hns基因对大肠埃希菌生物膜形成能力的影响

Effect of hns gene on biofilm formation of Escherichia coli

目的建立大肠埃希菌Red重组系统无痕敲除方法,探讨hns基因对大肠埃希菌生物膜形成能力的影响。方法以大肠埃希菌MG1655为研究对象,将p KD46质粒转化入MG1655感受态细胞。以质粒p KD3为模板扩增氯霉素抗性基因片段,并将其转化入MG1655/p KD46,氯霉素抗性平板筛选阳性克隆。利用p CP20质粒删除氯霉素抗性基因,构建MG1655 hns基因缺失菌株。96孔板结晶紫染色法分析MG1655 hns基因缺失株生物膜形成能力的变化。苯酚-硫酸法检测细菌胞外多糖含量。结果氯霉素抗性基因消除后的hns基因缺失株PCR产物长度为434 bp,测序鉴定正确,成功构建MG1655△hns基因缺失株。MG1655△hns菌株生物膜形成能力和胞外多糖产生显著低于MG1655(P<0.01)。结论利用Red重组技术成功构建出MG1655△hns菌株,hns基因对细菌生物膜的形成具有重要的调控作用。

Objective To establish the scarless knockout method in Escherichia coli（ E. coli） based on the Red recombination system,and study the effect of hns gene on biofilm formation of E. coli. Methods E. coli MG1655 was used as the target strain and transformed with plasmid p KD46 by electroporation. The chloramphenicol resistance gene was amplified using template plasmid p KD3,and then introduced into E. coli MG1655 / p KD46. The strains expressing chloramphenicol resistance gene were selected with chloramphenicol agar plate. The chloramphenicol resistance gene was deleted using plasmid p CP20,and then the MG1655 hns gene knockout（ △hns） strain was constructed. The effect of hns gene on biofilm formation of E. coli △hns strain was analyzed with 96-well crystal violet staining. The content of extracellular polymeric substance（ EPS） was determined by phenol-sulfuric acid testing. Results The 434 bp product was amplified from MG1655 △hns strain after the chloramphenicol resistance gene was deleted, and it was identified by sequencing,indicating that the MG1655 △hns strain was successfully constructed. The biofilm formation and EPS production of MG1655 △hns strain were significantly lower than that of MG1655 strain（ P〈0. 01）.Conclusion MG1655 △hns strain is successfully constructed by Red recombination technology. The hns gene plays an important regulatory role in the biofilm formation of E. coli.