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

Gruntzig, V., Nold, S.C., Zhou, J., Tiedje, J.M.
Applied and environmental microbiology 2001 v.67 no.2 pp. 760-768
Pseudomonas, nitrate reductase, genes, polymerase chain reaction, agricultural soils, groundwater, mine spoil, uranium, marine environment
We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 10(6) gene copies (r2 = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this 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.