Cao Dongmei, Ji Wenhui, Yan Yaxian, Wang Heng'an, Sun Jianhe, Lu Chengping
Wei Sheng Wu Xue Bao. 2014 Jul 4;54(7):737-45.
The effect of flhDC, fliA, fliD and fliE genes involved in moving of Escherichia coli (E. coli) on the motility of lysogened strain by Stx2-encoding phage phiMin27 was explored by gene knockout and phage lysogenic conversion.
Using the lambda Red recombinase system, the mutant strains of E. coli MG1655 named MG1655 deltaflhDC, MG1655 deltafliA, MG1655 deltafliD and MG1655 deltafliE 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 phiMin27, the lysogens for mutants named MG1655 deltaflhDCphiMin27, MG1655 deltafliAdeltaMin27, MG1655 deltafliDphiMin27 and MG1655 deltafliEphiMin27 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.
Lysogenic infection of Stx2-encoding phage phiMin27 could promote the expression of fliA and fliD gene and enhance the motility of MG1655. For flhDC deletion, higher expression of fliA and fliD gene of MG1655 appeared, but the motility had no change. However, lysogen for MG1655 deltaflhDC lost the swimming motility. By gene transcriptional level detection, the expression of fliA and fliD gene of MG1655 deltaflhDCphiMin27 was down-regulated significantly compared with MG1655 deltaflhDC, and no marked variation was observed for fliE gene. The single deletion of fliA, fliD and fliE gene had no effect on the motility of E. coli MG1655 and lysogened strain by Stx2-encoding phage phiMin27.
The results show that fliA 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.
通过基因敲除和噬菌体溶原性转化，探究参与大肠杆菌运动的flhDC、fliA、fliD和fliE基因对携带stx2编码噬菌体phiMin27的溶原菌运动性的影响。
利用λ Red重组酶系统构建大肠杆菌MG1655的突变株MG1655 deltaflhDC、MG1655 deltafliA、MG1655 deltafliD和MG1655 deltafliE。然后通过将扩增的目的基因在BamHI和Hind III位点连接到低拷贝载体pUC18中，并将这些质粒转化到突变株中，获得相应的互补菌株。通过对携带stx2编码噬菌体phiMin27进行溶原性感染，获得突变株的溶原菌MG1655 deltaflhDCphiMin27、MG1655 deltafliAdeltaMin27、MG1655 deltafliDphiMin27和MG1655 deltafliEphiMin27。随后，检测野生株、突变株、互补菌株和溶原菌的运动性。通过qRT-PCR分析flhDC缺失前后野生株和溶原菌中其他参与运动的基因表达的变化。
携带stx2编码噬菌体phiMin27的溶原性感染可促进fliA和fliD基因的表达并增强MG1655的运动性。对于flhDC缺失，MG1655的fliA和fliD基因表达升高，但运动性无变化。然而，MG1655 deltaflhDC的溶原菌失去了游动运动性。通过基因转录水平检测，与MG1655 deltaflhDC相比，MG1655 deltaflhDCphiMin27的fliA和fliD基因表达显著下调，fliE基因未观察到明显变化。fliA、fliD和fliE基因的单缺失对大肠杆菌MG1655和携带stx2编码噬菌体phiMin27的溶原菌的运动性无影响。
结果表明fliA和fliD基因共同参与鞭毛运动的调控，flhDC基因可影响噬菌体溶原菌的运动性。为进一步研究噬菌体溶原化与宿主基因之间的相互调控提供了理论依据。
