Niu Qiuhong, Tian Yunxia, Zhang Lin, Xu Xiao'e, Niu Xuemei, Xia Zhenyuan, Lei Liping, Zhang Ke-Qin, Huang Xiaowei
Laboratory for Conservation and Utilization of Bio-Resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, China.
J Mol Microbiol Biotechnol. 2011;21(3-4):130-7. doi: 10.1159/000332805. Epub 2012 Jan 31.
Proteases Bace16 and Bae16, an alkaline serine protease and a neutral protease, respectively, in the nematocidal bacterium Bacillus nematocida B16, have been identified as two key virulence factors and shown to have remarkable nematotoxic activities against the free-living nematode Panagrellus redivius and the plant parasite nematode Bursaphelenchus xylophilus. To facilitate the successful biological control application of this organism in the field, we genetically altered the strain B. nematocida B16 and optimized its growth condition to overexpress these two pathogenic proteases. The recombinant integration vectors of pAX01-Bace16 and pAX01-Bae16 for overexpressing the two proteases were constructed and successfully transformed into competent cells of the bacterium B. nematocida B16. The optimal induction condition for overexpressing Bace16 is 2% xylose at 37°C for 48 h. Our analyses showed that the proteolytic activity and nematocidal activity of the strain overexpressing Bace16 increased by about 62 and 80%, respectively, over the wild-type strain. However, our tested induction conditions could not significantly improve either the proteolytic activity or the nematocidal activity of the Bae16 overexpression mutant.
杀线虫芽孢杆菌B16中的蛋白酶Bace16和Bae16分别是一种碱性丝氨酸蛋白酶和一种中性蛋白酶，已被确定为两个关键毒力因子，并显示出对自由生活线虫雷氏帕纳线虫和植物寄生线虫松材线虫具有显著的杀线虫活性。为了促进该生物体在田间成功进行生物防治应用，我们对杀线虫芽孢杆菌B16菌株进行了基因改造，并优化了其生长条件以过量表达这两种致病蛋白酶。构建了用于过量表达这两种蛋白酶的重组整合载体pAX01-Bace16和pAX01-Bae16，并成功将其转化到杀线虫芽孢杆菌B16的感受态细胞中。过量表达Bace16的最佳诱导条件是在37°C下用2%木糖诱导48小时。我们的分析表明，过量表达Bace16的菌株的蛋白水解活性和杀线虫活性分别比野生型菌株提高了约62%和80%。然而，我们测试的诱导条件不能显著提高Bae16过表达突变体的蛋白水解活性或杀线虫活性。
