群体感应相关调控子对副溶血弧菌磷酸二酯酶GepA的表达调控

Quorum sensing regulators control the expression of phosphodiesterase GepA in Vibrio parahaemolyticus

摘要:

目的 研究群体感应系统(quorum sensing, QS)相关调控子AphA、ToxR和QsvR对副溶血弧菌磷酸二酯酶GepA编码基因gepA表达的调控关系。方法 提取野生株(wild type, WT)和aphA、toxR、qsvR突变株的总RNA，通过实时定量PCR (quantitative real-time PCR, qPCR)初步分析QS相关调控子对gepA的转录影响；将gepA上游调控区DNA序列克隆入pBBRlux质粒无启动子区的表达生物冷光基因luxCDABE上游，并将lux重组质粒分别导入WT和突变株中，采用lux报告基因融合实验进一步研究QS相关调控子对gepA的调控作用；采用引物延伸(primer extension)法定位gepA的转录起始位点和核心启动子区，并根据引物延伸产物丰度判断QS相关调控子对gepA的调控关系；将gepA的调控区DNA序列克隆入pHRP309质粒中β-半乳糖苷酶基因上游获得LacZ重组质粒，将LacZ重组质粒分别转化入含有pBAD33或pBAD33-qsvR的大肠杆菌EC100 λpir中，采用LacZ报告基因融合实验研究在异体宿主中QS相关调控子对gepA转录的调控方式；PCR扩增gepA上游调控区DNA序列，同时表达并纯化QS相关调控子的His重组蛋白，采用凝胶阻滞实验(electrophoresis mobility shift assay, EMSA)研究QS相关调控子是否直接调控gepA的表达。结果 低密度条件下，qPCR结果显示ΔaphA和ΔtoxR中gepA的转录水平明显低于WT，表明AphA和ToxR激活gepA的转录；lux报告基因融合实验显示，ΔaphA和ΔtoxR中gepA的启动子区转录活性明显低于WT，进一步表明AphA和ToxR促进gepA的转录；引物延伸结果显示，gepA的转录起始位点位于起始密码子ATG上游第30 bp的A，且其转录活性受到AphA的激活；EMSA结果显示，His-AphA和His-ToxR均不能与gepA的调控区DNA序列结合。高密度条件下，qPCR和引物延伸实验结果均显示QsvR抑制gepA的转录；EMSA实验结果显示，His-QsvR直接结合在gepA的启动子区DNA序列上；双质粒报告基因融合实验显示，QsvR可以抑制EC100 λpir中gepA的启动子区转录活性。结论 AphA和ToxR间接激活，而QsvR直接抑制gepA的转录。因此，gepA在低密度条件下转录水平较高，高密度时转录水平明显下降。

Abstract:

Objective To investigate the transcriptional regulation of quorum sensing (QS) regulators AphA, ToxR, and QsvR on the expression of the phosphodiesterase (GepA) gene gepA in Vibrio parahaemolyticus.Methods Total RNAs were extracted from the wild type (WT) and the mutant strains of aphA, toxR, and qsvR. Quantitative real-time PCR (qPCR) was carried out to calculate the transcriptional variation of gepA between WT and mutant strains. The regulatory DNA region of gepA was cloned into the upstream region of promoterless luxCDABE reporter gene in the pBBRlux plasmid. The recombinant plasmid was respectively transferred into the WT and mutant strains. Luminescence assay was used to test the regulatory effect of QS regulators on the expression of gepA. The primer extension assay was employed to detect the transcription start site and the promoter activity of gepA. The effects of QS regulators on gepA were evaluated based on the abundance of primer extension products. The regulatory DNA region of gepA was cloned into the upstream region of lacZ in the pHRP309 plasmid. The LacZ recombinant plasmid was transformed into EC100 λpir harboring pBAD33 or PBAD33-qsvR. Two-plasmid LacZ reporter assay was conducted to investigate the regulatory effects of QS regulators on the transcription of gepA in EC100 λpir. The regulatory DNA region of gepA was amplified by PCR, and the His recombinant proteins of QS regulators were purified. The electrophoretic mobility shift assay (EMSA) was performed to investigate whether QS regulators directly regulated the expression of gepA.Results At low cell density, the qPCR results showed that expression of gepA in ΔaphA and ΔtoxR were significantly lower than that in WT, indicating that AphA and ToxR activated the transcription of gepA. The luminescence assay showed that the transcriptional activity of the promoter region of gepA in ΔaphA and ΔtoxR was significantly lower than that in WT, further indicating that AphA and ToxR promoted the transcription of gepA. The primer extension assay detected that the transcription start site of gepA was located at the A nucleotide 30 bp upstream of the start codon ATG, and its transcriptional activity was activated by AphA. The EMSA result indicated that His-AphA and His-ToxR were unable to bind the promoter DNA region of gepA. At high cell density, both the qPCR and primer extension assay indicated that QsvR inhibited the transcription of gepA. The EMSA result demonstrated that His-QsvR directly bound to the promoter DNA region of gepA. Two-plasmid lacZ reporter assay demonstrated that QsvR inhibited the transcriptional activity of the promoter region of gepA in EC100 λpir.Conclusion AphA and ToxR indirectly activate while QsvR directly inhibits the transcription of gepA. Therefore, the transcription level of gepA is higher at low cell density and significantly decreases at high cell density.