鼠伤寒沙门菌rpoN基因缺失株构建及生物学特性研究

Construction and Biological Characterization of rpoN Gene Deletion Mutants of Salmonella Typhimurium

为了探索σ因子RpoN在鼠伤寒沙门菌生物被膜形成过程中的作用,对两株生物被膜形成能力较强的鼠伤寒沙门菌利用Red同源重组系统构建rpoN基因缺失株,利用原核表达载体构建回复株;比较野生株、缺失株和回复株的生物被膜形成能力和对环境应激抵抗力的差异,并通过建立的csgA和bcsA基因实时定量PCR方法检测编码生物被膜成分基因的表达差异。结果显示,与野生株相比,△rpoN缺失株生物被膜形成能力增强,主要由卷曲菌毛表达量显著增加引起。回复株生物被膜形成能力与野生株相似。△rpoN缺失株在酸性应激和碱性应激条件下对外界环境应激的抵抗力均显著增强。RpoN为鼠伤寒沙门菌中生物被膜形成相关σ因子之一,为进一步研究沙门菌生物被膜形成的调控机制提供理论基础。

The aim of this study was to understand the role of rpoN gene on biofilm formation of S.Typhimurium.Two S.Typhimuriumstrains with strong biofilm-forming ability were selected for construction of deletion mutants with deficiency of gene rpoN using the Red recombination system,and complemented strains were constructed by using prokaryotic expression vector.The biofilm-forming ability,and resistance to environmental stress of the wild-type,rpoN mutant and rpoNcomplemented strains were compared.A quantitative real-time PCR method based on csgA and bcsAgenes was established to compare the expression of their biofilm components.The results showed that the biofilm formation,mainly contributed by increased expression of curli,was significantly enhanced in the rpoN gene deletion mutants when compared with that of the wildtype strains.The biofilm formation of revertants was similar to that of the wild-type strains.Furthermore,deletion of rpoN gene of S.Typhimuriumstrains resulted in their increased resistance to acid and alkali environment.Therefore,RpoN was identified as one of the associated sigma factors involved in biofilm formation of S.Typhimurium.These data may be helpful for elucidatingthe regulatory mechanism of Salmonella biofilm formation.