Bacillus thuringiensis Bt185 and its insecticidal spectrum-expanded engineering strains are considered as potential biocontrol agents to soil insect Holotrichia parallela, Holotrichia oblita or Anomala corpulenta.  Here we reported the complete genome of strain Bt185, it harbors eight plasmids, and plasmid pBT1850294 carries three cry8 genes.

Bacillus thuringiensis subspecies morrisoni strain HD12, whose genome harbors multiple insecticidal protein-encoding genes, includes eight cry genes, as indicated by genome sequencing.  This strain produces crystals that are toxic to lepidopteran species.  These crystal inclusions were purified by sucrose gradients and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by liquid chromatography-mass spectrometry, and found to contain five proteins (Cry1Da, Cry1Ae, Cry1Bb, Cry1Fb, and Cry1Ja).  The transcriptional activities of the cry1Da, cry1Ae, cry1Bb, cry1Fb, and cry1Ja promoters indicated that transcription of cry1Da is controlled by SigE; however, the other four cry genes were found to be controlled by both SigE and SigK.  The activities of the cry1Ja and cry1Fb promoters were the strongest among the five genes studied.  These promoters were therefore used to direct the expression of the Cry1Ac- and Cry2Ab-encoding genes concurrently in a single strain.  Our findings indicate that promoters with the same transcriptional profile may be used to direct the expression of different cry genes in one strain.  Our results are expected to be valuable for the construction of strains with efficient expression of multiple cry genes in order to overcome current limitations associated with the application of B. thuringiensis-based insecticides.

Bacillus thuringiensis is one of the most widely used bioinsecticides, and cry gene is the major insecticidal gene. Because Cry1Ac protein shows strong toxicity against many lepidopteran species, it has been applied widely in spraying products and transgenic Bt-crops. The preparation of Cry protoxin is the first step in the very important processes of understanding the insecticidal mechanism, resistance screening, and biosafety assessments. The media for crystal production and the method for Cry protoxin preparation were varied, however, it was not clear which was better for preparing a larger amount of Cry protoxin. In this paper, three media for crystal production and the method for Cry1Ac protoxin preparation from HD73 strain were compared to find an efficacious way to prepare a large number of Cry1Ac protoxin. The results showed that the 1/2 LB (Luria-Bertani) medium was the ideal medium for crystal production, because the total yield of Cry1Ac protoxin in 300 mL 1/2 LB medium was (112.38±5.64) mg, the highest one among three media; the repeated crystal solubilization method was better for the preparation of the Cry protoxin comparing with the continuous crystal solubilization method. It will be a reference for other Cry protoxin preparation, especially for larger number.

The novel cry1Ai gene that cloned from Bacillus thuringiensis strain SC6H8 encoded a protein exhibiting strong toxicity against Plutella xylostella and Chilo suppressalis in our previous study. Using the available information for the active fragments of other Cry toxins, eight truncated fragments were constructed to identify the minimal active fragment of Cry1Ai. All truncated fragments were expressed in Escherichia coli strain BL21 (DE3), and the insecticidal activity against 2nd- instar P. xylostella larvae was assessed using full-length Cry1Ai as a positive control. The results indicate that the minimal active fragment of the Cry1Ai toxin against P. xylostella is located between amino acid residues 36I and 605I, which is smaller than the regions previously reported for Cry1A. The first two amino acids (34T and 35P) on helix α-1 and whole helix α-2 of domain I and sheet β-32 of domain III are necessary for Cry1Ai toxin to keep its toxicity against P. xylostella.

Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana, was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear cry1Ah gene (800 ng mL-1) and supercoiled plasmid DNA (800 ng mL-1) under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannon’s index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic cry1Ah maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.