Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soils

Abstract

Nitrous oxide (N2O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N2O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 10(5) to 10(7) target copies g(-1) of dry soil, whereas genes for 16S rRNA were found at 10(8) to 10(9) target copies g(-1) of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.

FIG. 1.

Sequences and binding positions of the nosZ1 and nosZ2 primers based on the nosZ sequence from Pseudomonas fluorescens (accession number AF197478). Degenerated sites are in boldface (W = AT, S = CG, Y = CT, M = AC, R = AG, K = GT, and V = ACG).

FIG. 2.

Standard curves of the nosZ1 and nosZ2 assays obtained by plotting the concentration of control DNA versus the cycle number required to elevate the fluorescence signal above the threshold.

FIG. 3.

Melting-curve profiles for the amplicons of environmental DNA obtained by nosZ1 (a) and nosZ2 (b) primer sets.

FIG. 4.

16S rRNA, narG, nirK, and nosZ (obtained with nosZ1 and nosZ2 assays) copy numbers g⁻¹ of dry soil in the different soils. Error bars indicate standard errors of the two independent PCRs of the three replicate DNA extractions. Significantly different values (P < 0.05) between different genes in the same soil are marked by capital letters (A to C) under the columns, and significantly different values (P < 0.05) between different soils for the same gene are marked by lowercase letters (a to c).

FIG. 5.

Phylogenetic tree of nosZ quantitative PCR products obtained with the nosZ1 primers and of nosZ genes from reference strains. Fragments of 259 nucleotides from the quantitative PCR products and from reference strains were used to calculate the tree. The tree is based on distance matrix analysis and the neighbor-joining method. Bootstrap values greater than 700 from 1,000 replicate trees are reported at the nodes. The sequence of nosZ from Wolinella succinogenes served as an outgroup to root the tree.

FIG. 6.

Phylogenetic tree of nosZ quantitative PCR products obtained with the nosZ2 primers and of nosZ genes from reference strains. Fragments of 267 nucleotides from the quantitative PCR products and from reference strains were used to calculate the tree. The tree is based on distance matrix analysis and the neighbor-joining method. Bootstrap values greater than 700 from 1,000 replicate trees are reported at the nodes. The sequence of nosZ from Wolinella succinogenes served as an outgroup to root the tree.