Diversity and abundance of Bacteria and nirS-encoding denitrifiers associated with the Juan de Fuca Ridge hydrothermal system

Denitrification, which results in the loss of bioavailable nitrogen-an essential macronutrient for all living organisms-may potentially affect chemosynthetic primary production in hydrothermal vent ecosystems where sub-oxic conditions favorable to denitrification are common. Here we describe the diversity and abundance of denitrifying bacteria in the subsurface biosphere at Axial Volcano and the Endeavour Segment on the Juan de Fuca Ridge using a combination of quantitative polymerase chain reaction assays, and small subunit ribosomal RNA (SSU or 16S rRNA) pyrotag and nitrite reductase (nirS) clone library sequencing methods. Bacterial communities were diverse and dominated by members of the epsilon- and gamma-proteobacteria, including taxonomic groups containing known denitrifiers. Assemblages of denitrifiers that could be evaluated by nirS gene sequence comparisons showed low diversity. The single nirS sequence shared by the two locations, affiliated with a gamma-proteobacteria isolated from estuarine sediments (Pseudomonas sp. BA2), represented more than half of all sequences recovered when clustered at 97 {%} identity. All other nirS sequences clustered into different taxonomic groups, indicating important differences in denitrifier community membership between the two sites. Total nirS gene abundance was at least two orders of magnitude lower than 16S rRNA abundance. Overall, our results demonstrate that the diversity and abundance of the nirS gene-containing bacterial community are rather low, as might be expected under the extreme conditions encountered in the subsurface biosphere of hydrothermal vent systems, and do not correlate clearly with any environmental variables investigated (i.e., pH, temperature, and H2S, NO3-, NH4+ concentrations).