CalR激活副溶血弧菌Ⅵ型分泌系统1相关基因的转录

CalR activates the transcription of type Ⅵ secretion system 1 genes in Vibrio parahaemolyticus

摘要:

【目的】研究副溶血弧菌群体感应（quorum sensing，QS）系统核心调控子AphA和OpaR对calR基因以及CalR对VI型分泌系统1（type VI secretion system 1，T6SS1；vp1386-1420）相关基因的转录调控关系。【方法】提取副溶血弧菌野生株（wild-type，WT）和调控子基因突变株的总RNA，采用实时定量PCR （quantitative real-time PCR，qPCR）研究调控子对靶基因的转录调控关系；采用引物延伸实验研究靶基因的转录起始位点，并根据产物的丰度判断调控子对靶基因的调控关系；将靶基因的启动子区克隆入pHRP309质粒的β-半乳糖苷酶基因上游，构建LacZ重组质粒，并将该重组质粒转入WT和调控子基因突变株中，获得LacZ菌株，通过LacZ报告基因融合实验进一步研究调控子对靶基因的调控关系。PCR扩增靶基因的上游调控区DNA序列，并纯化His-重组调控子蛋白，通过凝胶阻滞实验（electrophoretic mobility shift assay，EMSA）研究His-重组蛋白对靶基因调控区DNA序列是否具有直接的结合作用，若有结合作用，则进一步采用DNase I足迹实验研究具体的结合位点。【结果】在细菌生长密度（OD₆₀₀）从0.05依次增加到1.20时，CalR的表达水平呈梯度升高特征；QS系统在低细胞密度下的核心调控子AphA对calR基因的转录没有调控作用，而高细胞密度下的核心调控子OpaR对calR基因的转录具有间接的激活作用。此外，在非诱导条件下，CalR直接结合到vp1388-1390、vp1393-1406、vp1400-1406和vp1409-1407启动子区DNA序列上促进它们的转录。【结论】OpaR间接激活CalR的表达，而CalR是非诱导条件下维持T6SS1基础表达所必需的调控子。

Abstract:

[Objective] To study the transcriptional regulation of calR gene by the master quorum sensing (QS) regulators AphA and OpaR in Vibrio parahaemolyticus, and to investigate the regulatory actions of CalR on the transcription of type VI secretion system 1 (T6SS1) genes. [Methods] Total RNAs were extracted from the wild-type (WT) strain and the regulatory gene mutants. Quantitative real-time PCR was carried out to calculate the transcriptional variation of each target gene between WT and the mutants. Primer extension assay was employed to detect the transcription start site and the expression variation of each target gene between WT and the mutants. The promoter region of each target gene was cloned into the restriction endonuclease sites of pHRP309, which harbors a gentamicin resistance gene and a promoterless lacZ reporter gene. Thereafter, each recombinant pHRP309 plasmid was transferred into WT and the mutants. The transformants were cultured and then lysed, and a β-Galactosidase Enzyme Assay System (Promega) was used for quantifying the β-galactosidase activity in cell lysates. The over-expressed His-recombinant regulatory proteins were purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay (EMSA) and DNase I footprinting were adopted to analyze the binding activity of each His-recombinant protein to its target promoter DNA region in vitro. [Results] The expression level of calR gene was up-regulated gradiently with the increase in OD₆₀₀ value from 0.05 to 1.20. AphA operating at a low cell density had no regulatory activity on calR gene transcription, whereas OpaR operating at a high cell density indirectly activated the transcription of calR gene. Moreover, CalR bound to the promoter regions of the T6SS1 operons vp1388-1390, vp1393-1406, vp1400-1406, and vp1409-1407 to activate their transcription under the non-inducing growth condition. [Conclusion] The transcription of calR gene was indirectly activated by OpaR. CalR was required for the basal transcription of T6SS1 genes under the non-inducing growth condition.