Niu Qiuhong, Huang Xiaowei, Zhang Lin, Lian Lihui, Li Yunxia, Li Juan, Yang Jinkui, Zhang Keqin
Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, PR China.
Appl Microbiol Biotechnol. 2007 May;75(1):141-8. doi: 10.1007/s00253-006-0794-7. Epub 2007 Feb 14.
Bacillus nematocida is a Gram-positive bacterium capable of killing nematodes. Our recent studies identified an extracellular serine protease Bace16 in B. nematocida as a candidate of pathogenic factor in the infection against nematodes, which displayed a high similarity with the serine protease family subtilisin BPN', and the MEROPS ID is S08.034. To further confirm the roles that bace16 played in the mechanism of nematocidal pathogenesis, recombinant mature Bace16 (rm-Bace16) was expressed in Escherichia coli strain BL21 using pET-30 vector system. Bioassay experiments demonstrated that the purified recombinant protease had the ability to degrade nematode cuticles and kill nematodes. In addition, a bace16 knockout mutant of B. nematocida constructed by homologous recombination showed considerably lower proteolytic activity and less than 50% nematocidal activity than the wild-type strain. These results confirmed that Bace16 could serve as an important virulence factor during the infectious process.
杀线虫芽孢杆菌是一种能够杀死线虫的革兰氏阳性细菌。我们最近的研究确定，杀线虫芽孢杆菌中的一种细胞外丝氨酸蛋白酶Bace16是感染线虫过程中致病因子的候选物，它与丝氨酸蛋白酶家族枯草杆菌蛋白酶BPN'具有高度相似性，其蛋白酶分类编号为S08.034。为了进一步证实bace16在杀线虫致病机制中所起的作用，使用pET-30载体系统在大肠杆菌BL21菌株中表达了重组成熟Bace16（rm-Bace16）。生物测定实验表明，纯化的重组蛋白酶具有降解线虫表皮和杀死线虫的能力。此外，通过同源重组构建的杀线虫芽孢杆菌bace16基因敲除突变体的蛋白水解活性明显低于野生型菌株，杀线虫活性也低于野生型菌株50%以上。这些结果证实，Bace16在感染过程中可作为一种重要的毒力因子。
