Metagenomic analysis revealed treatment differences both for functional and taxanomic EGTs between our +NO3- and –N metagenomes. These differences were apparent even though the metagenome sequencing conducted here returned a lower number of sequences than are typically reported for shotgun metagenome studies [20–22]. However, a shotgun metagenomic sequencing effort conducted by Fierer et al. , where comparable sequence numbers to ours are reported, was able to elucidate increases in functional genes with increased N fertilization, suggesting that our sequence numbers are adequate for determining relative metabolic and taxonomic changes.
A somewhat surprising result was no proportional abundance change in any of the N metabolism EGTs between our treatments with the BLASTX comparison to the SEED database. Particularly surprising was no change in the denitrification EGTs (determined with the BLASTX) between treatments and no detection of denitrification genes with the BLASTN, other than two sequence matches to nitrate reductase in the +NO3- treatment. The two sequence matches with the BLASTN in the +NO3- metagenome were to the nitrate reductase genes napA and napB. Because the periplasmic nitrate reductases, which are the products of napA and napB, are used in both denitrification and DNRA , no conclusions can be drawn on which of these microbial groups grew to a level where they could be detected in the +NO3- microcosms. This lack of EGT response was despite the fact that we observed denitrification rate responses to our treatments , where the microcosms receiving NO3- displayed a denitrification rate near or higher than the upper range of what has been measured in flooded soils in the field . This result is consistent with a number of other studies that have found no link between function (including measurements of denitrification rate and denitrifying enzyme activity) and denitrifier gene copy number using QPCR [13, 25–27]. We previously suggested that, in the absence of NO3- addition, denitrifiers in our microcosms used other electron acceptors for respiration when NO3- was not available , since denitrifiers are known to use other respiratory pathways [see review 10]. There were proportionally higher EGTs in the iron acquisition and metabolism category in the –N metagenome, and the specific EGT match was to a TonB-dependent receptor (Table 1). TonB-dependent receptors are a category of energy-coupling proteins, which are known to be involved in iron uptake by members of the genus Pseudomonas[28, 29], and there is some evidence that one specific TonB-dependent receptor is involved in dissimilatory iron reduction by Shewanella oneidensis. This suggests that the microbial community in the –N microcosms contained a greater number of organisms capable of acquiring iron and, perhaps, utilizing it for energy, which may have been a potential survival strategy in the absence of the NO3- addition. To our knowledge, evidence to support this hypothesis is sparse (but see Hauck et al. , who found that denitrifiers can also perform anaerobic ferrous iron oxidation). It is accepted, however, that denitrifying organisms primarily perform aerobic respiration and then switch to denitrification under anoxic conditions where NO3- supply is sufficient . There is a category available through MG-RAST for respiration genes. There were close to 400 EGT matches from the two metagenomes to this category for genes involved in both aerobic and anaerobic respiratory pathways. However, there were no proportional changes in respiration EGT abundance between the +NO3- and the –N conditions (data not shown), likely because the microcosms were made anoxic prior to the metagenome creation, which could negate any advantage to aerobic organisms in either treatment. Though we did not observe proportional changes for EGTs involved in a known alternative respiratory pathway for denitrifiers, the observed proportional increase in iron acquisition and metabolism EGTs in the –N metagenome suggests that iron might be biogeochemically important under anoxic N-limited conditions.
