts in biotechnology. The formation of bioinsecticides was established by hydrolysis methods, followed by determination of the component of β-exotoxin – ribose, the formation of octagonal crystals of exotoxin – by microscopic method. Research results. Three variants of nutrient media, which include yeast-polysaccharide complex: corn flour, corn oil, yeast autolysate were developed. The parameters of the bacterial cultivation process were studied. The final product is a paste or powder with a titer of 35 .109 spores in 1 g of the bioproduct. Scope of research results. Microbiological preparations based on Bacillus thuringiensis are highly specific and act only on insect larvae from the classes Lepidoptera and Diptera. The resulting biopesticide can be used against pests of a wide range of vegetable and fruit crops.

scholarly journals Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 543
Author(s):  
Oihane Simón ◽  
Leopoldo Palma ◽  
Ana Beatriz Fernández ◽  
Trevor Williams ◽  
Primitivo Caballero
Keyword(s):  
Bacillus Thuringiensis ◽  
Actin Polymerization ◽  
Consensus Sequence ◽  
Cell Polarization ◽  
Sf9 Cells ◽  
Culture Cells ◽  
Control Virus ◽  
Occlusion Bodies ◽  
Recombinant Bacmid ◽  
Adp Ribosyltransferase

The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpa1/Vpa2 is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two novel Vpa2-like genes, Vpa2-like1 and Vpa2-like2, under the baculovirus p10 promoter in transfected Sf9 cells. Pairwise amino acid analyses revealed a higher percentage of identity and a lower number of gaps between Vpa2Ac1 and Vpa2-like2 than to Vpa2-like1. Moreover, Vpa2-like1 lacked the conserved Ser-Thr-Ser motif, involved in NAD binding, and the (F/Y)xx(Q/E)xE consensus sequence, characteristic of the ARTT toxin family involved in actin polymerization. Vpa2Ac1, Vpa2-like1 and Vpa2-like2 transcripts and proteins were detected in Sf9 culture cells, but the signals of Vpa2Ac1 and Vpa2-like2 were weak and decreased over time. Sf9 cells infected by a recombinant bacmid expressing Vpa2-like1 showed typical circular morphology and produced viral occlusion bodies (OBs) at the same level as the control virus. However, expression of Vpa2Ac1 and Vpa2-like2 induced cell polarization, similar to that produced by the microfilament-destabilizing agent cytochalasin D and OBs were not produced. The presence of filament disrupting agents, such as nicotinamide and nocodazole, during transfection prevented cell polarization and OB production was observed. We conclude that Vpa2Ac1 and Vpa2-like2 proteins likely possess ADP-ribosyltransferase activity that modulated actin polarization, whereas Vpa2-like1 is not a typical Vpa2 protein. Vpa2-like2 has now been designated Vpa2Ca1 (accession number AAO86513) by the Bacillus thuringiensis delta-endotoxin nomenclature committee.

INVITATION PAPER (C.P. ALEXANDER FUND): HISTORY OF BACILLUS THURINGIENSIS BERLINER RESEARCH AND DEVELOPMENT

1992 ◽  
Vol 124 (4) ◽  
pp. 587-616 ◽  
Author(s):  
Clayton C. Beegle ◽  
Takashi Yamamoto
Keyword(s):  
Bacillus Thuringiensis ◽  
Ostrinia Nubilalis ◽  
Microbial Control ◽  
The United States ◽  
Control Agent ◽  
Turn Of The Century ◽  
Insect Larvae ◽  
Commercial Development ◽  
Japanese Workers ◽  
History Of

AbstractThis review article starts with the discovery of Bacillus thuringiensis Berliner in Japan at the turn of the century and notes that the observations of the early Japanese workers clearly show that they were aware of the toxin-mediated nature of the activity of B. thuringiensis toward insect larvae. The early work in Europe with B. thuringiensis against Ostrinia nubilalis (Hubner) showed that the bacterium had promise as a microbial control agent. The commercial development of B. thuringiensis in France in the late 1930s, and in Eastern Europe and the United States in the 1950s, is traced.

scholarly journals Four of the seven zinc fingers of the Evi-1 myeloid-transforming gene are required for sequence-specific binding to GA(C/T)AAGA(T/C)AAGATAA.

