Laboratory of Hygienic Chemistry, College of Pharmaceutical Sciences, Matsuyama University.
Department of Microbiology and Infection Control, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University.
Biol Pharm Bull. 2021;44(11):1790-1795. doi: 10.1248/bpb.b21-00372.
Vibrio vulnificus can utilize the xenosiderophore desferrioxamine B (DFOB) as an iron source under iron-restricted conditions. We previously identified in V. vulnificus that transcription of the desA gene encoding the outer membrane receptor for ferrioxamine B (FOXB) is activated by the AraC-type transcriptional regulator encoded by desR together with DFOB. In this study, we overexpressed and purified DesR as a glutathione S-transferase-fused protein and examined interaction between the promoter region of desA and DesR. Electrophoretic mobility shift assay (EMSA) revealed that DesR directly binds to the regulatory region of desA, and this binding was enhanced by the presence of DFOB in a concentration-dependent manner, while the presence of FOXB did not affect the potentiation of their binding. Moreover, EMSA identified that DNA fragments lacking a probable DesR binding sequence were unable to form complexes with DesR. Finally, deoxyribonuclease I footprinting assay demonstrated that the DNA binding sequence of DesR is located between -27 and -50 nucleotides upstream of the desA transcription start site. These results strongly indicate that DesR can directly activate the transcription of desA in cooperation with DFOB, which acts as a coactivator for DesR.
创伤弧菌可以在缺铁条件下利用外源性铁载体去铁胺 B（DFOB）作为铁源。我们之前在创伤弧菌中发现，编码 FOXB（DFOB 的外膜受体）的 desA 基因的转录受 desR 编码的 AraC 型转录调节剂与 DFOB 共同激活。在这项研究中，我们过表达并纯化了作为谷胱甘肽 S-转移酶融合蛋白的 DesR，并研究了 desA 的启动子区域与 DesR 之间的相互作用。电泳迁移率变动分析（EMSA）显示 DesR 直接与 desA 的调节区结合，这种结合可被 DFOB 以浓度依赖性方式增强，而 FOXB 的存在并不影响它们结合的增强。此外，EMSA 表明缺乏可能的 DesR 结合序列的 DNA 片段无法与 DesR 形成复合物。最后，脱氧核糖核酸酶 I 足迹分析表明 DesR 的 DNA 结合序列位于 desA 转录起始位点上游的-27 到-50 个核苷酸处。这些结果强烈表明 DesR 可以直接与 DFOB 协同激活 desA 的转录，DFOB 作为 DesR 的共激活剂发挥作用。
