The transport of nucleosides and their homeostasis within the cell are essential for growth and proliferation. Here, we have identified a novel transcription factor, NupR, which, to our knowledge, is the first GntR family transcription factor primarily involved in the regulation of nucleoside transport.
Background: Vip3Aa is an insecticidal protein secreted by some Bacillus thuringiensis strains during vegetative growth. It has excellent insecticidal activity, its mechanism of action is different from that of Cry protein, and it can delay the development of pest resistance. To date, Vip3Aa has been widely used in genetically modified Bt crops. However, the secretion of Vip3Aa by industrial production strains is usually very low. Moreover, most of the Vip3Aa in the medium is degraded by proteases, limiting its application as a biopesticide.Results: We report a novel constitutive strong promoter from B. thuringiensis, P rsi , which directs the abundant expression of vip3Aa in B. thuringiensis BMB171. Furthermore, to reduce the degradation of Vip3Aa caused by proteases, we constructed B. thuringiensis mutants in which different protease genes were knocked out. We found that the degradation of Vip3Aa was greatly inhibited and its yield was significantly improved in a mutant that lacked all three protease genes. Conclusion: Our results provide a new strategy to enhance the production of Vip3Aa in B. thuringiensis and have reference value for the research and development of novel bioinsecticides.
Vegetative insecticidal protein Vip3Aa, secreted by many Bacillus thuringiensis (Bt) strains during the vegetative growth stage, represents the second-generation insecticidal toxin. In recent years, significant progress has been made on its structure and action mechanism. However, how it is translocated across the cytoplasmic membrane into the environment remains a mystery. This work demonstrates that Vip3Aa is not secreted by the General Secretion (Sec) System. To reveal the secretory pathway of Vip3A, we purified the membrane vesicles (MVs) of B. thuringiensis BMB171 and observed by TEM. The size of MVs was determined by the dynamic light scattering method, and their diameter was approximately 40–200 nm, which is consistent with the vesicles in Gram-negative bacteria. Moreover, Vip3A could be detected in the purified MVs by Western blot, and immunoelectron microscopy reveals Vip3A antibody-coated gold particles located in the MVs. After deleting its signal peptide, chitinase B (ChiB) failed to be secreted. However, the recombinant ChiB, whose signal peptide was substituted with the N-terminal 39 amino acids from Vip3A, was secreted successfully through MVs. Thus, this sequence is proposed as the signal region responsible for vesicle transport. Together, our results revealed for the first time that Vip3Aa is transported to the medium via MVs.
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