1993 ◽  
Vol 13 (7) ◽  
pp. 4291-4300 ◽  
Author(s):  
R Delwel ◽  
T Funabiki ◽  
B L Kreider ◽  
K Morishita ◽  
J N Ihle
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
Zinc Fingers ◽  
Consensus Sequence ◽  
Myelogenous Leukemia ◽  
Chromosome Band ◽  
Aberrant Expression ◽  
Relative Specificity ◽  
Acute Myelogenous ◽  
Binding Sequence

Expression of the Evi-1 gene is activated in murine myeloid leukemias by retroviral insertions and in human acute myelogenous leukemia by translocations and inversions involving chromosome band 3q26 where the gene resides. Aberrant expression of the Evi-1 gene has been shown to interfere with myeloid differentiation, which is proposed to be the basis for its role in leukemias. The Evi-1 gene encodes a 145-kDa DNA-binding protein containing two domains of seven and three Cys2-His2 zinc fingers. Previous studies identified a portion of the consensus DNA-binding sequence for the first domain of zinc fingers. The experiments presented here extend these studies and demonstrate that the first domain recognizes a consensus of 15 nucleotides consisting of GA(C/T)AAGA(T/C)AAGATAA. The first three fingers of the first domain do not detectably bind DNA but contribute to the binding by conferring a relative specificity for GACAA verses GATAA in the first position. The first three fingers also contribute to optimal binding of the 15-nucleotide consensus sequence.

scholarly journals High-Salt Stress Conditions Increase the pAW63 Transfer Frequency in Bacillus thuringiensis

2012 ◽  
Vol 78 (19) ◽  
pp. 7128-7131 ◽  
Author(s):  
Elise Beuls ◽  
Pauline Modrie ◽  
Cédric Deserranno ◽  
Jacques Mahillon
Keyword(s):  
Salt Stress ◽  
Bacillus Thuringiensis ◽  
Positive Impact ◽  
Brain Heart Infusion ◽  
Luria Bertani ◽  
Plasmid Transfer ◽  
High Salt ◽  
Content Type ◽  
Luria Bertani Broth ◽  
High Salt Stress

ABSTRACTConjugation experiments withBacillus thuringiensisand transfer kinetics demonstrated that salt stress has a positive impact on plasmid transfer efficiency. Compared to standard osmotic conditions (0.5% NaCl), plasmid transfer occurred more rapidly, and at higher frequencies (>100-fold), when bacteria were exposed to a high-salt stress (5% NaCl) in liquid brain heart infusion (BHI). Under milder salt conditions (2.5% NaCl), only a 10-fold effect was observed in Luria-Bertani broth and no difference was detected in BHI. These observations are particularly relevant in the scope of potential gene exchanges among members of theBacillus cereusgroup, which includes food-borne contaminants and pathogens.

scholarly journals Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

2008 ◽  
Vol 74 (9) ◽  
pp. 2565-2572 ◽  
Author(s):  
Frédéric Girard ◽  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Lucie Marceau ◽  
Yanhui Su ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Insect Larvae ◽  
Insecticidal Toxin ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis ◽  
Pore Lining

ABSTRACT Helix α4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the α4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

HIGH-DOSAGE TREATMENT OF A QUEBEC STREAM WITH BACILLUS THURINGIENSIS SEROVAR. ISRAELENSIS: EFFICACY AGAINST BLACK FLY LARVAE (DIPTERA: SIMULIIDAE) AND IMPACT ON NON-TARGET INSECTS

