副溶血弧菌密度感应系统调控exsA和exsD的分子机制

Regulation of exsA and exsD by quorum sensing in Vibrio parahaemolyticus

目的研究副溶血弧菌密度感应系统核心调控子AphA和OpaR对exsA和exsD的调控机制。方法提取野生株(WT)和调控子基因突变株(ΔaphA或ΔopaR)总RNA,采用引物延伸实验确定exsA和exsD的转录起始位点,并根据WT和突变株中引物延伸产物的丰度,判定AphA和OpaR对exsA和exsD的调控关系;将exsA和exsD的启动子区DNA序列克隆入pHRP309质粒无启动子区的β-半乳糖苷酶基因的上游,构建重组质粒,并将其分别转入WT、ΔaphA和ΔopaR中得到LacZ菌株,用β-半乳糖苷酶检测试剂盒检测并比较各LacZ菌株中β-半乳糖苷酶活性的差异来判断AphA和OpaR对exsA和exsD的调控关系;PCR扩增exsA和exsD的上游调控序列,并表达纯化His-AphA和His-OpaR重组蛋白,利用凝胶阻滞实验(EMSA)探究重组蛋白是否可结合到exsA和exsD的上游调控序列上,并采用DNase I足迹实验鉴定具体的结合位点。结果引物延伸实验结果显示,exsA和exsD各有一个转录起始位点,分别为C(-440)和C(-105)(翻译起始位点为+1),且在低密度生长条件下,AphA可激活它们的转录活性,而在高密度生长条件下,OpaR抑制它们的转录活性,随后的LacZ报告基因融合实验进一步确证了这种调控关系。体外的EMSA和DNaseⅠ足迹实验结果表明,His-AphA和His-OpaR均不能与exsA和exsD启动子DNA序列结合。结论低密度生长时,AphA间接激活exsA和exsD的转录;高密度生长时,OpaR间接抑制exsA和exsD的转录。

Objective To study the transcriptional regulation of exsA and exsD by quorum sensing(QS)master regulators AphA and OpaR in Vibrio parahaemolyticus.Methods Total RNAs were extracted from the WT and AaphA(or AopaR)strains.The primer extension assay was employed to detect the transcription start sites of exsA and exsD.The entire promoter DNA region of each target gene was cloned into the upstream of the promoterless lacZ reporter gene in pHRP309.The recombinant pHRP309 plasmid was then transferred into WT,△aphA and△opaR,respectively,to measure theβ-galactosidase activities in cellular extracts.The upstream regulatory DNA regions of exsA and exsD were amplified by PCR.The over-expressed His-AphA and His-OpaR were also purified under native conditions.The electrophoretic mobility shift assay(EMSA)and the DNase I footprinting assay were applied to analyze the DNA-binding activities of His-AphA and His-OpaR to the target promoters in vitro.Results The primer extension assay detected one transcriptional start site for each target gene,which was located at 440 and 105 bp upstream of the translation start site of exsA and exsD,respectively.The results of primer extension and LacZ fusion showed that AphA activated the transcription of exsA and exsD at a low cell density,whereas OpaR inhibited their transcription at a high cell density.The results of EMSA and DNase I footprinting assays showed that neither His-AphA nor His-OpaR was able to bind to the regulatory DNA regions of exsA and exsD.Conclusion AphA indirectly activates the transcription of exsA and exsD at a low cell density,while OpaR indirectly represses their transcription at a high cell density.