Abstract Abstract Bacillus thuringiensis (Bt) has become one of the most widely uses and most successful microbial insecticides in the world because of its insecticidal effect, specificity to the target organism, harmlessness to humans and animals, non-polluting environment, no harm to the natural enemies of pests, and high efficiency. However, there is a serious problem that Bt and its ICPs sprayed on crops have a short survival period under sunlight, which seriously hinders the further utilization of Bt. Therefore, in order to enhance the insecticidal effect of Bt, it is a key measure to prolong the persistence time of Bt. Bacterial biofilm (BBF) can improve the UV resistance of bacteria and regulate the adaptability of bacteria to environmental stress. Based on a previous comparative genomic analysis of our lab, a putative biofilm gene 00940 from Bt XL6-was identified (GenBank No. CP013000.1). According to bioinformatics analysis, 00940 gene is likely to be a glutamine synthase, which is regulated by SigL, CcpA, DegU and LexA. In Bacillus subtilis, SigL is an enhancer located downstream of the B. subtilis glutamate dehydrogenase gene and transcribes the rocG gene encoding glutamate dehydrogenase;CcpA transcription factor is involved in the breakdown of metabolites; DegU controls the gene expression of flagella formation and biofilm formation; LexA protein is induced in the case of DNA damage and is transcription of the bacterial SOS DNA repair system inhibitor. It is speculated that these transcription factors also play a similar role in Bt. The 1 kb upstream sequence and 1 kb downstream sequence of 00940 gene were obtained using PCR, as well as the kanamycin resistant gene. The 00940 gene knockout box containing the above three sequences was inserted into the thermosensitive vector pMAD, and the resulting recombinant plasmid was transformed into Bt XL6-. A 00940 gene knockout mutant Bt XL6-Δ00940ΩKm was then obtained. Bt XL6- was used as a control for the phenotypic analysis of the knockout mutants. Growth curve measured data showed that the lack of 00940 gene did not affect the growth of strains.The swarming motility of Bt XL6-Δ00940ΩKm was significantly higher than that of Bt XL6-. Based on laser scanning confocal microscopy, the biofilm thickness of the mutant was higher than that of Bt XL6-. Herein, lack of the 00940 gene improve the biofilm formation of Bt XL6-. The results lay a good foundation for the elucidation of the 00940 gene function.
YAO Jun-Min,ZHANG Shao-Rui,JIN Xin, et al. Sequence Analysis of A Putative Biofilm Gene 00940 from Bacillus thuringiensis XL6- and Construction of Its Gene Knockout Mutant [J]. , 2018, 26(8): 1401-1409.
