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

Abstract

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.

FIG. 1

Generation of standard curve. (A) Increase of fluorescence intensity with cycle number for serially diluted P. stutzeri DNA. Symbols, from left to right: ▾, 17.8 ng; ●, 5.9 ng; ▴, 0.59 ng; ⧫, 59 pg; ■, 5.9 pg; ▾, 0.59 pg; , 59 fg; ○, no-template control. C_T, cycle at which the fluorescence intensity crosses an arbitrary threshold value. (B) Standard curve. Values represent means ± 95% confidence interval (n = 3).

FIG. 2

Limit of detection of the P. stutzeri nirS gene using real-time PCR. The denoted volumes of serially diluted P. stutzeri DNA were added to different reaction mixtures. (A) Low concentrations. (B) High concentrations. Values represent means ± 95% confidence interval (n = 3).

FIG. 3

Relationship between C_T and error. The horizontal line represents the MAE to discriminate between samples containing a twofold difference in P. stutzeri nirS copy number. Values shown represent the means from 14 independent standard curves. The 95% confidence interval is shown as a dashed line.

FIG. 4

Quantification of nirS DNA in artificial mixtures of P. stutzeri KC, P. aeruginosa, and E. coli cells. (A) Comparison between P. stutzeri nirS gene copies per μg of DNA measured values by real-time PCR (gray bars) and calculated values based on cell counts (black bars). (B) Correlation between calculated and measured values (slope = 0.98, r² = 0.992). Error bars represent 95% confidence intervals (n = 3) for both panels.

FIG. 5

Quantification of nirS in KBS soil (●) and Wintergreen Lake sediment (▴) samples spiked with P. stutzeri cells. The line shows the correlation between P. stutzeri nirS gene copies per microgram of DNA measured by real-time PCR and calculated values. Error bars represent 95% confidence intervals (n = 3).

FIG. 6

P. stutzeri nirS copy number measured by real-time PCR without (black bars) and with (gray bars) the addition of 10⁶ P. stutzeri nirS copy numbers to DNA extracted from the following environmental samples: KBS, soil from KBS (Mich.); SCH, groundwater from Schoolcraft bioremediation site (Mich.); SHI, groundwater from Shiprock (N.Mex.); WIN, freshwater sediment from Wintergreen Lake (Mich.); WAS, marine sediment from Washington margin (Wash.); PUG, marine sediment from Puget Sound (Wash.).