AphA蛋白促进副溶血弧菌c-di-GMP合成和生物膜形成

AphA is an activator of c-di-GMP synthesis and biofilm formation in Vibrio parahaemolyticus

摘要:

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。

Abstract:

Abstract: ［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 (ΔaphA) and the wide-type (WT) parent strain． The intracellular levels of c-di-GMP in the ΔaphA and WT strains were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS /MS) method． Total ＲNAs were extracted from ΔaphA and WT．Quantitative ＲT-PCＲ was applied to calculate the transcriptional variation of scrABC and scrG between ΔaphA 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 ΔaphA and WT，respectively，to measure the promoter activity (the β-Galactosidase activity) of the target genes in ΔaphA 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．