Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR

Denitrification, the reduction of nitrate to nitrous oxide or dinitrogen, is the major biological mechanism by which fixed nitrogen returns to the atmosphere from soil and water. Microorganisms capable of denitrification are widely distributed in the environment but little is known about their abundance since quantification is performed using fastidious and time-consuming MPN-based approaches. We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirK), a key enzyme of the denitrifying pathway catalyzing the reduction of soluble nitrogen oxide to gaseous form. The real-time PCR assay was linear over 7 orders of magnitude and sensitive down to 10(2) copies by assay. Real-time PCR analysis of different soil samples showed nirK densities of 9.7x10(4) to 3.9x10(6) copies per gram of soil. Soil real-time PCR products were cloned and sequenced. Analysis of 56 clone sequences revealed that all cloned real-time PCR products exhibited high similarities to previously described nirK. However, phylogenetic analysis showed that most of environmental sequences are not related to nirK from cultivated denitrifiers.