Isabella Vincent M, Lapek John D, Kennedy Edward M, Clark Virginia L
Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue, Rochester, NY, USA.
Mol Microbiol. 2009 Jan;71(1):227-39. doi: 10.1111/j.1365-2958.2008.06522.x.
Nitric oxide (NO) has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2-type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope-tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe-2S] cluster. NsrR/DNA interactions were thoroughly analysed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA and nsrR upstream regions with a K(d) of 7, 19 and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97 or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in co-ordination of a NO-sensitive Fe-S centre required for DNA binding.
一氧化氮（NO）已被证明是人类免疫反应的重要组成部分，因此，了解致病生物如何对其存在做出反应很重要。在淋病奈瑟菌中，最近的研究表明，Rrf2型转录抑制因子NsrR可以感知NO并控制负责NO代谢的基因的表达。分离出了一种高度纯化的表位标记NsrR提取物，质谱分析表明该蛋白质含有一个[2Fe-2S]簇。在体外对NsrR/DNA相互作用进行了全面分析。使用电泳迁移率变动分析（EMSA），显示NsrR::FLAG与norB、aniA和nsrR上游区域的预测操纵子相互作用，其解离常数（K(d)）分别为7、19和35 nM。对norB和nsrR的上游区域进行了DNase I足迹分析，结果显示NsrR可保护预测的29 bp结合位点。外源添加的NO的存在会抑制NsrR与DNA的结合。NsrR中C90、C97或C103的丙氨酸取代消除了对norB::lacZ的抑制并抑制了DNA结合，这与其在协调DNA结合所需的NO敏感铁硫中心中的假定作用一致。
