Department of Bioproducts and Biosystems Engineering, University of Minnesota, 1390 Eckles Avenue, St. Paul, MN, 55108-6130, USA.
Biotechnology Institute, University of Minnesota, St. Paul, MN, 55108, USA.
Appl Microbiol Biotechnol. 2022 Aug;106(13-16):5051-5061. doi: 10.1007/s00253-022-12059-x. Epub 2022 Jul 9.
The enzyme nitrogenase performs the process of biological nitrogen fixation (BNF), converting atmospheric dinitrogen gas into the biologically accessible ammonia, which is rapidly protonated at physiological pH to yield ammonium. The reduction of dinitrogen requires both ATP and electrons. Azotobacter vinelandii is an aerobic nitrogen-fixing microbe that is a model organism for the study of BNF. Previous reports have described strains of A. vinelandii that are partially deregulated for BNF, resulting in the release of large quantities of ammonium into the growth medium. Determining the source of the electrons required to drive BNF is complicated by the existence of several protein complexes in A. vinelandii that have been linked to BNF in other species. In this work, we used the high-ammonium-accumulating strains of A. vinelandii to probe the source of electrons to nitrogenase by disrupting the Rnf1 and Fix complexes. The results of this work demonstrate the potential of these strains to be used as a tool to investigate the contributions of other enzymes or complexes in the process of BNF. These results provide strong evidence that the Rnf1 complex of A. vinelandii is the primary source of electrons delivered to the nitrogenase enzyme in this partially deregulated strain. The Fix complex under native regulation was unable to provide sufficient electrons to accumulate extracellular ammonium in the absence of the Rnf1 complex. Increased ammonium accumulation could be attained in a strain lacking the Rnf1 complex if the genes of the Fix protein complex were relocated behind the strong promoter of the S-layer protein but still failed to achieve the levels found with just the Rnf1 complex by itself. KEY POINTS: • The Rnf1 complex is integral to ammonium accumulation in A. vinelandii. • The Fix complex can be deleted and still achieve ammonium accumulation in A. vinelandii. • A. vinelandii can be engineered to increase the contribution of the Fix complex to ammonium accumulation.
固氮酶进行生物固氮（BNF）过程，将大气中的氮气气体转化为生物可利用的氨，氨在生理 pH 值下迅速质子化生成铵。还原氮气需要 ATP 和电子。维氏固氮菌是一种需氧固氮微生物，是 BNF 研究的模式生物。以前的报告描述了部分解除 BNF 调控的维氏固氮菌菌株，导致大量铵释放到生长培养基中。确定驱动 BNF 所需的电子来源很复杂，因为在维氏固氮菌中存在几种与其他物种中的 BNF 相关的蛋白质复合物。在这项工作中，我们使用高铵积累的维氏固氮菌菌株通过破坏 Rnf1 和 Fix 复合物来探测氮酶的电子来源。这项工作的结果表明，这些菌株有可能成为研究 BNF 过程中其他酶或复合物贡献的工具。这些结果提供了强有力的证据，表明维氏固氮菌的 Rnf1 复合物是该部分解除调控的菌株中氮酶电子的主要来源。在没有 Rnf1 复合物的情况下，天然调节下的 Fix 复合物无法提供足够的电子来积累细胞外铵。如果 Fix 蛋白复合物的基因被重新定位到 S-层蛋白的强启动子后面，在缺乏 Rnf1 复合物的菌株中可以获得增加的铵积累，但仍未能达到仅 Rnf1 复合物本身的水平。关键点：• Rnf1 复合物是维氏固氮菌中铵积累的必要组成部分。• Fix 复合物可以被删除，并且仍然可以在维氏固氮菌中实现铵积累。• 可以对维氏固氮菌进行工程改造以增加 Fix 复合物对铵积累的贡献。
