Overcoming urea and nitrate inhibition of biological nitrogen fixation in Azotobacter vinelandii versus natural tolerance in Gluconacetobacter diazotrophicus.

Nitrogen is often a limiting nutrient for agricultural crops. Most microbes also require an external fixed-nitrogen source for optimal growth. Even for microbes capable of biological nitrogen fixation (BNF), it is generally the case that they will regulate their metabolism to prioritize assimilation of fixed-nitrogen. Microbes employ various strategies and pathways to take advantage of available fixed-nitrogen compounds found in their natural environments. Azotobacter vinelandii is a model microbe for the study of BNF. Due to the high energetic cost of the process, BNF in A. vinelandii is repressed in the presence of ammonium, urea and nitrate. Prior studies indicated that strong inhibition of nitrogen fixation by urea and nitrate in A. vinelandii is actually the result of intracellular conversions of these metabolites into ammonium. In this study, we demonstrate a strategy to eliminate BNF inhibition by both urea and nitrate at concentrations ranging as high as 15 mM in a strain lacking the genes for urease and nitrate reductase, resulting in continued nitrogenase activity in the presence of these common fertilizer inputs. In contrast to the properties of A. vinelandii, the diazotroph Gluconacetobacter diazotrophicus naturally lacks these pathways, prompting the question of whether urea or nitrate inhibit BNF or support growth in G. diazotrophicus. To probe this observation, we developed experiments to demonstrate that while the presence of urea and nitrate delay the initial growth rate in G. diazotrophicus, nitrogenase activity and ammonium accumulation occurs at a similar rate in the presence of these metabolites. These results indicate that biological nitrogen fixation in G. diazotrophicus is somewhat insensitive to these nitrogen sources. This illustrates that alternative pathways in diazotrophic strains should be carefully considered in any efforts to optimize extracellular nitrogen production for biofertilizer applications and strain optimization, and that additional design strategies can be effective to assure that diazotrophs continue to fix nitrogen in the presence of specific nitrogen compounds common to industrial fertilizers.