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Title: Resource concentration modulates the fate of dissimilated nitrogen in a dual-pathway actinobacterium

Author
item VUONO, DAVID - Desert Research Institute
item READ, ROBERT - Desert Research Institute
item HEMP, JAMES - California Institute Of Technology
item SULLIVAN, BENJAMIN - University Of Nevada
item ARNONE, JOHN - Desert Research Institute
item NEVEUX, IVA - Desert Research Institute
item Blank, Robert - Bob
item STAUBE, CARL - Agtron, Inc
item LONEY, EVAN - Desert Research Institute
item MICELI, DAVID - Desert Research Institute
item WINKLER, MARI - University Of Washington
item CHAKRABORTY, ROMY - Lawrence Berkeley National Laboratory
item STAHL, DAVID - University Of Washington
item GRZYMSKI, JOSEPH - Desert Research Institute

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2019
Publication Date: 1/22/2019
Publication URL: https://handle.nal.usda.gov/10113/6471132
Citation: Vuono, D.C., Read, R.W., Hemp, J., Sullivan, B., Arnone, J.A., Neveux, I., Blank, R.R., Staube, C., Loney, E., Miceli, D., Winkler, M., Chakraborty, R., Stahl, D., Grzymski, J. 2019. Resource concentration modulates the fate of dissimilated nitrogen in a dual-pathway actinobacterium. Frontiers in Microbiology. 10:3. https://doi.org/10.3389/fmicb.2019.00003.
DOI: https://doi.org/10.3389/fmicb.2019.00003

Interpretive Summary: Carbon to nitrate (C:NO3-) ratios are thought to control pathway selection between respiratory ammonification and denitrification, two processes vital to the global N budget. However, the molecular mechanisms that enable the selection of these pathways remains unclear. Using Intrasporangium calvum C5, a gram-positive menaquinone-based dual-pathway denitrifier/respiratory ammonifier, we show that C:NO3- control theory is insufficient to explain pathways selection. The bacterium disproportionately utilizes ammonification rather than denitrification when grown under carbon or nitrate limitation, not C:NO3- ratio. The ammonification pathway also promoted higher bacterial growth rates. Although lactate was the only carbon source provided to the organism, we detected formate production during growth, a five-fold upregulation in formate transporters, and a simultaneous up-regulation of formate dehydrogenase used to translocate protons via a quinol-loop. These results suggest that additional reducing equivalents can be obtained from a single carbon source and used for ammonification during resource limitation. Mechanistically, pathway selection may be driven by intracellular redox potential. Our work advances our understanding of the conditions and underlying mechanisms that select for denitrification and respiratory ammonification in environmental systems.

Technical Abstract: Respiratory ammonification and denitrification are two evolutionarily unrelated dissimilatory nitrogen (N) processes central to the global N cycle, the activity of which is thought to be controlled by carbon (C) to nitrate (NO3-) ratio. Here we find that Intrasporangium calvum C5, a novel dual-pathway denitrifier/respiratory ammonifier,disproportionately utilizes ammonification rather than denitrification when grown under low C concentrations, even at low C:NO3- ratios. This finding is in conflict with the paradigm that high C:NO3- ratios promote ammonification and low C:NO3- ratios promote denitrification. We find that the protein atomic composition for denitrification modules (NirK) are significantly cost minimized for C and N compared to ammonification modules (NrfA), indicating that limitation for C and N is a major evolutionary selective pressure imprinted in the architecture of these proteins. The evolutionary precedent for these findings suggests ecological importance for microbial activity as evidenced by higher growth rates when I. calvum grows predominantly using its ammonification pathway and by assimilating its end-product (ammonium) for growth under ammoniumfree conditions. Genomic analysis of I. calvum further reveals a versatile ecophysiology to cope with nutrient stress and redox conditions. Metabolite and transcriptional profiles during growth indicate that enzyme modules, NrfAH and NirK, are not constitutively expressed but rather induced by nitrite production via NarG. Mechanistically, our results suggest that pathway selection is driven by intracellular redox potential (redox poise), which may be lowered when resource concentrations are low, thereby decreasing catalytic activity of upstream electron transport steps (i.e., the bc1 complex) needed for denitrification enzymes. Our work advances our understanding of the biogeochemical flexibility of N-cycling organisms, pathway evolution, and ecological food-webs.