Relative Abundances of Proteobacterial Membrane-Bound and Periplasmic Nitrate Reductases in Selected Environments

Abstract

Dissimilatory nitrate reduction is catalyzed by a membrane-bound and a periplasmic nitrate reductase. We set up a real-time PCR assay to quantify these two enzymes, using the narG and napA genes, encoding the catalytic subunits of the two types of nitrate reductases, as molecular markers. The narG and napA gene copy numbers in DNA extracted from 18 different environments showed high variations, with most numbers ranging from 2 ؋ 10 2 to 6.8 ؋ 10 4 copies per ng of DNA. This study provides evidence that, in soil samples, the number of proteobacteria carrying the napA gene is often as high as that of proteobacteria carrying the narG gene. The high correlation observed between narG and napA gene copy numbers in soils suggests that the ecological roles of the corresponding enzymes might be linked.Nitrate in the environment can be either assimilated by plants and microorganisms or reduced to nitrite by one of two microbial dissimilatory processes: denitrification or dissimilatory reduction of nitrate to ammonium. Nitrate reduction by denitrification is of great importance since the produced nitrite is then reduced to N 2 O or N 2 gases, which can lead to considerable nitrogen losses in agriculture and emissions of greenhouse gases (6,13,28). The reduction of nitrate present in contaminated water caused by the nitrate-reducing bacteria living in the human digestive tract is a potential health problem. As nitrite enters the bloodstream, it reacts with hemoglobin to form methemoglobin, blocking oxygen transport and causing a disease commonly called "blue baby syndrome" (26). Two types of dissimilatory nitrate reductase, differing in their locations, were characterized: a membrane-bound (Nar) and a periplasmic (Nap) nitrate reductase (2,16,29). The membrane-bound nitrate reductase is present in proteobacteria, firmicutes, actinobacteria, and even archaea, whereas the periplasmic nitrate reductase is present only in proteobacteria (21, 24). Nitrate-reducing proteobacteria can harbor Nar or Nap or both (18,29). In contrast to that of Nar, the physiological role of Nap is still unclear and seems to differ between bacteria (11, 24). Thus, one proposed role for Nap is to support anaerobic metabolism as an alternative to Nar (1, 8). It has also been proposed that Nap facilitates the switch from aerobic respiration to denitrification (27) or scavenges nitrate in some pathogenic bacteria (23). The importance and diversity of the bacteria containing Nar have been extensively studied using both cultivation-based and direct molecular approaches (3,5,7,17,19,22). However, only a few studies have focused on bacteria containing Nap (4, 9, 25). In this study, we investigated the relative abundances of the two types of nitrate reductases in various environments, using a real-time PCR-based assay.Primer design, standard curves, and real-time PCR procedures. In order to quantify the two types of nitrate reductases, a new real-time PCR assay was set up, using the narG and napA genes, encoding the catalytic subunits of the membranebound and...

Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra

“…Pyrosequencing and quantitative kinetic real-time PCR of denitrification-associated genes substantiated the previous conclusion (Figure 4, Tables 4 and 5). narG copy numbers of cryoturbated peat soil were higher than or in the same range as in agricultural soils or glacier forelands Kandeler et al, 2006;Bru et al, 2007), and significantly higher than in unturbated peat soil (Table 5). Such findings are in agreement with the high in situ concentrations of nitrate in cryoturbated peat soil (Repo et al, 2009), and the inability of unturbated peat soil to cope with high nitrate concentrations (Figure 2).…”

Nitrogen cycling processes and microbial community composition in bed sediments in the Yukon River at Pilot Station

“…Reaction protocols and primers for the analyzed genes have been described previously [Bru et al, 2007;Henry et al, 2006;Rotthauwe et al, 1997]. Melting curve analyses confirmed fluorescent signals resulted from specific PCR products rather than primer dimers or other artifacts.…”

Determination of Denitrification Genes Abundance in Environmental Samples

“…The method has been unanimously approved by the ISO as an international standard method (ISO standard 11063) [80]. The method has also been used to extract DNA from river sediments and agricultural soil, waters, biofilms and glacier soils [10,11], polychlorinated biphenyls-contaminated sites [79], constructed wetlands [21], and technosols [40]. Essentially, the method is as follows.…”

“…The nirK, nirS, and nosZ genes were the first denitrification genes to be partially amplified [7,42,70,101,133]; and primers were also developed for amplification of the narG, napA, and nosB genes [8,17,31,37,85]. With the exponential increase in the databases of the amount of DNA sequences corresponding to denitrification genes, new primers were developed with broader amplification range, including nirK [45], nirS [113], nosZ [46], narG [59], and napA [11]. A list of commonly used PCR primers for denitrification genes has been published [41].…”