However, a sequencing effort in cultured strains of Acidobacteria recently found that these organisms possess NO3- and NO2- reducing genes [40]. Alphaproteobacteria[41], and likely Acidobacteria[40], are adapted to low nutrient conditions. While this seems counterintuitive to our microcosm study, vernal pools

in nature are known to be oligotrophic [7]. The Alphaproteobacteria and Acidobacteria in vernal pools, then, may be adapted to AR-13324 ic50 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 OSI-906 molecular weight in a previous examination of general bacteria or general fungi in these microcosms with TRFLP [17]. 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 [17] 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 learn more 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 Cytoskeletal Signaling inhibitor 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 [17]. Therefore, we expect community changes in response to the 30 day incubation to be minimal compared to the NO3- addition.