The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages
The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages
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DOI：
10.1046/j.1365-2958.2001.02684.x
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发表时间：
2001-11-01
影响因子：
3.6
通讯作者：
Smith, I
中科院分区：
文献类型：
--
作者：
Gold, B;Rodriguez, GM;Smith, I
在这项工作中，我们描述了结核分枝杆菌中受IdeR调控的基因（（i）bar下的罗恩-（de）bar下的pendent（r）bar下的egulator），DtxR家族的一种铁响应性DNA结合蛋白，已被证明可调控耻垢分枝杆菌中的铁获得。为了鉴定构成IdeR调节子的一些基因，我们搜索了M。结核病基因组中含有共有IdeR/DxR结合序列的启动子区域。IdeR盒之前的基因包括一组编码铁获得所必需的蛋白质，如铁载体的生物合成（mbtA，mbtB，mbtI），芳香族氨基酸（pheA，hisE，hisB样）和其他注释为参与铁储存蛋白合成的蛋白质（bfrA，bfrB）。一些推定的IdeR调节基因在此搜索中确定编码蛋白质预测参与脂多糖（LPS）样分子（rv 3402 c），脂质（acpP）和肽聚糖（murB）的生物合成。我们分析了四个启动子区域含有推定的IdeR盒，mbtA-mbtB，mbl，rv 3402 c和bfrA-bfd，与IdeR的相互作用和铁依赖性表达。凝胶阻滞实验和DNA酶足迹分析纯化的IdeR表明，IdeR结合这些IdeR盒在体外。通过引物延伸对启动子的分析表明，IdeR盒位于每个启动子的-10位置附近，表明IdeR通过阻断RNA聚合酶结合而充当转录阻遏物。使用定量逆转录-聚合酶链反应（RT-PCR）结合分子信标，我们发现，在M.结核病患者缺乏铁，而bfrA的mRNA水平下降了约三倍。我们目前的证据表明，ldeR不仅作为一个转录抑制因子，但也作为一个激活剂的bfrA的功能。三个IdeR和铁抑制基因，mbtB，mbtl和rv 3402 c，在M。结核感染的人THP-1巨噬细胞。
In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR ((i) under bar ron-(de) under bar pendent (r) under bar egulator), an iron-responsive DNA-binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB-like) and others annotated to be involved in the synthesis of iron-storage proteins (bfrA, bfrB). Some putative IdeR-regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA-mbtB, mbl, rv3402c and bfrA-bfd, for interaction with IdeR and for iron-dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the -10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtl, rv3402c and bfd are induced 14- to 49-fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that ldeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR- and iron-repressed genes, mbtB, mbtl and rv3402c, were induced during M. tuberculosis infection of human THP-1 macrophages.