Dataset Information

Role of hsp20 in the Production of Spores and Insecticidal Crystal Proteins in Bacillus thuringiensis.

ABSTRACT: The small heat shock protein plays an important role in response to stresses. We wanted to investigate how Hsp20 affects sporulation and production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis (Bt) at the stationary growth phase when cells are starved. The hsp20 gene was knocked out in Bt4.0718 (wide type), which is a B. thuringiensis strain screened in our laboratory, using endonuclease I-SceI mediated unmarked gene replacement method. Deletion of Hsp20 resulted in a decrease in both sporulation and ICPs production. Bt4-?hsp20 cells and its ICP did not have a significant difference in shape and size but entered the decline phase 2 h earlier than the Bt4.0718. In order to find the mechanism that underlies these phenotypes, we completed a proteomic study of differentially expressed proteins (DEPs). In Bt4-?hsp20 cells, 11 DEPs were upregulated and 184 DEPs downregulated. These affected DEPs are involved in multiple metabolic pathways: (1) six DEPs (two upregulated and four downregulated) are directly related to the sporulation and ICPs synthesis; (2) supply of amino acids including amino acid synthesis and protein recycling; (3) the energy supplementation (the tricarboxylic acid cycle and glycolysis); (4) purine metabolism and mRNA stability. These results suggest that hsp20 may be critical in maintaining the homeostasis of B. thuringiensis during the production of spores and ICPs, and could provide new sight into the sporulation and ICPs formation in B. thuringiensis.

SUBMITTER: Xie J

PROVIDER: S-EPMC6737285 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Role of hsp20 in the Production of Spores and Insecticidal Crystal Proteins in Bacillus thuringiensis.

Xie Junyan J Peng Jinli J Yi Zixian Z Zhao Xiaoli X Li Shuiming S Zhang Tong T Quan Meifang M Yang Shuqing S Lu Jiaoyang J Zhou Pengji P Xia Liqiu L Ding Xuezhi X

Frontiers in microbiology 20190904

The small heat shock protein plays an important role in response to stresses. We wanted to investigate how Hsp20 affects sporulation and production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis (Bt) at the stationary growth phase when cells are starved. The hsp20 gene was knocked out in Bt4.0718 (wide type), which is a B. thuringiensis strain screened in our laboratory, using endonuclease I-SceI mediated unmarked gene replacement method. Deletion of Hsp20 ...[more]

PMID: 31551991

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Project description:Bacillus thuringiensis (Bt) is the most widely used active ingredient for biological insecticides. The composition of δ-endotoxins (Cry and Cyt proteins) in the parasporal crystal determines the toxicity profile of each Bt strain. However, a reliable method for their identification and quantification has not been available, due to the high sequence identity of the genes that encode the δ-endotoxins and the toxins themselves. Here, we have developed an accurate and reproducible mass spectrometry-based method (liquid chromatography-tandem mass spectrometry-multiple reaction monitoring [LC-MS/MS-MRM]) using isotopically labeled proteotypic peptides for each protein in a particular mixture to determine the relative proportion of each δ-endotoxin within the crystal. To validate the method, artificial mixtures containing Cry1Aa, Cry2Aa, and Cry6Aa were analyzed. Determination of the relative abundance of proteins (in molarity) with our method was in good agreement with the expected values. This method was then applied to the most common commercial Bt-based products, DiPel DF, XenTari GD, VectoBac 12S, and Novodor, in which between three and six δ-endotoxins were identified and quantified in each product. This novel approach is of great value for the characterization of Bt-based products, not only providing information on host range, but also for monitoring industrial crystal production and quality control and product registration for Bt-based insecticides.IMPORTANCEBacillus thuringiensis (Bt)-based biological insecticides are used extensively to control insect pests and vectors of human diseases. Bt-based products provide greater specificity and biosafety than broad-spectrum synthetic insecticides. The biological activity of this bacterium resides in spores and crystals comprising complex mixtures of toxic proteins. We developed and validated a fast, accurate, and reproducible method for quantitative determination of the crystal components of Bt-based products. This method will find clear applications in the improvement of various aspects of the industrial production process of Bt. An important aspect of the production of Bt-based insecticides is its quality control. By specifically quantifying the relative proportion of each of the toxins that make up the crystal, our method represents the most consistent and repeatable evaluation procedure in the quality control of different batches produced in successive fermentations. This method can also contribute to the design of specific culture media and fermentation conditions that optimize Bt crystal composition across a range of Bt strains that target different pestiferous insects. Quantitative information on crystal composition should also prove valuable to phytosanitary product registration authorities that oversee the safety and efficacy of crop protection products.

Dataset Information

Role of hsp20 in the Production of Spores and Insecticidal Crystal Proteins in Bacillus thuringiensis.

Publications

Role of <i>hsp20</i> in the Production of Spores and Insecticidal Crystal Proteins in <i>Bacillus thuringiensis</i>.

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