Töwe Stefanie, Leelakriangsak Montira, Kobayashi Kazuo, Van Duy Nguyen, Hecker Michael, Zuber Peter, Antelmann Haike
Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, F.-L.-Jahn-Str. 15, D-17487 Greifswald, Germany.
Mol Microbiol. 2007 Oct;66(1):40-54. doi: 10.1111/j.1365-2958.2007.05891.x. Epub 2007 Aug 27.
Catechol and 2-methylhydroquinone (2-MHQ) cause the induction of the thiol-specific stress response and four dioxygenases/glyoxalases in Bacillus subtilis. Using transcription factor arrays, the MarR-type regulator YkvE was identified as a repressor of the dioxygenase/glyoxalase-encoding mhqE gene. Transcriptional and proteome analyses of the DeltaykvE mutant revealed the upregulation of ykcA (mhqA), ydfNOP (mhqNOP), yodED (mhqED) and yvaB (azoR2) encoding multiple dioxygenases/glyoxalases, oxidoreductases and an azoreductase. Primer extension experiments identified sigma(A)-type promoter sequences upstream of mhqA, mhqNOP, mhqED and azoR2 from which transcription is elevated after thiol stress. DNase I footprinting analysis showed that YkvE protects a primary imperfect inverted repeat with the consensus sequence of tATCTcgaAtTCgAGATaaaa in the azoR2, mhqE and mhqN promoter regions. Analysis of mhqE-promoter-bgaB fusions confirmed the significance of YkvE binding to this operator in vivo. Adjacent secondary repeats were protected by YkvE in the azoR2 and mhqN promoter regions consistent with multiple DNA-protein binding complexes. DNA-binding activity of YkvE was not directly affected by thiol-reactive compounds in vitro. Mutational analyses showed that MhqA, MhqO and AzoR2 confer resistance to 2-MHQ. Moreover, the DeltaykvE mutant displayed a 2-MHQ and catechol resistant phenotype. YkvE was renamed as MhqR controlling a 2-MHQ and catechol-resistance regulon of B. subtilis.
儿茶酚和2-甲基对苯二酚(2-MHQ)可诱导枯草芽孢杆菌中的硫醇特异性应激反应以及四种双加氧酶/乙二醛酶。利用转录因子阵列,鉴定出MarR型调节因子YkvE是双加氧酶/乙二醛酶编码基因mhqE的阻遏物。对ΔykvE突变体的转录组和蛋白质组分析显示,编码多种双加氧酶/乙二醛酶、氧化还原酶和一种偶氮还原酶的ykcA(mhqA)、ydfNOP(mhqNOP)、yodED(mhqED)和yvaB(azoR2)上调。引物延伸实验确定了mhqA、mhqNOP、mhqED和azoR2上游的σ(A)型启动子序列,硫醇应激后转录从这些序列升高。DNase I足迹分析表明,YkvE在azoR2、mhqE和mhqN启动子区域保护一个主要的不完全反向重复序列,其共有序列为tATCTcgaAtTCgAGATaaaa。对mhqE启动子-bgaB融合体的分析证实了YkvE在体内与该操纵子结合的重要性。在azoR2和mhqN启动子区域,相邻的二级重复序列受到YkvE的保护,这与多个DNA-蛋白质结合复合物一致。YkvE的DNA结合活性在体外不受硫醇反应性化合物的直接影响。突变分析表明,MhqA、MhqO和AzoR2赋予对2-MHQ的抗性。此外,ΔykvE突变体表现出对2-MHQ和儿茶酚的抗性表型。YkvE被重新命名为MhqR,它控制枯草芽孢杆菌的一个2-MHQ和儿茶酚抗性调节子。