Ebert Matthias, Schweyen Peter, Bröring Martin, Laass Sebastian, Härtig Elisabeth, Jahn Dieter
From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig.
the Institute for Inorganic and Analytical Chemistry, Technische Universität Braunschweig, Hagenring 30, D-38106 Braunschweig.
J Biol Chem. 2017 Sep 15;292(37):15468-15480. doi: 10.1074/jbc.M117.798728. Epub 2017 Aug 1.
Under oxygen-limiting conditions, the marine bacterium DFL12 generates energy via denitrification, a respiratory process in which nitric oxide (NO) is an intermediate. Accumulation of NO may cause cytotoxic effects. The response to this nitrosative (NO-triggered) stress is controlled by the Crp/Fnr-type transcriptional regulator DnrF. We analyzed the response to NO and the mechanism of NO sensing by the DnrF regulator. Using reporter gene fusions and transcriptomics, here we report that DnrF selectively repressed nitrate reductase () genes, preventing further NO formation. In addition, DnrF induced the expression of the NO reductase genes (), which promote NO consumption. We used UV-visible and EPR spectroscopy to characterize heme binding to DnrF and subsequent NO coordination. DnrF detects NO via its bound heme cofactor. We found that the dimeric DnrF bound one molecule of heme per subunit. Purified recombinant apo-DnrF bound its target promoter sequences (, , , and ) in electromobility shift assays, and we identified a specific palindromic DNA-binding site 5'-TTGATNATCAA-3' in these target sequences via mutagenesis studies. Most importantly, successive addition of heme as well as heme and NO to purified recombinant apo-DnrF protein increased affinity of the holo-DnrF for its specific binding motif in the promoter. On the basis of these results, we propose a model for the DnrF-mediated NO stress response of this marine bacterium.
在氧气限制条件下，海洋细菌DFL12通过反硝化作用产生能量，反硝化是一种呼吸过程，其中一氧化氮（NO）是中间产物。NO的积累可能会导致细胞毒性作用。对这种亚硝化（NO引发）应激的反应由Crp/Fnr型转录调节因子DnrF控制。我们分析了对NO的反应以及DnrF调节因子对NO的感应机制。利用报告基因融合和转录组学技术，我们在此报告DnrF选择性地抑制硝酸盐还原酶（）基因，阻止进一步的NO形成。此外，DnrF诱导NO还原酶基因（）的表达，促进NO的消耗。我们使用紫外可见光谱和电子顺磁共振光谱来表征血红素与DnrF的结合以及随后的NO配位。DnrF通过其结合的血红素辅因子检测NO。我们发现二聚体DnrF每个亚基结合一分子血红素。在电泳迁移率变动分析中，纯化的重组脱辅基DnrF结合其靶启动子序列（、、、和），并且我们通过诱变研究在这些靶序列中鉴定出一个特定的回文DNA结合位点5'-TTGATNATCAA-3'。最重要的是，向纯化的重组脱辅基DnrF蛋白中连续添加血红素以及血红素和NO会增加全酶DnrF对其在启动子中特定结合基序的亲和力。基于这些结果，我们提出了这种海洋细菌中DnrF介导的NO应激反应模型。
