Gao Rongsui, Lin Jingxia, Zhang Han, Feng Youjun
Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
Appl Environ Microbiol. 2016 Apr 18;82(9):2819-2832. doi: 10.1128/AEM.00293-16. Print 2016 May.
Recently, our group along with others reported that the Vibrio FadR regulatory protein is unusual in that, unlike the prototypical fadR product of Escherichia coli, which has only one ligand-binding site, Vibrio FadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of the vc2105 gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of the vc2105 gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates the vc2105 promoter from the FadR regulator. The expression level of the Vibrio cholerae vc2105 gene was elevated 2- to 3-fold in a fadR null mutant strain, validating that FadR is a repressor for the vc2105 gene. The β-galactosidase activity of a vc2105-lacZ transcriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike the fadD gene, a member of the Vibrio fad regulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression of ctxAB (cholera toxin A/B) and tcpA (toxin coregulated pilus A). Given that the transcriptional regulation of vc2105 fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of the Vibrio fad regulon.
The Vibrio FadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterized in vitro and in vivo. An auxiliary fad gene (vc2105) from Vibrio was proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Further in vitro and in vivo experiments revealed that the Vibrio FadR is a repressor for the vc2105 gene. Unlike fadD, a member of the Vibrio fad regulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxAB and tcpA). Therefore, since transcriptional regulation of vc2105 fits the criteria for fad genes, it seems likely that vc2105 acts as a new auxiliary member of the Vibrio fad regulon.
最近，我们团队与其他团队报告称，弧菌FadR调节蛋白不同寻常，与大肠杆菌典型的FadR产物不同，后者只有一个配体结合位点，而弧菌FadR有两个配体结合位点，代表了一种新的脂肪酸传感机制。对编码一种假定硫酯酶的vc2105基因的启动子区域进行了定位，并提出了一个假定的FadR结合位点（AA CTG GTA AGA GCA CTT）。制备了不同版本的FadR调节蛋白并纯化至同质。电泳迁移率变动分析（EMSA）和表面等离子体共振（SPR）均确定了vc2105基因与各种来源的FadR蛋白之间的直接相互作用。此外，EMSA表明添加长链酰基辅酶A（CoA）能有效使vc2105启动子与FadR调节因子解离。霍乱弧菌vc2105基因在fadR缺失突变株中的表达水平提高了2至3倍，证实FadR是vc2105基因的阻遏物。在用油酸补充生长培养基后，vc2105 - lacZ转录融合体的β - 半乳糖苷酶活性增加了2倍多。与弧菌fad操纵子的成员fadD基因不同，VC2105蛋白在细菌生长以及ctxAB（霍乱毒素A/B）和tcpA（毒素共调节菌毛A）的毒力相关基因表达中不起作用。鉴于vc2105的转录调控符合脂肪酸降解（fad）基因的标准，我们认为它是弧菌fad操纵子的一个新成员。
弧菌FadR调节因子不同寻常之处在于它有两个配体结合位点。制备了不同版本的FadR调节蛋白并在体外和体内进行了表征。提出了一个来自弧菌的辅助fad基因（vc2105），它编码一种假定硫酯酶并具有一个预测的FadR结合位点（AAC TGG TA A GAG CAC TT）。使用EMSA和SPR都证明了这个假定结合位点的功能。进一步的体外和体内实验表明弧菌FadR是vc2105基因的阻遏物。与弧菌fad操纵子的成员fadD不同，VC2105在细菌生长以及两个毒力相关基因（ctxAB和tcpA）的表达中不起作用。因此，由于vc2105的转录调控符合fad基因的标准，vc2105似乎很可能作为弧菌fad操纵子的一个新的辅助成员发挥作用。
