Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating Pseudomonas in the Rhizosphere of Perennial Grasses Grown in Field Conditions

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221

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...

supporting

confidence: 93%

mentioning

confidence: 99%

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

Bru

Sarr

Philippot

2007

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221

“…These results also suggest that the napA community was more affected by the type and amount of plant residues than the narG community. This difference between the effects of residues on the napA and narG genes can be explained by the fact that the narG and napA communities do not fully overlap, since nitrate reducers can possess either Nar, Nap, or both (47). In contrast to our results, no significant difference in denitrifier abundance between soils amended with soybean, red clover, and barley residues was observed previously by Henderson et al (21).…”

Section: Vol 76 2010 Impact Of Plant Residue Types On Nitrate Reduccontrasting

confidence: 99%

“…Two types of dissimilatory nitrate reductases, differing in location, have been characterized: a membranebound nitrate reductase (Nar) and a periplasmic nitrate reductase (Nap) (9,53). Nitrate reducers can harbor either Nar, Nap, or both (40,47). Nitrate reducers are probably the most taxonomically diverse functional community within the nitrogen cycle, with members in most bacterial phyla and also archaea (42).…”

mentioning

confidence: 99%

Role of Plant Residues in Determining Temporal Patterns of the Activity, Size, and Structure of Nitrate Reducer Communities in Soil

Chèneby

Bru

Pascault

et al. 2010

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The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20-to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.

“…However, since potential nitrate reductase activities are expressed per gram of soil, only the gene copy numbers per gram of soil were used for further analyses of the relationships between the size, structure, and activity of the nitrate reducer community. Besides, while most nitrate reducing bacteria possess either narG or napA, a significant proportion of nitrate reducing bacteria also possess both narG and napA (39,47), which makes it difficult to convert the narG and napA gene copy numbers into nitrate reducer cell numbers. Therefore, we used the narG and napA real-time PCR data separately but also the sum of the narG and napA gene copy numbers to investigate relationships between the size, structure, and activity of the nitrate reducer community.…”

Section: Discussionmentioning

confidence: 99%

Differential Responses of Nitrate Reducer Community Size, Structure, and Activity to Tillage Systems

Chèneby

Brauman²,

Rabary

et al. 2009

Self Cite

The main objective of this study was to determine how the size, structure, and activity of the nitrate reducer community were affected by adoption of a conservative tillage system as an alternative to conventional tillage. The experimental field, established in Madagascar in 1991, consists of plots subjected to conventional tillage or direct-seeding mulch-based cropping systems (DM), both amended with three different fertilization regimes. Comparisons of size, structure, and activity of the nitrate reducer community in samples collected from the top layer in 2005 and 2006 revealed that all characteristics of this functional community were affected by the tillage system, with increased nitrate reduction activity and numbers of nitrate reducers under DM. Nitrate reduction activity was also stimulated by combined organic and mineral fertilization but not by organic fertilization alone. In contrast, both negative and positive effects of combined organic and mineral fertilization on the size of the nitrate reducer community were observed. The size of the nitrate reducer community was a significant predictor of the nitrate reduction rates except in one treatment, which highlighted the inherent complexities in understanding the relationships the between size, diversity, and structure of functional microbial communities along environmental gradients.