Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon
Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon
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DOI：
10.1111/j.1365-2958.2005.04599.x
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发表时间：
2005-05-01
影响因子：
3.6
通讯作者：
Wolf, RE
中科院分区：
文献类型：
--
作者：
Griffith, KL;Becker, SM;Wolf, RE
在大肠杆菌中，SoxS、MarA 和 Rob 形成 AraC/XylS 正调节子家族的密切相关子集，在 SoxS 长度上具有相似的 42% 氨基酸序列同一性，并且能够激活一组常见靶基因的转录，这些靶基因提供对氧化还原循环化合物和抗生素的抗性。基于其与 SoxS、MarA 和 Rob 相似的 43% 氨基酸序列同一性，TetD（编码位转座子 Tn 10）似乎是该子集的第四个成员。然而，尽管已证明其表达受 TetC 负向调节且不受四环素诱导，但 TetD 的生理功能尚不清楚。因此，在此介绍的工作中，我们启动了 TetD 的分子表征。我们发现 TetD 的表达激活了 SoxS/MarA/Rob 调节子基因子集的转录，并赋予对氧化还原循环化合物和抗生素的抗性。我们发现，TetD 可激活启动子的假定 TetD 结合位点的突变和蛋白质 N 端 DNA 识别螺旋的突变会干扰转录激活，从而表明 TetD 直接激活靶基因转录。最后，我们证明 TetD 与 SoxS 和 MarA 一样，本质上是不稳定的；然而，与 SoxS 和 MarA 不同，TetD 不会被 Lon 或任何细胞已知的细胞质 ATP 依赖性蛋白酶降解。因此，我们得出结论，TetD 是正调节因子 SoxS/MarA/Rob 亚家族的真正成员。
In Escherichia coli, SoxS, MarA and Rob form a closely related subset of the AraC/XylS family of positive regulators, sharing similar to 42% amino acid sequence identity over the length of SoxS and the ability to activate transcription of a common set of target genes that provide resistance to redox-cycling compounds and antibiotics. On the basis of its similar to 43% amino acid sequence identity with SoxS, MarA and Rob, TetD, encoded bit transposon Tn 10, appears to be a fourth member of the subset. However, although its expression has been shown to be negatively regulated by TetC and not inducible by tetracycline, the physiological function of TetD is unknown. Accordingly, in the work presented here, we initiate a molecular characterization of TetD. We show that expression of TetD activates transcription of a subset of the SoxS/MarA/Rob regulon genes and confers resistance to redox-cycling compounds and antibiotics. We show that mutations in the putative TetD binding site of a TetD-activatable promoter and a mutation in the protein's N-terminal DNA recognition helix interfere with transcription activation, thereby indicating that TetD directly activates target gene transcription. Finally, we show that TetD, like SoxS and MarA, is intrinsically unstable; however, unlike SoxS and MarA,TetD is not degraded by Lon or any of the cell's known cytoplasmic ATP-dependent proteases. Thus, we conclude that TetD is a bona fide member of the SoxS/MarA/Rob subfamily of positive regulators.