肺炎克雷伯菌KP1_0324基因突变株构建及其对生物膜形成的影响

Construction of KP1_0324 mutant strain of Klebsiella pneumoniae and its effect on biofilm formation

目的探究肺炎克雷伯菌NTUH-K2044中KP1_0324基因对菌株生物膜形成的影响。方法构建肺炎克雷伯菌KP1_0324基因缺失的突变株。先分别扩增KP1_0324基因的上下游同源臂,通过融合PCR得到缺失此目的基因的融合片段,将该片段克隆到自杀质粒pKO3-Km上得到重组质粒,再将重组质粒转入肺炎克雷伯菌野生株中,利用同源重组的原理,筛选得到目的基因缺失的突变株Kp-Δ0324。构建回补株,利用PCR扩增得到KP1_0324基因片段,并克隆到表达载体pBAD33上,再将得到的重组质粒转入突变株Kp-Δ0324中,得到回补株Kpc-Δ0324。利用生物膜结晶紫染色实验和扫描电镜观察野生株、突变株和回补株的生物膜相对形成量及生物膜的表面结构。结果成功构建突变株Kp-Δ0324和回补株Kpc-Δ0324;与野生株相比,突变株Kp-Δ0324的生物膜形成量明显降低(P<0.01)。结论肺炎克雷伯菌NTUH-K2044中KP1_0324基因与细菌生物膜的形成有关。

Objective To explore the effect of KP1_0324 gene on the biofilm formation in Klebsiella pneumoniae NTUH-K2044.Methods Firstly,the mutant strains of Klebsiella pneumoniae with deletions of KP1_0324 gene were constructed.The upstream and downstream flanking DNA fragments of KP1_0324 gene were amplified by PCR,and then cloned into plasmid pKO3-Km.The recombinant plasmid was transformed into wild-type Klebsiella pneumoniae strain.The mutant strains with deletions of KP1_0324 gene were screened by PCR after homologous recombination.Secondly,the complemented strains were constructed.The KP1_0324 gene fragment was amplified and cloned into the vector pBAD33,then the recombinant plasmid was transformed into mutant strains with deletions of KP1_0324 gene to get the complemented strains.The biofilm formation of wild-type strain,mutant and complemented strain were tested by using crystal violet staining and scanning electron microscopy.Results The mutant Kp-Δ0324 and the complemented Kpc-Δ0324 strains were successfully constructed.The The quantity of biofilm formed by the mutant Kp-Δ0324 strain was significantly lower than that formed by the wild-type strain.Conclusion The KP1_0324 gene might be related to the formation of bacterial biofilm in Klebsiella pneumoniae NTUH-K2044.