Institute of Biological Chemistry, Washington State Universitygrid.30064.31, Pullman, Washington, USA.
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Appl Environ Microbiol. 2022 Mar 22;88(6):e0187621. doi: 10.1128/AEM.01876-21. Epub 2022 Feb 9.
The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting gene expression as an additional requirement for ammonium excretion. These ammonium-excreting mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.
无处不在的固氮土壤细菌 Azotobacter vinelandii 一直被广泛研究作为生物固氮 (BNF) 的模式生物。在 A. vinelandii 中，BNF 受到 NifL-NifA 双组分系统的调控，其中 NifL 作为一种反激活剂，根据氧化还原、氮和碳状态，紧密控制氮固定特异性转录激活剂 NifA 的活性。虽然有几项研究报道 A. vinelandii 中的突变导致氮酶表达失调并释放大量铵，但缺乏对这种铵排泄表型的特定决定因素的了解。在这项工作中，我们报告只有特定的突变导致大量铵在液体培养中积累（约 12mM）。铵排泄表型仅与 NifL 结构域的缺失以及在转录方向相反的位置插入启动子序列有关。我们进一步证明，插入启动子的强度可以通过影响 基因表达作为铵排泄的附加要求来影响铵的排泄量。这些排泄铵的 突变体显著刺激固定氮向水稻的转移。这项工作定义了导致 A. vinelandii 铵排泄的离散决定因素，并证明可以通过简单的基因编辑产生菌株，提供有前途的生物肥料，能够将氮转移到作物中。人们对工程化铵排泄细菌在农业中的应用产生了浓厚的兴趣，以促进在固定氮限制条件下植物的生长。这项工作定义了导致 A. vinelandii 铵排泄的离散决定因素，并证明可以通过简单的基因编辑产生菌株，提供有前途的生物肥料，能够将氮转移到作物中。
