Regulation of insecticidal crystal protein production in <i>Bacillus thuringiensis</i>

Baum, James A.; Malvar, Thomas

doi:10.1111/j.1365-2958.1995.mmi_18010001.x

Cited by 147 publications

(121 citation statements)

References 29 publications

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“…Therefore, the intrinsic mechanisms of high-level ICP syntheses and parasporal crystal formation have drawn extensive attention of biologists. The high expression levels of ICP genes appear to result from regulatory mechanisms coordinately occurring at the transcriptional (7,8), posttranscriptional (9), translational and posttranslational levels (10). However, the regulatory mechanisms underlying the metabolic pathways that provide amino acids, carbon sources, and energy for the high-level syntheses of ICPs remain unclear.…”

mentioning

confidence: 99%

The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

Wang

Han

Cao

et al. 2013

Molecular & Cellular Proteomics

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Bacillus thuringiensis is a well-known entomopathogenic bacterium used worldwide as an environmentally compatible biopesticide. During sporulation, B. thuringiensis accumulates a large number of parasporal crystals consisting of insecticidal crystal proteins (ICPs) that can account for nearly 20 -30% of the cell's dry weight. However, the metabolic regulation mechanisms of ICP synthesis remain to be elucidated. In this study, the combined efforts in transcriptomics and proteomics mainly uncovered the following 6 metabolic regulation mechanisms: (1) proteases and the amino acid metabolism (particularly, the branched-chain amino acids) became more active during sporulation; (2) stored poly-␤-hydroxybutyrate and acetoin, together with some low-quality substances provided considerable carbon and energy sources for sporulation and parasporal crystal formation; (3) the pentose phosphate shunt demonstrated an interesting regulation mechanism involving gluconate when CT-43 cells were grown in GYS medium; (4) the tricarboxylic acid cycle was significantly modified during sporulation; (5) an obvious increase in the quantitative levels of enzymes and cytochromes involved in energy production via the electron transport system was observed; (6) most F 0 F 1 -ATPase subunits were remarkably up-regulated during sporulation. This study, for the first time, systematically reveals the metabolic regulation mechanisms involved in the supply of amino acids, carbon substances, and energy for B. thuringiensis spore and parasporal crystal formation at both the transcriptional and translational levels. Molecular & Cellular Proteomics

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confidence: 99%

The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

Wang

Han

Cao

et al. 2013

Molecular & Cellular Proteomics

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“…On the other hand, no nuclear occlusion bodies should be seen in the nucleus of cells infected with the recombinant baculovirus generated by transposition ( Figure 2C), since the polyhedrin gene is not present in these viruses. The C-terminal portions of Bt 130 kDa Cry proteins like Cry1A, Cry4A and Cry4B are suggested to have an important role in the crystallization and/or stabilization of the proteins (Baum & Malvar, 1995;Park, 2000). This region of Cry proteins contains a higher concentration of cysteine residues, which are responsible for disulfide bond formation and are believed by some authors to contribute to crystal formation (Ge et al, 1998).…”

Section: Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp Israelementioning

confidence: 99%

Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp. Israelensis Expressed in Insect Cells by Recombinant Baculoviruses Are Toxic to Aedes Aegypti Larvae

Corrêa¹,

Aguiar²,

Monnerat³

et al. 2013

VR&R

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Bacillus thuringiensis subsp. israelensis (Bti) is highly toxic to mosquito larvae. Most of its toxicity relies on δ-endotoxins (Cry and Cyt) crystalline inclusions produced at the sporulation phase of growth. Obtention of individual Bti mosquitocidal toxins is crucial for the understanding of Bti´s potential activity against dipteran larvae. A Cry toxin expression system based on baculovirus was developed to produce two of the Cry toxins comprised in the crystals of Bti. Th e cry4Aa and cry4Ba genes from two Brazilian strains of Bacillus thuringiensis were amplifi ed by PCR, cloned into a plasmid cloning vector and sequenced. Sequence analysis of the cry4Aa and cry4Ba genes showed high identity to previous known cry genes. Both cry4Aa and cry4Ba genes were further cloned into transfer vectors for construction of recombinant baculoviruses.. Aft er isolation of the recombinant viruses, they were used to infect insect cells (BTI-TN5B1-4) that were analyzed by light microscopy at diff erent times post infection. Putative crystals consisting of either Cry4Aa or Cry4Ba were observed in the cytoplasm of infected insect cells. RT-PCR was performed with mRNA from insect cell extracts (72 h.p.i.) in order to confi rm the presence of the genes specifi c transcripts. Recombinant virus-infected insect extracts (120 h.p.i.) were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) showing the presence of polypeptide bands of around 128 and 130 kDa, corresponding, respectively to the sizes of the proteins Cry4Aa and Cry4Ba. Bioassays with virus-infected insect extracts were shown to be toxic to second instar Aedes aegypti larvae, confi rming the usefulness of this expression system for the study of Cry proteins.

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“…This aerobic spore-forming bacterium produces a variety of plasmid-encoded crystal proteins known as δ-endotoxins or Cry proteins, which exhibit high toxicity against larvae of certain agricultural and forest pests as well as vectors diseases. This toxic activity is highly specific, usually to a particular insect order, and is determined by the type of Cry proteins produced by each Bt subspecies (Baum and Malvar 1995;Schnepf et al 1998). Crystals produced by Bacillus thuringiensis var.…”

Section: Introductionmentioning

confidence: 99%

Oxygen supply in Bacillus thuringiensis fermentations: bringing new insights on their impact on sporulation and δ-endotoxin production

Boniolo

Rodrigues

Prata

et al. 2012

Appl Microbiol Biotechnol

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The growth kinetics, sporulation, and toxicity of Bacillus thuringiensis var. israelensis were evaluated through the analysis of batch cultures with different dissolved oxygen (DO) profiles. Firstly, DO was maintained constant at 5%, 20%, or 50% throughout fermentation in order to identify the most suitable one to improve the main process parameters. Higher biomass concentration, cell productivity, and cell yield based on glucose were obtained with 50% DO. The higher aeration level also resulted in higher spore counts and markedly improved the toxic activity of the fermentation broth, which was 9-fold greater than that obtained with 5% DO (LC 50 of 39 and 329 mg/L, respectively). Subsequently, using a two-stage oxygen supply strategy, DO was kept at 50% during the vegetative and transition phases until the maximum cell concentration was achieved. Then, DO was changed to 0%, 5%, 20%, or 100% throughout sporulation and cell lysis phases. The interruption of oxygen supply strongly reduced the spore production and thoroughly repressed the toxin synthesis. On the contrary, when DO was raised to 100% of saturation, toxic activity increased approximately four times (LC 50 of 8.2 mg/L) in comparison with the mean values reached with lower DO levels, even though spore counts were lower than that from the 50% DO assay. When pure oxygen was used instead of normal air, it was possible to obtain 70% of the total biomass concentration achieved in the air assays; however, cultures did not sporulate and the toxin synthesis was consequently suppressed.

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Regulation of insecticidal crystal protein production in Bacillus thuringiensis

Cited by 147 publications

References 29 publications

The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp. Israelensis Expressed in Insect Cells by Recombinant Baculoviruses Are Toxic to Aedes Aegypti Larvae

Oxygen supply in Bacillus thuringiensis fermentations: bringing new insights on their impact on sporulation and δ-endotoxin production

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