QsvR对副溶血弧菌VI型分泌系统1相关基因的转录调控

Transcriptional regulation of type VI secretion system 1 genes by QsvR in Vibrio parahaemolyticus

摘要:

【目的】研究调控蛋白QsvR对副溶血弧菌VI型分泌系统1 （type VI secretion system 1，T6SS1）相关基因的转录调控关系。【方法】提取野生株（wild type，WT）和qsvR突变株（ΔqsvR）的总RNA，采用实时定量PCR （quantitative real-time PCR，qPCR）研究QsvR对靶基因的调控关系；进而采用引物延伸法定位靶基因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QsvR对靶基因的调控关系；将靶基因的调控区DNA序列克隆入pHRP309质粒中的β-半乳糖苷酶基因上游（LacZ重组质粒），并将重组质粒转化入WT和ΔqsvR中，通过LacZ报告基因融合试验研究QsvR对靶基因的调控关系；将LacZ重组质粒分别转化入含有pBAD33或pBAD33-qsvR的大肠杆菌100lpir中，进一步采用LacZ报告基因融合试验研究在异体宿主中QsvR对靶基因的调控关系；PCR扩增靶基因调控区DNA序列，同时表达并纯化His-QsvR重组蛋白，采用凝胶阻滞试验（electrophoresis mobility shift assay，EMSA）研究His-QsvR对靶基因调控区DNA序列是否具有直接的结合作用。【结果】qPCR结果显示，与WT相比，ΔqsvR中T6SS1相关基因VP1388 （操纵子VP1388-1390首基因）和hcp1 （操纵子VP1393-1406首基因）的转录水平显著性升高，表明QsvR抑制VP1388和hcp1的转录；引物延伸结果显示VP1388和hcp1各有一个转录起始位点，分别为C （-64）和T （-62），且它们的转录活性受QsvR的抑制；LacZ报告基因融合试验结果显示QsvR可以抑制副溶血弧菌和EC100lpir中VP1388和hcp1的启动子区转录活性；EMSA结果显示His-QsvR对VP1388和hcp1的启动子区DNA序列具有直接的结合活性。【结论】QsvR对T6SS1相关操纵子VP1388-1390和VP1393-1406的转录具有直接的抑制作用。

Abstract:

[Objective] To study the transcriptional regulation of type VI secretion system 1 (T6SS1) genes by QsvR in Vibrio parahaemolyticus. [Methods] Total RNA was extracted from the wild type (WT) and qsvR mutant (ΔqsvR). Quantitative real-time PCR (qPCR) was employed to investigate the transcriptional regulation of target genes by QsvR. Primer extension was carried out to detect the transcription initiation site and core promoter for each target gene and calculate the transcriptional variations between WT and ΔqsvR. The regulatory DNA region of each target gene was cloned into the restriction endonuclease sites of pHRP309 harboring a promoterless gene lacZ, and then each recombinant plasmid was transferred into WT and ΔqsvR, respectively. A β-Galactosidase Enzyme Assay System (Promega) was used to measure the β-galactosidase activity in cell lysates. The recombinant pHRP309 vector containing the regulatory DNA region of one of the target gene was transferred into Escherichia coli 100λpir harboring an empty pBAD33 or pBAD33-qsvR to test whether QsvR can regulate the target genes in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and His-QsvR was over-expressed and then purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay (EMSA) was employed to determine the DNA-binding activity of His-QsvR to each target DNA fragment in vitro. [Results] The mRNA levels of T6SS1-associated genes, VP1388 (the first gene of VP1388-1390 operon) and hcp1 (the first gene of VP1393-1406 operon), were significantly up-regulated in ΔqsvR relative to those in WT, indicating that QsvR activated the transcription of VP1388 and hcp1. Only one transcription initiation site was detected for VP1388 or hcp1, locating at 64 bp upstream of VP1388 and 62 bp upstream of hcp1, respectively, and their transcriptional activities were all repressed by QsvR. QsvR repressed the promoter activities of VP1388 and hcp1 in both V. parahaemolyticus and E. coli 100λpir. His-QsvR was able to bind to the regulatory DNA regions of VP1388 and hcp1. [Conclusion] QsvR directly repressed the transcription of T6SS1-associated operons, VP1388-1390 and VP1393-1406, in V. parahaemolyticus.