Mus Florence, Tseng Alex, Dixon Ray, Peters John W
Institute of Biological Chemistry, Washington State University, Pullman, Washington, USA.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, USA.
Appl Environ Microbiol. 2017 Jun 16;83(13). doi: 10.1128/AEM.00808-17. Print 2017 Jul 1.
Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of , attempts to obtain deletion mutants in the gene encoding GS () have been unsuccessful. We have generated a deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance. In this study, the characterization of the knockout mutant of the model diazotroph provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.
克服过量环境铵对固氮酶合成或活性的抑制作用以及防止铵同化，已被视为在联合或共生植物 - 固氮菌关系中增加从细菌转移至植物伙伴的固定氮量的策略。谷氨酰胺合成酶（GS）腺苷酰转移酶/腺苷去除酶催化谷氨酰胺合成酶（GS）的可逆腺苷酸化，从而影响铵同化的翻译后调控，这对于碳和氮同化的适当协调至关重要。由于GS是[具体生物]唯一的铵同化途径的关键，因此尝试获得编码GS（[具体基因]）的基因缺失突变体一直未成功。我们构建了一个[具体基因]缺失菌株，从而防止了GS的翻译后调控。如先前在其他生物体中观察到的那样，所得含有组成型活性GS的菌株在含铵培养基上生长不佳，并且只能在低铵浓度下培养。这种表型是由于缺乏GS活性的下调导致的，在过量氮条件下，细胞内谷氨酰胺水平升高，谷氨酰胺与2 - 酮戊二酸的比例受到严重干扰。有趣的是，该突变体能够以与野生型相当的速率进行固氮生长。这一观察结果表明，通过NifA响应2 - 酮戊二酸在转录水平上对固氮特异性基因表达的控制足够严格，足以单独将铵产生调节到适合最佳碳氮平衡的水平。在本研究中，对模式固氮菌[具体菌株]的[具体基因]敲除突变体的表征为固氮过程中铵产生调控机制与铵同化调控机制的整合提供了重要见解。这项工作揭示了固氮调控在提供足以实现最大生长的铵但限制能量消耗巨大的过量产生方面的高度精确性。对铵产生调控与同化之间相互作用的详细基础理解对于开发现有的以及潜在地工程化新的植物 - 固氮菌关系以改善农业至关重要。