1985 ◽  
Vol 117 (12) ◽  
pp. 1523-1534 ◽  
Author(s):  
C. Back ◽  
J. Boisvert ◽  
J.O. Lacoursière ◽  
G. Charpentier
Keyword(s):  
Bacillus Thuringiensis ◽  
Aquatic Insects ◽  
Artificial Substrates ◽  
High Dose ◽  
Artificial Turf ◽  
Insect Larvae ◽  
Commercial Formulation ◽  
Lake Outlet ◽  
Pre Treatment ◽  
Drifting Larvae

AbstractA typical lake outlet of the Canadian Shield was treated for 15 min with a high dose (5.28 g/L s−1 of discharge) of Teknar®, a commercial formulation of Bacillus thuringiensis serovar. israelensis. Efficacy on Simuliidae larvae and impact on non-target aquatic insects of this stream were monitored using drift nets, counting plates, and artificial turf substrates along a 1000-m section downstream of the site of application. Compared with a 4-day pre-treatment average for 12-h sampling periods, drift of Simuliidae increased from 64 to 92 ×, with shorter peaks of 133–184 ×, 2–6 h after treatment. There was no evident drift increase in larvae of Ephemeroptera, Plecoptera, Trichoptera, Chironomidae, or dipterous pupae, but larvae of Blephariceridae (Diptera) were severely affected as their drift was increased by up to 50 × and remained high for 3 days. After 30 h the mortality of Simuliidae on counting plates ranged from 95 to 82% in the first 300 m, with detachment rates of 78.5–46.5%. Densities of non-target insect larvae were not reduced on the artificial substrates, except for 2 genera of Chironomidae (Eukiefferella and Polypedilum) which were reduced 26 to 39% of their original density. Drifting larvae of 1 chironomid genus (Phaenopsectra) also showed symptoms of toxemia by B.t.i. The main impact of the treatment was thus seen in 2 Nematocera families (Chironomidae and Blephariceridae) which were mainly exposed to B.t.i. sedimented on the bottom of the stream or attached to periphyton growing on rocks.

scholarly journals Regulation by Overlapping Promoters of the Rate of Synthesis and Deposition into Crystalline Inclusions ofBacillus thuringiensis δ-Endotoxins

2000 ◽  
Vol 182 (3) ◽  
pp. 734-741 ◽  
Author(s):  
Mira Sedlak ◽  
Thomas Walter ◽  
Arthur Aronson
Keyword(s):  
Bacillus Thuringiensis ◽  
Northern Blotting ◽  
Sigma Factors ◽  
Wild Type ◽  
Insect Larvae ◽  
Crystalline Inclusions ◽  
Major Class ◽  
Ofbacillus Thuringiensis ◽  
Wild Type Promoter ◽  
Stimulation Of

ABSTRACT During sporulation, Bacillus thuringiensis produces intracellular, crystalline inclusions comprised of a mixture of protoxins active on insect larvae. A major class of these protoxin genes, designated cry1, is transcribed from two overlapping promoters (BtI and BtII) utilizing RNA polymerase containing sporulation sigma factors ςE and ςK, respectively. Fusions of these promoters to lacZ were constructed in order to analyze transcription patterns. Mutations within the −10 region of the BtII promoter (within the spacer region of the BtI promoter) which departed from the consensus −10 sequence for either ςE or ςK resulted in inactivation of transcription from BtII and a fivefold stimulation of transcription from BtI. In contrast, transcription from both promoters was inhibited with a change to the ςE consensus. One of the “promoter-up” mutations was fused to the cry1Ac1gene, and enhanced transcription was confirmed by Northern blotting. There was an increase in the accumulation of Cry1Ac antigen at early but not later times in sporulation in the mutant. This shift was due to the rapid turnover of much of the excessively accumulated protoxin at the early times as measured by pulse-chase labeling. As a result of the turnover and the inactivation of the BtII promoter, the mutant produced smaller inclusions which contained two- to threefold-less protoxin than inclusions from the wild type. Promoter overlap is a mechanism for modulating protoxin synthesis, thus ensuring the efficient packaging of these protoxins into inclusions.

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