Integration host factor is required for anaerobic pyruvate induction of pfl operon expression in Escherichia coli.
Integration host factor is required for anaerobic pyruvate induction of pfl operon expression in Escherichia coli.
复制标题
整合宿主因子是厌氧丙酮酸诱导大肠杆菌中 pfl 操纵子表达所必需的。
DOI：
10.1128/jb.175.18.5769-5777.1993
复制
发表时间：
1993
影响因子：
3.2
通讯作者：
Sawers,G
中科院分区：
文献类型：
--
作者：
Sirko,A;Zehelein,E;Freundlich,M;Sawers,G
丙酮酸甲酸裂解酶基因 (pfl) 的表达是由厌氧生长诱导的，并且通过在丙酮酸中生长进一步增加。先前的工作表明，厌氧诱导强烈依赖于激活剂 FNR，并部分依赖于第二个转录因子 ArcA，而丙酮酸诱导仅需要 FNR。厌氧和丙酮酸调节都需要存在跨越大约 500 bp DNA 的 5' 非翻译调节序列。开发了迁移率变化测定法来识别与该调节区域结合的蛋白质。通过肝素琼脂糖色谱法分离了几种结合活性，其中一种活性被表征并显示为整合宿主因子（IHF）。迁移率变化和 DNase I 足迹实验确定了 pfl 启动子调控区域中的单个 IHF 结合位点。通过pfl-lacZ融合，可以证明himD突变的引入消除了体内丙酮酸依赖性厌氧表达的诱导。当 pfl IHF 结合位点突变时观察到相同的结果。此外，在fnr菌株中发现的部分厌氧表达诱导在fnr HimD双突变体和fnr IHF结合位点双突变体中被完全阻断。总而言之，这些数据表明 IHF 对于丙酮酸诱导和 ArcA 介导的厌氧诱导都是必需的。
The expression of the pyruvate formate-lyase gene (pfl) is induced by anaerobic growth, and this is increased further by growth in pyruvate. Previous work has shown that anaerobic induction is strongly dependent on the activator FNR and partially dependent on a second transcription factor, ArcA, while pyruvate induction only required FNR. Anaerobic and pyruvate regulation both require the presence of a 5' nontranslated regulatory sequence which spans approximately 500 bp of DNA. A mobility shift assay was developed to identify proteins that bind to this regulatory region. Several binding activities were separated by heparin agarose chromatography, and one of these activities was characterized and shown to be integration host factor (IHF). Mobility shift and DNase I footprinting experiments defined a single IHF binding site in the pfl promoter-regulatory region. With pfl-lacZ fusions, it could be shown that introduction of a himD mutation abolished pyruvate-dependent induction of anaerobic expression in vivo. The same result was observed when the pfl IHF binding site was mutated. In addition, the partial anaerobic induction of expression found in an fnr strain was completely blocked in an fnr himD double mutant and in an fnr IHF binding site double mutant. Taken together, these data suggest that IHF is necessary for both pyruvate induction and the anaerobic induction mediated by ArcA.