嗜水气单胞菌生物被膜形成突变株的筛选及其特性

Construction and character of biofilm mutants of Aeromonas hydrophica by the transposon of mTn5gusA-pgfp21

为研究调控基因对嗜水气单胞菌生物被膜的形成机制,本研究利用二亲本结合转座的方法,将携带mTn5gusA-pgfp21的载体pFAJ1819导入嗜水气单胞菌野生株AH7中进行转座突变。通过对153株突变株的生物被膜测定,筛选出形成被膜能力较高的AHΔ82和形成被膜能力较低的AHΔ148。扫描电镜观察显示AHΔ82生物被膜最致密,其次是AH7,AHΔ148较疏松。SDS-PAGE电泳表明,与AH7相比,两个突变株在25 ku和35 ku之间缺少一条带,AHΔ82比野生株多3条蛋白条带。毒力测定表明3株细菌的毒力强度为:AH7>AHΔ148>AHΔ82,突变株毒力降低。实验结果提示Tn5转座子的插入突变影响细菌生物被膜的形成和毒力。

Characterization of the functional genes and related regulation elements is the basis to understand the mechanism of bacterial biofilm formation. In the present study, the suicide plasmid pFAJ1819, carrying a transposon mTn5gusA-pgfp21, was transferred to the recipient Aeromona hydrophila AH7 strain to generate an insertional mutant library by cell conjugation between the donor Escherichia coli S17-1hpir （pFAJ1819） and the recipient A.hydrophila. A total of 153 mutants were screetied and two mutants named AH△148 and AH△82 stably exhibited differences in biofilm formation. The morphological differences of biofilms of AH△148, AH△82 and AH7 were observed by scanning electron microscopy, of which AH7 and AH△82 developed higher density biofilm than AH△148. In addition, SDS-PAGE analysis showed that the protein patterns had a slightly difference between the mutant and parental bacteria. However, the pathogenicity tests displayed a trend of attenuated virulence for the mutated A.hydrophila, which the medial lethal dose were 2.828×10^3 cfu for AH7, 1.027×10^4 cfu for AH△148 and 1.027×10^5 cfu for AH△82 tested in zebra fish. These results demonstrated that the insertion of Tn5-transposon affected the formation of bacterial biofilm and virulence.