肺炎克雷伯菌KP1＿4563基因突变株的构建及其生物膜表型分析

Construction and Biofilm Phenotype Analysis of KP1＿4563 Gene Mutant of Klebsiella Pneumoniae

KP1_4563基因是肺炎克雷伯菌NTUH-K2044中假设的蛋白编码基因,与Ⅲ型菌毛的功能有关。本实验首先采用同源重组基因敲除方法构建肺炎克雷伯菌KP1_4563基因缺失的突变株(Kp-△4563),然后PCR扩增KP1_4563基因片段,克隆到质粒p BAD33上,将重组质粒导入Kp-△4563获得回补株(Kpc-△4563)。分别测定野生株、突变株、回补株用普通LB培养基,改良Minka培养基以及含胆汁盐的LB培养基培养时生物膜形成能力,以此来探讨KP1_4563基因以及不同培养基对肺炎克雷伯菌体外生物膜形成的影响。我们成功构建KP1_4563基因缺失的突变株和回补株Kpc-△4563。与野生株相比,突变株Kp-△4563生物膜形成能力减弱,回补株介于野生株和突变株之间。使用改良Minka培养基使菌株菌毛化以及加入胆汁盐可以增加生物膜的形成能力。这些分析表明肺炎克雷伯菌KP1_4563基因能正调控细菌生物膜的形成。体外培养使细菌菌毛化以及加入胆汁盐可以促进生物膜的形成。

KP1_4563 of K.pneumoniae NTUH-K2044 is a hypothetical protein-encoding gene,and is related to the function of type Ⅲ fimbria.Firstly,the KP1_4563 gene deletion mutant of Klebsiella pneumoniae(Kp-△4563)was constructed by homologous recombination gene knock-out method.Then,the KP1_4563 gene fragment was amplified and cloned into plasmid pBAD33,and then the recombinant plasmid was transferred into Kp-△4563 to construct the complemented strain(Kpc-△4563).The biofilm formation ability of wild strain,mutant strain,complemented strain which were cultured with LB medium or Minka medium or LB medium with 1.0% bile salt were tested respectively,to determine the effects of KP1_4563 gene and different culture mediums on biofilm formation ability of Klebsiella pneumoniae.In this study,we constructed the KP1_4563-deletion mutant and complemented strain Kpc-△4563 successfully.Compared with wild strain,the biofilm formation ability of mutant KP1_4563 decreased while that of complemented strain Kpc-△4563 was between the wild starin and the mutant strain.The biofilm formation ability of strains could be increased by using modified Minka medium to fimbriate the strains and by adding bile salt.These results indicated that the KP1_4563 gene of Klebsiella pneumoniae could positively regulates biofilm formation and the biofilm formation could be promoted by culture in vitro to fimbriate the strains and by the addition of bile salts.