Sangwan Indu, Small Sandra K, O'Brian Mark R
Department of Biochemistry, 140 Farber Hall, State University of New York at Buffalo, Buffalo, New York 14214, USA.
J Bacteriol. 2008 Aug;190(15):5172-7. doi: 10.1128/JB.00495-08. Epub 2008 Jun 6.
The Irr protein is a global regulator of iron homeostasis in Bradyrhizobium japonicum, and a subset of genes within the Irr regulon are negatively controlled under iron limitation. However, repressor function, high-affinity DNA binding in vitro, or promoter occupancy in vivo of Irr for a negatively regulated gene has not been demonstrated. Here, we show that the blr7895 and bll6680 genes are negatively regulated by Irr as determined by derepression of transcript levels in iron-limited cells of an irr mutant strain. Electrophoretic gel mobility shift analysis showed that a component in extracts of wild-type cells grown under iron limitation bound the iron control elements (ICE) within the promoters of blr7895 and bll6680 identified previously (G. Rudolph, G. Semini, F. Hauser, A. Lindemann, M. Friberg, H. Hennecke, and H. M. Fischer, J. Bacteriol. 188:733-744, 2006). Binding was not observed with extracts of cells from the parent strain grown under high iron conditions or with those from an irr mutant. Furthermore, gel mobility supershift experiments identified Irr as a component of the binding complex. Purified recombinant Irr bound to ICE DNA with high affinity in the presence of divalent metal, with K(d) values of 7 to 19 nM, consistent with a physiological role for Irr as a transcriptional regulator. In addition, in vitro transcription initiated from the blr7895 promoter was inhibited by Irr. Whole-cell cross-linking and immunoprecipitation experiments showed that Irr occupies the promoters of blr7895 and bll6680 in vivo in an iron-dependent manner. The findings demonstrate that Irr is a transcriptional repressor that binds DNA with high affinity.
Irr蛋白是日本慢生根瘤菌中铁稳态的全局调节因子，在铁限制条件下，Irr调控子中的一部分基因受到负调控。然而，尚未证实Irr对负调控基因具有阻遏功能、体外高亲和力DNA结合能力或体内启动子占据能力。在此，我们发现，通过irr突变株铁限制细胞中转录水平的去阻遏作用确定，blr7895和bll6680基因受Irr负调控。电泳凝胶迁移率变动分析表明，在铁限制条件下生长的野生型细胞提取物中的一种成分与先前鉴定的blr7895和bll6680启动子内的铁控制元件（ICE）结合（G. Rudolph、G. Semini、F. Hauser、A. Lindemann、M. Friberg、H. Hennecke和H. M. Fischer，《细菌学杂志》188:733 - 744，2006年）。在高铁条件下生长的亲本菌株细胞提取物或irr突变体细胞提取物中未观察到结合。此外，凝胶迁移超迁移实验确定Irr是结合复合物的一个成分。纯化的重组Irr在二价金属存在下以高亲和力结合ICE DNA，解离常数（K(d)）值为7至19 nM，这与Irr作为转录调节因子的生理作用一致。此外，从blr7895启动子起始的体外转录受到Irr的抑制。全细胞交联和免疫沉淀实验表明，Irr在体内以铁依赖的方式占据blr7895和bll6680的启动子。这些发现证明Irr是一种能以高亲和力结合DNA的转录阻遏物。
