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Background  Human activities have greatly increased nitrogen (N) levels in natural habitats through atmospheric N deposition and nutrient leaching, which can have large effects on N cycling and other ecosystem processes. Because of the significant role microorganisms play in N cycling, high inputs of nitrogenous compounds, such as nitrate (NO 3 -), into natural ecosystems could have cascading effects on microbial community structure and the metabolic processes that microbes perform. To investigate the multiple effects of NO 3 - pollution on microbial communities, we created two shotgun metagenomes from vernal pool microcosms that were either enriched with a solution of 10 mg NO 3 --N (+NO 3 -) or received distilled water as a control (−N).    Results  After only 20 hours of exposure to NO 3 -, the initial microbial community had shifted toward one containing a higher proportional abundance of stress tolerance and fermentation environmental gene tags (EGTs). Surprisingly, we found no changes to N metabolism EGTs, even though large shifts in denitrification rates were seen between the +NO 3 - and –N microcosms. Thus, in the absence of NO 3 - addition, it is plausible that the microbes used other respiratory pathways for energy. Respiratory pathways involving iron may have been particularly important in our –N microcosms, since iron acquisition EGTs were proportionally higher in the –N metagenome. Additionally, we noted a proportional increase in  Acidobacteria  and  Alphaproteobacteria  EGTs in response to NO 3 - addition. These community shifts in were not evident with TRFLP, suggesting that metagenomic analyses may detect fine-scale changes not possible with community profiling techniques.    Conclusions  Our results suggest that the vernal pool microbial communities profiled here may rely on their metabolic plasticity for growth and survival when certain resources are limiting. The creation of these metagenomes also highlights how little is known about the effects of NO 3 - pollution on microbial communities, and the relationship between community stability and function in response to disturbance.

Shotgun metagenomic analysis of metabolic diversity and microbial community structure in experimental vernal pools subjected to nitrate pulse