[The functions of yhcZ gene during Bacillus thuringiensis growth].

yhcZ and yhcY genes constitute a two-component system in Bacillus subtilis and B. cereus that regulates bacterial growth. However, the exact biological function of yhcZ gene in B. thuringiensis has not been fully elucidated. In this study, we proved that HD73_5824 is an yhcZ gene in B. thuringiensis subsp. kurstaki HD73 strain by combining gene functional annotation, analysis of upstream and downstream genes arrangement, and amino acid sequence alignment. This yhcZ gene may co-regulate bacterial growth with HD73_5825 gene (yhcY gene) by constituting a two-component system. Homologous recombination technology was employed to knock out yhcZ gene of HD73, resulting in a mutant strain HD (ΔyhcZ). The HD (ΔyhcZ) strain grew slower than wild-type strain HD73 in both LB and SSM medium. Re-introduction of yhcZ gene in HD (ΔyhcZ) strain can partially restore the growth, indicating that the deletion of yhcZ gene impacts the cell growth of HD73 strain. HD (ΔyhcZ) strain grew faster than HD73 strain in M9 medium with 0.4% glucose as the sole carbon source, implying that the yhcZ gene plays an important role in glucose utilization by HD73 strain. The results of biolog assay showed that HD (ΔyhcZ) exhibits a lower average well color development compared to HD73. HD(ΔyhcZ) cells also demonstrated a decreased capacity for absorbing and utilizing D/L-serine, formic acid, D-gluconic acid, L-histamine, D-methyl lactate, and citric acid, indicating that yhcZ gene could dramatically influence carbon source utilization of HD73 strains. Additionally, HD (ΔyhcZ) was less resistant to 8% NaCl, suggesting that yhcZ gene may be involved in the expression and regulation of genes related to high-salt stress response in bacterial cells. The results above show that the yhcZ gene significantly promotes glucose and other carbon sources utilization of HD73 strain during growth. Our findings will lay a foundation not only for analyzing the regulatory mechanisms of glucose and carbon sources utilization by yhcZ gene, but also providing a reference for the further research on bacterial growth and fermentation.

[Deletion of spoIVF operon affects the sporulation and the production of crystal in Bacillus thuringiensis G03].

The spoIVF operon exists in Bacillus universally. Two proteins encoded by the spoIVF operon are essential for the sporulation of Bacillus subtilis. In this study, a spoIVF operon disruption mutant G03 (spoIVF-), in which the spoIVF operon was deleted, was constructed by homologous recombination. The result showed that the mutant strain lost the ability of sporulation. At the same time, the expression of Insecticidal Crystal Protein (ICP) was severely reduced in G03 (spoIVF-) mutant strain and resulted in no crystals. The lacZ gene was fused with the promoter of the cry1Aa gene and expressed in mutant strain G03 (spoIVF-) and G03 wild strain. The activity of beta-galactosidase much lower in mutant G03 (spoIVF-) strain than in the wild-type strain. This further suggested that the activity of sigmaE and sigmaK factors was affected in mutant G03 (spoIVF-) strain. The ability of sporulation and production of Insecticide Crystal Protein was complemented by the expression of spoIVF operon through the vector of pSTK in the mutant strain. In all, The spoIVF operon is essential for the sporulation and the expression of cry gene controlled by sigmaE and sigmaK factors.

Microbial control and biotechnology research on Bacillus thuringiensis in China.

The current status of production and application of biopesticides for pest control in China is briefly reviewed, with a focus on research advances in microbial control with Bacillus thuringiensis (Bt). These have led to improvements in Bt production, exploitation of Bt gene resources, and development of engineered Bt insecticides and transgenic Bt crops that have expanded host ranges and increased efficacy against target pests. Both conventional and biotechnology approaches need to be employed to achieve further progress in discovery, production technology, formulation processing, development of quality standards and recommended use patterns.

[Expression and insecticidal activity of a novel gene cry2ab4 from Bacillus thuringiensis strain B-Pr-88].

The full length cry2Ab gene was cloned by PCR-RFLP method from Bt strain B-Pr-88, which was isolated in China with high toxicity to the Lepidopteran insect pests. Nucleic acid sequence analysis showed that this gene was 1902 base pairs encoding 633 amino acids. This cry gene was named cry2Ab4 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The full open reading frame sequence of the cry2Ab4 gene was amplified with a pair of PCR primers L2ab5/L2ab3 designed according to its DNA sequence,and inserted into the BamH I /EcoR I sites of E. coli expression vector pET21b to obtain the recombinant plasmid pET-2Ab4. The result of SDS-PAGE proved that Cry2Ab4 could be expressed as a 60 kD protein in E. coli BL21 (DE3)strain induced by IPTG. Bioassay of the expressed product of the cry2Ab4 gene showed that Cry2Ab4 was highly toxic to the larvae of Helicoverpa armigera and Leguminivora glycinivorella, moderately active to the larvae of Plutella xylostella and Chilo suppressalis, but not insecticidal to the larvae of Spodotera exigua and Ostrinia furnacalis. Our result indicated that cry2Ab4 gene could be used as a novel gene for generation of transgenic plants and engineered microorganism.

Expression of a modified Cry1Ie gene in E. coli and in transgenic tobacco confers resistance to corn borer.

The wild-type Cry1Ie gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Cry1Ie gene (designated as Cry1Iem) was cloned into prokaryotic expression vector pET28b and its expression in E. coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E. coli revealed that Cry1Iem protein had a similar toxicity to corn borer as wild-type Cry1Ie. Cry1Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cry1Iem showed insecticidal activity against corn borer.