ArcA and AppY Antagonize IscR Repression of Hydrogenase-1 Expression under Anaerobic Conditions, Revealing a Novel Mode of O2 Regulation of Gene Expression in Escherichia coli
ArcA and AppY Antagonize IscR Repression of Hydrogenase-1 Expression under Anaerobic Conditions, Revealing a Novel Mode of O2 Regulation of Gene Expression in Escherichia coli
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DOI：
10.1128/jb.01757-12
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发表时间：
2012-12-01
影响因子：
3.2
通讯作者：
Kiley, P. J.
中科院分区：
文献类型：
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作者：
Nesbit, A. D.;Fleischhacker, A. S.;Kiley, P. J.
大肠杆菌氢化酶-1操纵子（hyaABCDEF）的转录增加的转录因子ArcA和AppY在厌氧生长条件下。然而，在有氧条件下抑制hyaA启动子（P-hyaA）转录的IscR，在厌氧条件下抑制该启动子的转录是未知的。在这里，我们报告说，ArcA和AppY增加P-hyaA的表达在厌氧条件下，通过拮抗IscR结合在P-hyaA，因为IscR抑制时观察到ArcA或AppY被消除。ArcA和AppY作为P-hyaA的IscR阻遏的抗阻遏物的能力依赖于IscR水平，表明IscR与ArcA和/或AppY竞争结合。为了支持这种竞争模型，电泳迁移率变动分析和DNA酶I足迹法表明，ArcA和IscR的结合位点重叠，ArcA和IscR的结合是相互排斥的。出乎意料的是，具有C92A突变的IscR（IscR-C92A）（其模拟主要存在于有氧条件下的蛋白质的无簇形式）在厌氧条件下是P-hyaA和含有来自P-hyaA的IscR结合位点的组成型启动子两者的更好的阻遏物，而野生型IscR在厌氧条件下主要以[2Fe-2S]形式存在。这一观察结果不能用DNA结合亲和力或IscR水平的差异来解释，因此我们得出结论，[2Fe-2S]-IscR是比无簇IscR更弱的P-hyaA阻遏物。总之，ArcA和AppY对IscR功能的抗阻遏、较低水平的IscR和[2Fe-2S]-IscR的弱阻遏的组合导致在厌氧条件下P-hyaA表达增加。
Transcription of the Escherichia cob hydrogenase-1 operon (hyaABCDEF) is increased by the transcription factors ArcA and AppY under anaerobic growth conditions. However, IscR, which represses transcription of the hyaA promoter (P-hyaA) under aerobic conditions, was not known to repress transcription of this promoter under anaerobic conditions. Here, we report that ArcA and AppY increase P-hyaA expression under anaerobic conditions by antagonizing IscR binding at P-hyaA, since IscR repression is observed when either ArcA or AppY is eliminated. The ability of ArcA and AppY to act as antirepressors of IscR repression of P-hyaA depended on IscR levels, suggesting that IscR competes with ArcA and/or AppY for binding. In support of this competition model, electrophoretic mobility shift assays and DNase I footprinting showed that the ArcA and IscR binding sites overlap and that binding of ArcA and IscR is mutually exclusive. Unexpectedly, IscR with a C92A mutation (IscR-C92A), which mimics the clusterless form of the protein that is present predominantly under aerobic conditions, was a better repressor under anaerobic conditions of both P-hyaA and a constitutive promoter containing the IscR binding site from P-hyaA than wild-type IscR, which is predominantly in the [2Fe-2S] form under anaerobic conditions. This observation could not be explained by differences in DNA binding affinities or IscR levels, so we conclude that [2Fe-2S]-IscR is a weaker repressor of P-hyaA than clusterless IscR. In sum, a combination of ArcA and AppY antirepression of IscR function, lower levels of IscR, and weak repression by [2Fe-2S]-IscR leads to increased P-hyaA expression under anaerobic conditions.