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

Abstract

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 (NO3-), into natural ecosystems could have cascading effects on microbial community structure and the metabolic processes that microbes perform. To investigate the multiple effects of NO3- pollution on microbial communities, we created two shotgun metagenomes from vernal pool microcosms that were either enriched with a solution of 10 mg NO3--N (+NO3-) or received distilled water as a control (-N).

Results: After only 20 hours of exposure to NO3-, 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 +NO3- and -N microcosms. Thus, in the absence of NO3- 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 NO3- 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 NO3- pollution on microbial communities, and the relationship between community stability and function in response to disturbance.

Figure 1

Subsystem matches in the nitrogen metabolism category. The proportional numbers of environmental gene tags that matched with level 2 sequences within the nitrogen metabolism subsystem category for the +NO₃- (solid bars) and –N (open bars) metagenomes. No significant differences were found when these sequences were analyzed with Fisher exact tests in the Statistical Analysis of Metagenomic Profiles program.

Figure 2

Significant subsystem differences between the +NO₃- and –N metagenomes. Results of a Fisher exact test (conducted with the Statistical Analysis of Metagenomic Profiles program) showing the significant differences of subsystem environmental gene tag (EGT) matches between treatments. Higher EGT relative abundance in the +NO₃- metagenome have a positive difference between proportions (closed circles), while higher EGT relative abundance in the –N metagenome have a negative difference between proportions (open circles).

Figure 3

Significant phylum differences between the +NO₃- and –N metagenomes. Results of a Fisher exact test (conducted with the Statistical Analysis of Metagenomic Profiles program) showing the significant differences of environmental gene tag (EGT) matches to phyla between treatments. Higher EGT relative abundance in the +NO₃- metagenome have a positive difference between proportions (closed circles), while higher EGT relative abundance in the –N metagenome have a negative difference between proportions (open circles).

Figure 4

Significant class differences in the domain bacteria between the +NO₃- and –N metagenomes. Results of a Fisher exact test (conducted with the Statistical Analysis of Metagenomic Profiles program) showing the significant differences of environmental gene tag (EGT) matches to class between treatments. Higher EGT relative abundance in the +NO₃- metagenome have a positive difference between proportions (closed circles), while higher EGT relative abundance in the –N metagenome have a negative difference between proportions (open circles).