Nitrous oxide (N 2 O) 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 N 2 O 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.Nitrous oxide (N 2 O), with a global warming potential approximately 300 times higher than that of carbon dioxide, is an important greenhouse gas, contributing up to 6% of global warming. N 2 O also participates in depletion of the stratospheric ozone layer through stratospheric nitric oxide (NO) production. At present, the N 2 O concentration in the atmosphere is increasing at a rate of about 0.3% per year. The soil is the dominant source of atmospheric nitrous oxide, contributing about 57% (9 Tg year Ϫ1 ) of the total annual global emission (12). N 2 O emissions are highly variable in soils and are primarily produced by biological nitrification and denitrification, although the latter is considered to be the main source (31). N 2 O is an intermediate product in the denitrification pathway, which consists of the sequential reduction of NO 3 Ϫ to N 2 via the metalloenzymes nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase (32). Therefore, N 2 O emission by denitrification is the net result of the balance between production and reduction of N 2 O by denitrifying bacteria. The N 2 O reductase (EC 1.7.99.6) is a homodimeric multicopper enzyme, which has been purified from numerous gram-negative denitrifiers but not yet from a gram-positive bacterium (4,5,11,17,27). Production of N 2 O by denitrifying isolates as an end product of denitrification has been reported by several authors (2, 3, 7). Sequencing of the complete genome of Agrobacterium tumefaciens C58 revealed the presence of a denitrification cluster with genes encoding the periplasmic nitrate reductase, the copper nitrite reductase...

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To determine to which extent root-derived carbon contributes to the effects of plants on nitrate reducers and denitrifiers, four solutions containing different proportions of sugar, organic acids and amino acids mimicking maize root exudates were added daily to soil microcosms at a concentration of 150 microg C g(-1) of soil. Water-amended soils were used as controls. After 1 month, the size and structure of the nitrate reducer and denitrifier communities were analysed using the narG and napA, and the nirK, nirS and nosZ genes as molecular markers respectively. Addition of artificial root exudates (ARE) did not strongly affect the structure or the density of nitrate reducer and denitrifier communities whereas potential nitrate reductase and denitrification activities were stimulated by the addition of root exudates. An effect of ARE composition was also observed on N(2)O production with an N(2)O:(N(2)O + N(2)) ratio of 0.3 in microcosms amended with ARE containing 80% of sugar and of 1 in microcosms amended with ARE containing 40% of sugar. Our study indicated that ARE stimulated nitrate reduction or denitrification activity with increases in the range of those observed with the whole plant. Furthermore, we demonstrated that the composition of the ARE affected the nature of the end-product of denitrification and could thus have a putative impact on greenhouse gas emissions.

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The plasmid pADP1::Tn5 derived from pADP1[Atr+] carrying a Tn5 transposon conferring kanamycin and streptomycin resistances was constructed and introduced in Agrobacterium tumefaciens St96-4. This genetically modified strain was inoculated (approximately 10(8) cfu g(-1)) in potted soils planted with maize and treated or not with atrazine (1.5 mg kg(-1)). Bulk and maize rhizosphere soils were sampled 39 days after planting to look for soil indigenous bacteria that had acquired pADP1::Tn5. Four transconjugants were isolated from four different soil samples. The estimated transfer frequency of pADP1::Tn5 was 10(-4) per donor. Maize rhizosphere and atrazine treatment had no obvious effect on pADP1::Tn5 transfer frequency. The sequencing of the 16S rDNA sequences of the transconjugants revealed that they were almost identical and highly similar to that of Variovorax spp (97%). In addition, their characterization suggested that the atzA and atzB genes had been transferred from pADP1::Tn5 to the bacterial chromosome in two of the four transconjugants. These data suggest that the atz degrading genes are horizontally transferred in soil and possibly subjected to gene rearrangement.