Stx2噬菌体溶原对大肠杆菌运动性及其相关基因表达的影响

Effect of Stx2-encoding phage on the motility and gene expression involved in moving of Escherichia coli lysogen

【目的】通过基因缺失和噬菌体溶原转换研究大肠杆菌运动相关基因flhDC、fliA、fliD和fliE对Stx2噬菌体ΦMin27溶原菌的影响。【方法】本实验利用Red重组酶系统,构建了大肠杆菌MG1655的缺失株MG1655△flhDC、MG1655△fliA、MG1655△fliD及MG1655△fliE,并将flhDC片段、fliA片段、fliD片段和fliE片段连接pUC18后分别转化相应的突变株,得到相应的互补菌株。通过Stx2噬菌体ΦMin27的感染获得各缺失株的溶原株MG1655△flhDCФMin27、MG1655△fliAФMin27、MG1655△fliDФMin27及MG1655△fliEФMin27。随后测定了野生株、缺失株、互补株和溶原株的运动能力,并通过荧光定量PCR分析了flhDC缺失前后野生株和溶原株其他运动相关基因表达量的变化。【结果】Stx2噬菌体ФMin27溶原感染可促进MG1655的fliA和fliD基因的表达,增强宿主菌MG1655的运动特性;MG1655在flhDC基因缺失的状态下,fliA和fliD基因的表达同步出现上调,但运动性未发生变化,而MG1655△flhDC溶原菌丧失了运动特性,基因转录水平检测发现MG1655△flhDCФMin27与MG1655△flhDC相比,fliA和fliD基因的表达同步出现显著下调,而对fliE基因的表达几乎没有影响。fliA、fliD和fliE单个基因的缺失对大肠杆菌MG1655和Stx2噬菌体ΦMin27溶原菌的运动性几乎没有影响。【结论】提示fliA和fliD基因共同参与了鞭毛运动的调控,flhDC基因可影响噬菌体溶原菌株的运动性,为进一步研究噬菌体溶原与宿主基因之间的相互调节作用提供理论依据。

[ Objective ] The effect of flhDC, iliA, fliD and filE genes involved in moving of Escherichia coli （E. coli） on the motility of lysogened strain by Stx2-encoding phage qbMin27 was explored by gene knockout and phage lysogenic conversion. [ Methods] Using the lambda Red recombinase system, the mutant strains of E. coli MG1655 named MG1655 △flhDC, MG1655 △fliA, MG1655 △fliD and MG1655 △fliE were constructed. Then the corresponding complemented strains by ligating amplified targeted genes into the low copy vector pUC18 at the BamHI and Hind Ⅲ sites and transforming these plasmids into mutant strains were acquired. By lysogenic infection of Stx2-encoding phage ФMin27, the lysogens for mutants named MG1655 △flhDCФMin27, MG1655 △fliAФMin27, MG1655 △fliDФMin27 and MG1655 △fliEФMin27 were achieved. Subsequently, the motility of wild strain, the mutants, the complemented strains and the lysogens were detected. The changes of expression of the other genes involved in motility between wild strain and the lysogens before and after flhDC deletion by qRT-PCR were analyzed. [ Results] Lysogenic infection of Stx2-encoding phage ФMin27 could promote the expression of iliA and fliD gene and enhance the motility of MG1655. For flhDC deletion, higher expression of iliA andfliD gene of MG1655 appeared, but the motility had no change. However, lysogen for MG1655 △flhDC lost the swimming motility. By gene transcriptional level detection, the expression of iliA and fliD gene of MG1655 △flhDCФMin27 was down-regulated significantly compared with MG1655 △flhDC, and no marked variation was observed for fliE gene. The single deletion of iliA, fliD andfliE gene had no effect on the motility of E. coli MG1655 and lysogened strain by Stx2-encoding phage ФMin27. [ Conclusion] The results show that iliA and fliD gene together participated the regulation for flagella motility and flhDC gene could affect the motility of the lysogened strain by phage. It provides the theoretical basis for further research on the mutual regulation between phage lysogenization and host genes.