GENE EXPRESSION AS A PROXY FOR SUBSURFACE MICROBIAL RESPIRATION RATES: DENITRIFICATION AND THE NIRS GENE

Quantifiable levels of expressed functional genes may serve as proxies for rates of the reactions facilitated by those gene products. In this study, the rate of denitrification by subsurface microorganisms has been correlated with expression levels of the rate limiting nitrite reductase gene nirS. This novel technique uses the Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) to measure the number of copies of the nirS gene expressed in a sample, and correlates that level with known denitrification rates in a laboratory setting to build a calibration curve that can then be applied to environmental samples. This technique bypasses many of the usual limitations of quantifying subsurface microbial respiration rates. It does not depend on the diffusion of any substance to an active site, has no need of recovery of tracers, and can measure an instantaneous rate on a single sample of a solid matrix or extracted pore water without the need for repeated sampling. For the purposes of this study, the model organism Paracoccus denitrificans was grown in anaerobic batch culture on defined media. Denitrification rates were determined by mass balance of nitrate depletion, and RT-qPCR was carried out using primers and fluorogenic probes specifically designed for the NirS gene. Nitrate depletion was measured as a function of temperature and concentration to test the robustness of correlation between gene expression and denitrification rate under varying conditions.