Another possible reason for lack of denitrifier EGT treatment response is that denitrifiers may have been in low abundance compared to other microbial groups, making changes to their population undetectable relative to the background population numbers. For example, the denitrification gene nosZ is known to be in low abundance compared to 16S genes , and there are estimates that only 0.1 – 5% of culturable soil bacterial species can carry out denitrification . This conclusion is supported by our BLASTN results, which found only two sequences from either metagenome that matched with a N metabolism gene. With the BLASTX comparison to the SEED database, however, over 1% of our sequences from each metagenome matched with nitrogen metabolism subsystems. The fact that we found no differences in nitrogen metabolism EGT relative abundance after NO3- addition suggests that microbial populations involved in N cycling did not shift in the 20 hours following exposure to a NO3- pulse. This lack of treatment response could be due to insufficient time between treatment initiation and sampling (i.e. populations were slow to respond to the treatment). However, we did see other EGT changes, suggesting that some microbial populations grew and experienced a detectable community shift in response to acute changes in NO3- concentration. The initial microbial community response to NO3- in our metagenomes was toward organisms that contained stress response, carbohydrate, and fatty acids, lipids, and isoprenoid EGT matches (Figure 1). The stress response EGT that was higher in the +NO3- metagenome was for an alkyl hydroperoxide reductase subunit C-like protein. The gene for alkyl hydroperoxide reducates, subunit C is upregulated by NO3- exposure after only 30 minutes in Desulfovibrio vulgaris, suggesting that such increases in this and other oxidative stress genes may be a general stress response by the bacteria . Within the carbohydrates category, one EGT match that was higher in the +NO3- metagenome was for fermentation. Recently, there has been evidence for fermentation that is coupled to NO3- reduction in both bacteria and fungi [36, 37]. Fermentation in the +NO3- microcosms may have been particularly prominent for the fungi, because a switch to NO3- -coupled fermentation as the primary source of energy for soil fungi under anoxic conditions has been suggested .
The sequencing effort described here also showed changes to the proportional representation of taxonomic EGTs. There were highly significant increases in the relative abundance of Alphaproteobacteria and Acidobacteria EGTs in the +NO3- metagenome. Similarly, using freshwater microcosms, Barlett and Leff  found an increase in Alphaproteobacteria abundance when NO3- was present as a N source and suggested a competitive advantage to this group of organisms under these conditions. Under anoxic conditions, such as our microcosms, higher physiological activity and substrate uptake have been reported in several Alphaproteobacteria species when NO3- or NO2- were present as an electron acceptor . Therefore, in our microcosms, there could have been a competitive advantage to the Alphaproteobacteria due to greater growth compared to other facultative organisms in an anoxic environment with abundant NO3-. To our knowledge, there have been no other studies that found such an increase in Acidobacteria with NO3- addition. However, a sequencing effort in cultured strains of Acidobacteria recently found that these organisms possess NO3- and NO2- reducing genes . Alphaproteobacteria, and likely Acidobacteria, are adapted to low nutrient conditions. While this seems counterintuitive to our microcosm study, vernal pools in nature are known to be oligotrophic . The Alphaproteobacteria and Acidobacteria in vernal pools, then, may be adapted to survival in the disturbed, low nutrient conditions of these habitats and once NO3- becomes readily available they have a competitive advantage due to their growth capabilities in the presence of NO3-.
These taxonomic changes were not found in a previous examination of general bacteria or general fungi in these microcosms with TRFLP . The metagenomic analysis reported here provides a greater resolution than TRFLP, which is a coarse community profiling tool. Therefore, there may have been fine-scale changes in bacterial community structure that were not detected with TRFLP. Another reason for this discrepancy is that our previous TRFLP analyses used the gene regions of bacterial 16S and fungal ITS for profiling  and, in the current study, a nonredundant protein database was used for taxonomic comparisons. Therefore, the conclusions drawn here regarding taxonomic changes may be limited to the taxonomic groups that changed functionally. The fact that whole genome amplification (WGA) was used prior to 454 sequencing could also be contributing to the differences seen between the metagenomes that were not noted with TRFLP. This is because amplification techniques with the Phi29 DNA polymerase, which was used in the current study, have been shown to exclude the amplification of certain DNA sequences, particularly those in low abundance or those that are GC rich, and can skew the representation of certain OTUs compared to sequencing efforts of non-amplified DNA of the same sample [42–44]. Additionally, our study design cannot exclude the possibility that the communities changed between the treatments over the 30 day incubation period prior to our sample collection. Thus, differences seen between the metagenomes may not be only because of the NO3- addition, but could also be due to an incubation period that changed the communities in the separate microcosms. There were six replicate microcosms to help control for variability between each jar, and our previous TRFLP profiling of the bacterial and fungal communities and the nosZ gene showed no differences in community structure between the +NO3- and –N microcosms . Therefore, we expect community changes in response to the 30 day incubation to be minimal compared to the NO3- addition. Nevertheless, the observed proportional increase in Alphaproteobacteria and Acidobacteria in response to NO3- addition in the metagenomes requires more in depth study on the ecology of these groups and how they tolerate NO3- pollution.