Identification and Functional Characterization of Two Homologous SpoVS Proteins Involved in Sporulation of Bacillus thuringiensis.

Sporulation is an important part of the life cycle of Bacillus thuringiensis and the basis for the production of parasporal crystals. This study identifies and characterizes two homologous spoVS genes (spoVS1 and spoVS2) in B. thuringiensis, both of whose expression is dependent on the σH factor. The disruption of spoVS1 and spoVS2 resulted in defective B. thuringiensis sporulation. Similar to Bacillus subtilis, B. thuringiensis strain HD(ΔspoVS1) mutants showed delayed formation of the polar septa, decreased sporulation efficiency, and blocked spore release. Different from B. subtilis, B. thuringiensis HD(ΔspoVS1) mutants had disporic septa and failed to complete engulfment in some cells. Moreover, HD(ΔspoVS2) mutants had delayed spore release. The effect of spoVS1 deletion on polar septum delay and sporulation efficiency could be compensated by spoVS2. ß-Galactosidase activity analysis showed that the expression of pro-sigE and spoIIE decreased to different degrees in the HD(ΔspoVS1) and HD(ΔspoVS2) mutants. The different effects of the two mutations on the expression of sporulation genes led to decreases in Cry1Ac production of different levels. IMPORTANCE There is only one spoVS gene in B. subtilis, and its effects on sporulation have been reported. In this study, two homologous spoVS genes were found and identified in B. thuringiensis. The different effects on sporulation and parasporal crystal protein production in B. thuringiensis and their relationship were investigated. We found that these two homologous spoVS genes are highly conserved in the Bacillus cereus group, and therefore, the functional characterization of SpoVS is helpful to better understand the sporulation processes of members of the Bacillus cereus group.

Production and characterization of the 13 C/15 N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.

Synthetic Cry1Ab/Ac proteins expressed by genetically modified (GM) crops have a high potential to control insect pests without utilizing large amounts of chemical insecticides. Before these crops are used in agriculture, the environmental fate and interactions in the soil must be understood. Stable isotope-labeled Cry1Ab/Ac protein is a highly useful tool for collecting such data. We developed a protocol to produce 13 C/15 N single-labeled Cry proteins. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which has been certified by the Chinese government to be safe for human consumption, was subcloned into pUC57, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37°C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U-13 C] glucose (5% 13 C-enriched) or [15 N] ammonium chloride (5% 15 N-enriched). Then, Cry inclusion bodies were dissolved in urea and purified by affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The purities of 13 C/15 N-labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The δ 13 C and ä 15 N values of 13 C-labeled Cry protein and 15 N-labeled Cry protein were 3,269‰ and 2,854‰, respectively. A bioassay test revealed that the labeled Cry1Ab/Ac proteins had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies on Cry proteins in soil and the characteristics of nitrogen (N) and carbon (C) transformations. Our findings may also be employed as a reference for elucidating the environmental behavior and ecological effects of BT plants and expressed products.

Bacillus thuringiensis endotoxin production: a systematic review of the past 10 years.

Bacillus thuringiensis (Bt) is one of the most promising biological control agents used commercially. Its products can contribute to reducing ecological and environmental problems associated with the use of chemical pesticides. Among the limiting factors of using Bt as bioinsecticide are the costs and ensuring its biological activity, which may vary according to the strain and culture conditions. This systematic review aimed to collect state-of-the-art information on the production of Bt endotoxins and to score the methodological feasibility of the data obtained, thus highlighting possible incoherencies. In order to consolidate recent findings and guide future studies, a total of 47 original articles from the last 10 years was analysed, with special attention being given to corroborating data, identifying inconsistencies and suggesting future adjustments so as to increase data reliability. With a maximum score of 8 points, three production parameters were classified on the following scale: preferable (score: 2), adequate (score: 1) and inadequate (score: 0), and another two parameter were classified as adequate (score: 1) or inadequate (score: 0). No article scored more than 6 out of the maximum of 8, thus reflecting the need for more detailed studies regarding Bt endotoxin production. The lack of standardization of methods and units of measurement also have made a comparison of results and an overall analysis difficult. Standards are suggested in the present study. The inclusion of bioassays and quantifying toxin via alkaline dilution are strongly recommended for studies of this nature, along with LC50 expressed in mg/L. Sixteen articles (34%) did not use either of these suggested methods, which indicates the need for further supporting studies. These findings reinforce the need for robust studies in this area, which could include the development of more affordable and effective bioinsecticides, thus increasing their competitiveness against insecticides derived from unsustainable sources.

