Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant

Abstract

To optimize the expression of cry genes in a Bacillus thuringiensis sigK mutant failing in crystal releasing, the transcriptional activity of the cry promoters cry1A, cry3A, cry4A, and cry8E was compared using lacZ gene fusions. A beta-galactosidase assay indicated that the cry8E promoter showed the highest transcriptional activity. A novel Escherichia coli-B. thuringiensis shuttle vector pHT315-8E21b was constructed for cry gene expression using the cry8E promoter and the multiple cloning sites from vector pET21b, based on vector pHT315. SDS-PAGE analysis showed that the expression of the cry1Ac gene directed by the cry8E promoter was increased by approximately 2.4-fold over the expression directed by the cry3A promoter. The cry1Ba gene was expressed in the sigK mutant with the constructed vector pHT315-8E21b. Normal bipyramidal crystals encapsulated in mother cell were observed by transmission electron microscopy (TEM). The encapsulated Cry1Ba protein expressed in the sigK mutant showed activity against Ostrinia furnacalis and Plutella xylostella similar to that of the released Cry1Ba protein expressed in the acrystalliferous strain HD73 and can be protected from inactivation by UV light. All these results suggest that the cry8E promoter can be an efficient transcriptional element for cry gene expression in sigK mutants and can be utilized for the construction of a genetically engineered strain.

Characterization of CwlC, an autolysin, and its role in mother cell lysis of Bacillus thuringiensis subsp. israelensis

“…The antibiotic concentrations used for bacterial selection were 100 μ g ml −1 ampicillin (Amp) for E. coli strains and 25 μ g ml −1 erythromycin (Ery) for Bt strains (Zhou et al . 2014).…”

Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis

“…Bioassay of insecticidal activity. Biological assays were performed as described by Zhou et al (43), by using equivalent bacterial biomass concentrations of the HD73 and cwlC deletion mutant strains. Briefly, B. thuringiensis cells were grown to T 24 in 50 ml of fresh SSM at 30°C with shaking (220 rpm), centrifuged at 4°C for 10 min at 8,000 rpm, and then freeze-dried for ϳ48 h until the pellets became lyophilized powders.…”

Deletion of the novel gene mother cell lysis X results in Cry1Ac encapsulation in the Bacillus thuringiensis HD73

“…Encapsulation by extrusion is simple to use but low productivity, encapsulation by fluidized bed is high productivity but difficult to control, encapsulation by electrospinning is easy to expand production but the media may be toxic ( de Oliveira et al, 2021 ). Crystal protein encapsulation in mother cell by molecular biological techniques was proved to be a good strategy to improve Cry protein stability under ultraviolet (UV) radiation ( Sanchis et al, 1999 ; Zhou et al, 2014 ). The mother cell wall can protect Cry protein from inactivation by UV radiation.…”

“…This technology not only constructed spore-free strain, which had no pollution to the environment and no competition with wild B. thuringiensis strains, but also improved the UV resistance of crystal proteins in the field condition ( Sanchis et al, 1999 ). Encapsulation Cry1Ba in the mother cell of sigK mutant also increasing UV radiation resistance ( Zhou et al, 2014 ). However, some cry genes are regulated by σ K , and cry gene expressions were reduced in the sigK deletion mutant ( Adams et al, 1991 ; Bravo et al, 1996 ).…”