Pseudomonas stutzeri nitrite reductase gene abundance in environmental samples measured by real-time PCR

We used real-time PCR to quantify the denitrifying nitrite reductase gene ( nirS), a functional gene of biogeochemical significance. The assay was test ed in vitro and applied to environmental samples. The primer-probe set sele cted was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 10(6) gene co pies (r(2) = 0.999). Variability at low gene concentrations did not allow d etection of twofold differences in gene copy number at less than 100 copies . DNA spiking and cell-addition experiments gave predicted results, suggest ing that this assay provides an accurate measure of P. stutzeri nirS abunda nce in environmental samples. Although P. stutzeri abundance was high in la ke sediment and groundwater samples, we detected low or no abundance of thi s species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier.