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 nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, 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-ammoniumreleasing phenotype. The findings reported here indicated that cultures of this highammonium- 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 rnf1 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.
Bibliographical noteFunding Information:
This work is supported by a grant (RC-0007-12) from the Initiative for Renewable Energy & the Environment (Institute on the Environment), the MnDRIVE transdisciplinary research initiative through the University of Minnesota based on funding from the state of Minnesota, and grants from the National Institute of Food and Agriculture (project numbers MIN-12-070 and MIN-12-081) to B.M.B.; the National Science Foundation (grant number NSF-1331098) to J.W.P.; and the Biotechnology Institute at the University of Minnesota for fellowship funding to V.N. We thank Jon Bertram and Hanna Hondzo for assistance with performing specific experiments and Ying Zhang and the University of Minnesota Supercomputing Institute for assistance with data analysis using the pipeline to generate FPKM values. We thank Yaniv Brandvain for suggestions related to statistical analysis of our data.
- Rnf complex