Quantitative analyses of nitrogen cycling genes in soils

Soil nitrogen cycling is accomplished by a diverse microbial community using several nitrogen functional genes (NFGs). The relationship between the composition of these communities and nitrogen cycling processes is poorly understood. We developed techniques to quantify several important NFGs nitrogen transformations including fixation (nifH), ammonia oxidation (amoA), nitrogen reduction (nirS and nirK), and nitrous oxide reduction (nosZ) using quantitative real-time PCR with SybrGreen. Standard curves with known gene copies of each target molecule were developed using genomic DNA extracted from pure cultures. Primers from previously published studies were successfully used to amplify and quantify each NFGs in DNA extracted from two forest soils. Some soil samples inhibited PCR amplification of positive control DNA, and required further purification, which alleviated the problem. Two forest soils from Harvard Forest, MA and Duke Forest, NC differed greatly in their relative abundance of NFGs. The abundance of the amoA gene, associated with ammonia oxidation, was greater at Harvard Forest. The nifH gene, associated with N fixation, was more abundant at Duke Forest. These differences may reflect contrasting N cycling processes, however other published data do not indicate strong differences in gross N cycling rates. This technique can be widely adapted to quantify functional genes from a variety of complex environmental samples, and its utility could be further extended by amplifying mRNA using a reverse transcriptase-PCR procedure. (c) 2005 Elsevier GmbH. All rights reserved.