Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), C/Profesor Albareda 1, E-18008, Granada, Spain.
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), C/Profesor Albareda 1, E-18008 Granada, Spain.
Nitric Oxide. 2017 Aug 1;68:137-149. doi: 10.1016/j.niox.2017.02.002. Epub 2017 Feb 3.
Expression of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes requires low oxygen (O) tension and nitrate (NO), through a regulatory network comprised of two coordinated cascades, FixLJ-FixK-NnrR and RegSR-NifA. To precisely understand how these signals are integrated in the FixLJ-FixK-NnrR circuit, we analyzed β-Galactosidase activities from napE-lacZ, nirK-lacZ and norC-lacZ fusions, and performed analyses of NapC and NorC levels as well as periplasmic nitrate reductase (Nap) activity, in B. japonicum wildtype and fixK and nnrR mutant backgrounds. While microoxic conditions (2% O at headspace) were sufficient to induce expression of napEDABC and nirK genes and this control depends on FixK, norCBQD expression requires, in addition to microoxia, nitric oxide gas (NO) and both FixK and NnrR transcription factors. Purified FixK protein directly interacted and activated transcription in collaboration with B. japonicum RNA polymerase (RNAP) from the napEDABC and nirK promoters, but not from the norCBQD promoter. Further, recombinant NnrR protein bound exclusively to the norCBQD promoter in an O-sensitive manner. Our work suggest a disparate regulation of B. japonicum denitrifying genes expression with regard to their dependency to microoxia, nitrogen oxides (NOx), and the regulatory proteins FixK and NnrR. In this control, expression of napEDABC and nirK genes requires microoxic conditions and directly depends on FixK, while expression of norCBQD genes relies on NO, being NnrR the candidate which directly interacts with the norCBQD promoter.
根瘤菌 napEDABC、nirK 和 norCBQD 基因的表达需要低氧 (O) 张力和硝酸盐 (NO)，通过由两个协调级联组成的调控网络，FixLJ-FixK-NnrR 和 RegSR-NifA。为了精确理解这些信号如何在 FixLJ-FixK-NnrR 电路中整合，我们分析了 napE-lacZ、nirK-lacZ 和 norC-lacZ 融合物的β-半乳糖苷酶活性，并对 NapC 和 NorC 水平以及周质硝酸盐还原酶 (Nap) 活性进行了分析，在根瘤菌野生型和 fixK 和 nnrR 突变体背景下。虽然微氧条件（2%O 顶空）足以诱导 napEDABC 和 nirK 基因的表达，这种控制依赖于 FixK，但 norCBQD 基因的表达除了微氧外，还需要一氧化氮气体 (NO) 和 FixK 和 NnrR 转录因子。纯化的 FixK 蛋白直接相互作用并与根瘤菌 RNA 聚合酶 (RNAP) 一起激活转录，从 napEDABC 和 nirK 启动子，但不从 norCBQD 启动子。此外，重组 NnrR 蛋白仅以 O 敏感的方式特异性结合 norCBQD 启动子。我们的工作表明，根瘤菌脱氮基因的表达存在不同的调控，这与它们对微氧、氮氧化物 (NOx) 和调节蛋白 FixK 和 NnrR 的依赖有关。在这种控制下，napEDABC 和 nirK 基因的表达需要微氧条件并直接依赖于 FixK，而 norCBQD 基因的表达依赖于 NO，NnrR 是直接与 norCBQD 启动子相互作用的候选者。
