Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron-猫眼课题宝

白喉棒状杆菌受白喉毒素阻遏物 (DtxR) 和铁调节的两个启动子/操纵子的克隆、序列和足迹分析

DOI：

发表时间：

1994

期刊：

Journal of Bacteriology

影响因子：

3.2

通讯作者：

中科院分区：

生物学3区

文献类型：

作者：

M. Schmitt;R. Holmes

DtxR是一种铁依赖性序列特异性DNA结合蛋白，其结合至tox操纵子，该操纵子是位于白喉毒素基因上游的反向重复核苷酸序列。在这项研究中，两个额外的铁调节的启动子/操纵子序列（IRP 1和IRP 2）的DtxR控制的白喉棒状杆菌的染色体克隆和表征。操纵子融合lacZ被用来分析表达从IRP 1和IRP 2在大肠杆菌。从这两个启动子的转录强烈抑制在高铁培养基中的克隆的dtxR基因的存在下，然而，在没有dtxR的转录是50- 100倍以上，无论铁浓度。纯化的DtxR改变了携带IRP 1或IRP 2的DNA片段的电泳迁移率，并且两个启动子/操纵基因区的核苷酸序列表明它们都与tox操纵基因同源。DtxR保护IRP 1和IRP 2上约30 bp的区域免受DNA酶I消化。一个19 bp的共识DtxR结合位点来自各种DtxR调控的操作子/启动子序列的比较。使用羟基自由基和硫酸二甲酯的足迹实验表明，DtxR与这些运营商以对称的方式相互作用，可能是作为二聚体或多聚体。位于IRP 1下游的开放阅读框（ORF 1）的推导氨基酸序列与参与革兰氏阴性细菌铁转运的周质蛋白家族以及来自枯草杆菌的铁色素受体FhuD同源。这些发现表明，ORF 1编码的膜相关脂蛋白可能作为受体的铁铁载体复合物在C。白喉

DtxR is an iron-dependent sequence-specific DNA-binding protein that binds to the tox operator, an inverted-repeat nucleotide sequence located upstream from the diphtheria toxin gene. In this study, two additional iron-regulated promoter/operator sequences (IRP1 and IRP2) that are controlled by DtxR were cloned from the chromosome of Corynebacterium diphtheriae and characterized. Operon fusions to lacZ were used to analyze expression from IRP1 and IRP2 in Escherichia coli. Transcription from both promoters was strongly repressed in high-iron medium in the presence of the cloned dtxR gene; however, transcription in the absence of dtxR was 50- to 100-fold greater, regardless of the iron concentration. Purified DtxR altered the electrophoretic mobility of DNA fragments carrying IRP1 or IRP2, and the nucleotide sequences of the two promoter/operator regions indicated that they are both homologous with the tox operator. DtxR protected an approximately 30-bp region on both IRP1 and IRP2 from DNase I digestion. A 19-bp consensus DtxR-binding site was derived from a comparison of the various DtxR-regulated operator/promoter sequences. Footprinting experiments using hydroxyl radicals and dimethyl sulfate demonstrated that DtxR interacted with these operators in a symmetrical manner, probably as a dimer or multimer. The deduced amino acid sequence of an open reading frame (ORF1) located downstream from IRP1 was homologous with a family of periplasmic proteins involved in iron transport in gram-negative bacteria and with the ferrichrome receptor, FhuD, from Bacillus subtilis. These findings suggest that ORF1 encodes a membrane-associated lipoprotein that may serve as the receptor for a ferric-siderophore complex in C. diphtheriae.