Barney Brett M, Plunkett Mary H, Natarajan Velmurugan, Mus Florence, Knutson Carolann M, Peters John W
Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, Minnesota, USA
Biotechnology Institute, University of Minnesota, St. Paul, Minnesota, USA.
Appl Environ Microbiol. 2017 Sep 29;83(20). doi: 10.1128/AEM.01534-17. Print 2017 Oct 15.
Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene result in the deregulation of the transcriptional activator NifA in the nitrogen-fixing bacterium , triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.
生物固氮是由一类被称为固氮菌的多样生物体完成的，并且需要复杂的金属酶固氮酶发挥作用。固氮酶以及辅因子生物合成和整合到该酶中所需的许多辅助蛋白已得到表征，但生物固氮伴随的反应机制和关键细胞变化的完整图景仍有待充分阐明。研究表明，抗激活因子编码基因的特定破坏会导致固氮细菌中转录激活因子NifA的失调，在生长稳定期触发细胞外铵水平接近30 mM的产生。在这项工作中，我们表征了这种高铵释放表型的基因表达全局模式。此处报道的研究结果表明，当使用标准伯克培养基培养时，这种高铵积累菌株的培养物可能会经历金属限制，可通过增加钼水平来改善，以进一步提高铵产量。此外，固氮酶基因转录水平的升高并未伴随着氢化酶基因转录水平或氢气摄取速率的相应显著增加。在固氮酶的三种潜在电子供体系统中，只有基因簇与铵产量的增加呈现转录相关性。我们的结果还突出了几个可能在支持这种好氧固氮模式细菌中提高铵产量方面发挥作用的其他基因。在稳定期铵积累过程中发现的转录差异显示，失调（破坏）菌株与野生型菌株之间以及之前报道的野生型菌株在指数生长期条件下存在强烈对比。这些结果表明，通过增加培养基中可用钼的量，可以进一步提高这种固氮酶失调菌株的铵产量。这些结果还表明，该菌株中存在的两种ATP合酶之一可能具有潜在偏好，并且膜结合氢化酶在氢气循环中比可溶性氢化酶发挥更突出的作用。这些结果应为未来旨在阐明该表型的重要特征以及使该菌株的铵产量最大化的研究提供信息。