The signal peptide of Cry1Ia can improve the expression of eGFP or mCherry in Escherichia coli and Bacillus thuringiensis and enhance the host's fluorescent intensity.

BACKGROUND: The signal peptides (SPs) of secretory proteins are frequently used or modified to guide recombinant proteins outside the cytoplasm of prokaryotic cells. In the periplasmic space and extracellular environment, recombinant proteins are kept away from the intracellular proteases and often they can fold correctly and efficiently. Consequently, expression levels of the recombinant protein can be enhanced by the presence of a SP. However, little attention has been paid to the use of SPs with low translocation efficiency for recombinant protein production. In this paper, the function of the signal peptide of Bacillus thuringiensis (Bt) Cry1Ia toxin (Iasp), which is speculated to be a weak translocation signal, on regulation of protein expression was investigated using fluorescent proteins as reporters. RESULTS: When fused to the N-terminal of eGFP or mCherry, the Iasp can improve the expression of the fluorescent proteins and as a consequence enhance the fluorescent intensity of both Escherichia coli and Bt host cells. Real-time quantitative PCR analysis revealed the higher transcript levels of Iegfp over those of egfp gene in E. coli TG1 cells. By immunoblot analysis and confocal microscope observation, lower translocation efficiency of IeGFP was demonstrated. The novel fluorescent fusion protein IeGFP was then used to compare the relative strengths of cry1Ia (Pi) and cry1Ac (Pac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of Pi at the same level of sensitivity. The fluorescent signals of E. coli and Bt cells expressing the Iasp fused mCherry (ImCherry) were also enhanced. Importantly, the Iasp can also enhanced the expression of two difficult-to-express proteins, matrix metalloprotease-13 (MMP13) and myostatin (growth differentiating factor-8, GDF8) in E. coli BL21-star (DE3) strain. CONCLUSIONS: We identified the positive effects of a weak signal peptide, Iasp, on the expression of fluorescent proteins and other recombinant proteins in bacteria. The produced IeGFP and ImCherry can be used as novel fluorescent protein variants in prokaryotic cells. The results suggested the potential application of Iasp as a novel fusion tag for improving the recombinant protein expression.

No More Tears: Mining Sequencing Data for Novel Bt Cry Toxins with CryProcessor.

Bacillus thuringiensis (Bt) is a natural pathogen of insects and some other groups of invertebrates that produces three-domain Cry (3d-Cry) toxins, which are highly host-specific pesticidal proteins. These proteins represent the most commonly used bioinsecticides in the world and are used for commercial purposes on the market of insecticides, being convergent with the paradigm of sustainable growth and ecological development. Emerging resistance to known toxins in pests stresses the need to expand the list of known toxins to broaden the horizons of insecticidal approaches. For this purpose, we have elaborated a fast and user-friendly tool called CryProcessor, which allows productive and precise mining of 3d-Cry toxins. The only existing tool for mining Cry toxins, called a BtToxin_scanner, has significant limitations such as limited query size, lack of accuracy and an outdated database. In order to find a proper solution to these problems, we have developed a robust pipeline, capable of precise 3d-Cry toxin mining. The unique feature of the pipeline is the ability to search for Cry toxins sequences directly on assembly graphs, providing an opportunity to analyze raw sequencing data and overcoming the problem of fragmented assemblies. Moreover, CryProcessor is able to predict precisely the domain layout in arbitrary sequences, allowing the retrieval of sequences of definite domains beyond the bounds of a limited number of toxins presented in CryGetter. Our algorithm has shown efficiency in all its work modes and outperformed its analogues on large amounts of data. Here, we describe its main features and provide information on its benchmarking against existing analogues. CryProcessor is a novel, fast, convenient, open source (https://github.com/lab7arriam/cry_processor), platform-independent, and precise instrument with a console version and elaborated web interface (https://lab7.arriam.ru/tools/cry_processor). Its major merits could make it possible to carry out massive screening for novel 3d-Cry toxins and obtain sequences of specific domains for further comprehensive in silico experiments in constructing artificial toxins.