Construction and Characterization of Transposon Insertion Mutations in Corynebacterium diphtheriae That Affect Expression of the Diphtheria Toxin Repressor (DtxR)-猫眼课题宝

白喉棒杆菌中影响白喉毒素阻遏物 (DtxR) 表达的转座子插入突变的构建和表征

DOI：

发表时间：

2002

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：

D. M. Oram;Ana Avdalovic;R. Holmes

摘要噬菌体传播的白喉毒素基因 tox 的转录受到染色体编码白喉毒素阻遏物 (DtxR) 的负向调节，以响应细胞内 Fe2+ 浓度。由于缺乏工具，白喉棒状杆菌的遗传分析主要依赖于化学诱导和自发突变体的分析以及在大肠杆菌中克隆或体外分析的白喉棒状杆菌基因的实验结果。我们修改了一种基于 Tn5 的诱变技术，用于白喉杆菌，并用它在这种革兰氏阳性病原体的染色体中构建了第一个转座子插入文库。我们分离出了两个影响 DtxR 表达的插入，一个位于 dtxR 上游 121 bp，另一个位于 dtxR 编码序列的必需区域内，这首次表明 dtxR 是白喉棒状杆菌中的一个可有可无的基因。当在含有足够铁的培养基中生长时，两种突变菌株都会分泌白喉毒素，以抑制野生型白喉棒状杆菌分泌白喉毒素。上游插入突变体仍然产生减少量的 DtxR，并以类似于其野生型亲本的方式调节铁载体分泌以响应铁。 dtxR 内含有转座子插入的突变体不产生 DtxR，并且在铁存在的情况下过量产生铁载体。两种突变株在氧化应激下生存能力的差异也表明上游插入保留了轻微的 DtxR 活性，而 dtxR 内的插入消除了 DtxR 活性。这是 DtxR 在保护细胞免受氧化应激方面发挥作用的第一个证据。

ABSTRACT Transcription of the bacteriophage-borne diphtheria toxin gene tox is negatively regulated, in response to intracellular Fe2+ concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR). Due to a scarcity of tools, genetic analysis of Corynebacterium diphtheriae has primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments with C. diphtheriae genes cloned in Escherichia coli or analyzed in vitro. We modified a Tn5-based mutagenesis technique for use with C. diphtheriae, and we used it to construct the first transposon insertion libraries in the chromosome of this gram-positive pathogen. We isolated two insertions that affected expression of DtxR, one 121 bp upstream of dtxR and the other within an essential region of the dtxR coding sequence, indicating for the first time that dtxR is a dispensable gene in C. diphtheriae. Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-type C. diphtheriae. The upstream insertion mutant still produces DtxR in decreased amounts and regulates siderophore secretion in response to iron in a manner similar to its wild-type parent. The mutant containing the transposon insertion within dtxR does not produce DtxR and overproduces siderophore in the presence of iron. Differences in the ability of the two mutant strains to survive oxidative stress also indicated that the upstream insertion retained slight DtxR activity, whereas the insertion within dtxR abolished DtxR activity. This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.