摘要
【目的】综合运用表型和分子生化实验研究AphA蛋白对副溶血弧菌生物膜形成的调节机制。【方法】利用菌落褶皱和结晶紫染色实验比较aphA突变株(ΔaphA)和野生株(WT)的表型差异;进而利用色谱串联质谱(HPLC-MS/MS)的方法分别检测ΔaphA和WT中c-di-GMP分子含量;提取ΔaphA和WT的总RNA,采用实时定量RT-PCR的方法研究AphA对scrABC和scrG的调控关系;分别构建克隆有scrABC和scrG上游启动子区的LacZ重组质粒,并将重组质粒转入ΔaphA和WT中,通过测定并比较两株菌中β-半乳糖苷酶活性差异来进一步研究AphA对scrABC和scrG的调控关系;PCR扩增scrABC和scrG的整个启动子区DNA序列,并纯化His-AphA蛋白,通过凝胶阻滞实验(EMSA)验证AphA对靶基因启动子区是否具有直接的相互作用。【结果】表型结果显示AphA能促进c-di-GMP的合成和生物膜形成;实时定量RT-PCR和LacZ结果表明AphA能抑制scrABC和scrG的转录表达;EMSA结果证明AphA不能结合到scrABC和scrG的启动子区DNA上。【结论】AphA间接抑制scrABC和scrG的表达是其促进副溶血弧菌c-di-GMP合成及生物膜形成的机制之一。
[ Objective] To study the regulation mechanism of biofilm formation c-di-GMP synthesisby AphA in Vibrio parahaemolyticus, by using phenotypic and molecular biochemical experiments. [ Methods ] Colony morphology and crystal violet staining assays were used to analyze the phenotypic changes between the aphA null mutant （AaphA） and the wide-type （WT） parent strain. The intracellular levels of c-di-GMP in the AaphA and WT strains were determined by a chromatography-coupled tandem mass spectrometry （HPLC-MS/MS） method. Total RNAs were extracted from △aphA and WT. Quantitative RT-PCR was applied to calculate the transcriptional variation of scrABC and scrG between △aphA and WT. The promoter-proximal regions of scrABC and scrG were cloned into the pHRP309 vector containing a promoterless lacZ gene, respectively. Then, each of the two recombinant LacZ reporter plasmids was transformed into AaphA and WT, respectively, to measure the promoter activity （the β-Galactosidase activity） of the target genes in AaphA and WT by using the β-Galactosidase Enzyme Assay System. The over-expressed His-AphA was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns （Amersham）. Then, the electrophoretic mobility shift assay （EMSA） was applied to analyze the DNA-binding activity of His-AphA to scrABC and scrG promoter regions in vitro. [ Results] The phenotypic experiments disclosed that AphA was an activator of c-di-GMP synthesis and biofilm formation in Vibrio parahaemolyticus. The quantitative RT-PCR and LacZ fusion results showed that the transcription of scrABC and scrG was under negative control of AphA. However, the purified His-AphA could not bind to the upstream DNA regions of scrABC and scrG, as determined by EMSA. [ Conclusion ] The fact that AphA represses the transcription of scrABC and scrG will at least partially account for the positive regulation of c-di-GMP synthesis and biofilm formation by AphA in Vibrio parahaemolyticus.
出处
《微生物学报》
CAS
CSCD
北大核心
2014年第5期525-531,共7页
Acta Microbiologica Sinica
基金
国家自然科学基金项目(31170127)~~
