Functional gene pyrosequencing and network analysis: an approach to examine the response of denitrifying bacteria to increased nitrogen supply in salt marsh sediments

Functional gene pyrosequencing is emerging as a useful tool to examine the diversity and abundance of microbes that facilitate key biogeochemical processes. One such process, denitrification, is of particular importance because it converts fixed nitrate (NO(−)(3)) to N(2) gas, which returns to the atmosphere. In nitrogen limited salt marshes, removal of NO(−)(3) prior to entering adjacent waters helps prevent eutrophication. Understanding the dynamics of salt marsh microbial denitrification is thus imperative for the maintenance of healthy coastal ecosystems. We used pyrosequencing of the nirS gene to examine the denitrifying community response to fertilization in experimentally enriched marsh plots. A key challenge in the analysis of sequence data derived from pyrosequencing is understanding whether small differences in gene sequences are ecologically meaningful. We applied a novel approach from information theory to determine that the optimal similarity level for clustering DNA sequences into OTUs, while still capturing the ecological complexity of the system, was 88%. With this clustering, phylogenetic analysis yielded 6 dominant clades of denitrifiers, the largest of which, accounting for more than half of all the sequences collected, had no close cultured representatives. Of the 638 OTUs identified, only 11 were present in all plots and no single OTU was dominant. We did, however, find a large number of specialist OTUs that were present only in a single plot. The high degree of endemic OTUs, while accounting for a large proportion of the nirS diversity in the plots, were found in lower abundance than the generalist taxa. The proportion of specialist taxa increased with increasing supply of nutrients, suggesting that addition of fertilizer may create conditions that expand the niche space for denitrifying organisms and may enhance the genetic capacity for denitrification.