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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-Δhsp20 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-Δhsp20 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.

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Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.

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