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首页> 美国卫生研究院文献>Journal of Bacteriology >Cloning sequence and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron.
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Cloning sequence and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron.

机译：白喉棒状杆菌的两个启动子/操纵子的克隆序列和足迹分析这些启动子/操纵子受白喉毒素阻遏物（DtxR）和铁的调控。

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摘要

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.
机译：DtxR是一种铁依赖性序列特异性DNA结合蛋白，与tox操纵子结合，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，从白喉棒状杆菌的染色体上克隆了两个由DtxR控制的铁调控的启动子/操纵子序列（IRP1和IRP2）。 Operon与lacZ的融合蛋白用于分析大肠杆菌中IRP1和IRP2的表达。在存在克隆的dtxR基因的情况下，在高铁培养基中都强烈抑制了两个启动子的转录。但是，无论铁浓度如何，在没有dtxR的情况下转录都提高了50到100倍。纯化的DtxR改变了携带IRP1或IRP2的DNA片段的电泳迁移率，两个启动子/操纵子区域的核苷酸序列表明它们都与tox操纵子同源。 DtxR保护了DNase I消化的IRP1和IRP2上大约30 bp的区域。从各种DtxR调节的操纵子/启动子序列的比较中得出一个19 bp的DtxR结合位点。使用羟基自由基和硫酸二甲酯的足迹实验表明，DtxR与这些操纵子以对称方式相互作用，可能是二聚体或多聚体。推导的位于IRP1下游的开放阅读框（ORF1）的氨基酸序列与参与革兰氏阴性细菌铁运输的周质蛋白家族以及枯草芽孢杆菌的铁色素受体FhuD同源。这些发现表明，ORF1编码一种与膜相关的脂蛋白，可作为白喉衣原体铁-铁载体复合体的受体。

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