肺炎克雷伯菌突变株ΔrcsB的构建及其菌株超粘性与生物膜表型

Construction of Klebsiella pneumoniae mutant ΔrcsB and its hypermucoviscosity and biofilm formation ability

目的构建肺炎克雷伯菌rcs B基因缺失的无痕突变株和回补株,通过对突变株的超粘性和生物膜表型进行分析,探讨转录调控子Rcs B对细菌超粘性和生物膜形成能力的影响。方法首先构建突变株Δrcs B,PCR扩增rcs B基因上下游同源臂片段,克隆到质粒p KO3-Km上,将重组质粒导入肺炎克雷伯菌NTUH-K2044中,经同源重组后筛选得到无痕突变株。然后构建回补株,扩增rcs B基因片段,克隆到质粒p BAD33上,将重组质粒导入突变株Δrcs B中,得到回补株。测定野生株、突变株、回补株的超粘性和生物膜形成能力。结果成功构建rcs B基因缺失的无痕突变株Δrcs B和回补株C-Δrcs B。与野生株相比,突变株超粘性和生物膜形成能力均减弱,回补株介于野生株和突变株之间。结论转录调控子Rcs B对肺炎克雷伯菌超粘性和生物膜形成具有正向调控作用。

Objective To determine the effect of rcsB gene on hypermucoviscosity and biofilm formation ability of Klebsiella pneumoniae by constructing unmarked rcsB-deletion mutant and rcsB complementation strain. Methods First, unmarked rcsB-deletion mutant was constructed. The upstream and downstream flanking DNA fragments of rcsB gene were amplified by PCR and then cloned into plasmid pKO3-Km. The recombinant plasmid was transferred into Klebsiella pneumoniae NTUH-K2044. After homologous recombination, unmarked rcsB-deletion mutant was screened. Second, the complementation strain was constructed. The rcsB fragment was amplified and cloned into plasmid pBAD33, and then the recombinant plasmid was transferred into rcsB-deletion mutant to get the complementation strain. The hypermucoviscosity and biofilm formation ability were tested. Results The unmarked rcsB-deletion mutant and complementation strain were constructed successfully. The results showed that the hypermucoviscosity and biofilm formation ability of the mutant were decreased, while the hypermucoviscosity and biofilm formation of the complementation strain was between the wild strain and the mutant. Conclusion The transcription factor RcsB positively regulates the hypermucoviscosity and biofilm formation ability of Klebsiella pneumonia.