Molecular Analysis of the Nitrate-Reducing Community from Unplanted and Maize-Planted Soils

Abstract

Microorganisms that use nitrate as an alternative terminal electron acceptor play an important role in the global nitrogen cycle. The diversity of the nitrate-reducing community in soil and the influence of the maize roots on the structure of this community were studied. The narG gene encoding the membrane bound nitrate reductase was selected as a functional marker for the nitrate-reducing community. The use of narG is of special interest because the phylogeny of the narG gene closely reflects the 16S ribosomal DNA phylogeny. Therefore, targeting the narG gene provided for the first time a unique insight into the taxonomic composition of the nitrate-reducing community in planted and unplanted soils. The PCR-amplified narG fragments were cloned and analyzed by restriction fragment length polymorphism (RFLP). In all, 60 RFLP types represented by two or more clones were identified in addition to the 58 RFLP types represented by only one clone. At least one clone belonging to each RFLP type was then sequenced. Several of the obtained sequences were not related to the narG genes from cultivated bacteria, suggesting the existence of unidentified nitrate-reducing bacteria in the studied soil. However, environmental sequences were also related to NarG from many bacterial divisions, i.e., Actinobacteria and ␣, ␤, and ␥ Proteobacteria. The presence of the plant roots resulted in a shift in the structure of the nitrate-reducing community between the unplanted and planted soils. Sequencing of RFLP types dominant in the rhizosphere or present only in the rhizosphere revealed that they are related to NarG from the Actinobacteria in an astonishingly high proportion.Nitrate-reducing prokaryotes constitute a wide group with members among the ␣, ␤, and ␥ Proteobacteria, gram-positive Bacteria, and even Archea, sharing the ability to obtain energy from dissimilatory reduction of nitrate into nitrite. The nitrite can then be reduced into gaseous nitrogen compounds by denitrification or into NH 4 by dissimilatory nitrate reduction into ammonia. Denitrification is the dominant process in soils and lake sediment, whereas dissimilatory nitrate reduction into ammonia occurs mainly in the rumen and in digested sludge (30). Due to nitrogen losses in agricultural soils and production of the greenhouse gases NO and N 2 O, denitrification has received a lot of attention over the last 20 years (7). There are two distinct forms of dissimilatory nitrate reductase; a periplasmic nitrate reductase and a membrane-bound nitrate reductase (reviewed in references 16 and 22). Only the nitrate reductase is found universally in prokaryotes and can therefore be used as a functional marker for these microorganisms. In addition, many microorganisms can contain both nitrate reductases (22). The membrane-bound nitrate reductase is composed of a soluble ␤ subunit containing four [Fe-S] clusters and the ␥ subunit containing two b-type hemes. The last subunit, encoded by the narG gene, contains a [4Fe-4S] cluster and a molybdopterin guanine dinucleotide cofact...

Effect of pyrene on denitrification activity and abundance and composition of denitrifying community in an agricultural soil

“…However, this did not mean that these nirKharboring denitrifiers belonged to Rhizobiales or Burkholderiales, since the nirK phylogeny was incompatible with the 16S rDNA phylogeny (Heylen et al, 2006;Jones et al, 2008;Philippot et al, 2002;Yoshida et al, 2009). Our results showed that over 80% of NirS clones distributed in clusters I and VI (unknown branch), cluster II (Rhodobacterales).…”

“…However, Saito et al (2008) reported that NirS clones related to Burkholderiales and Rhodocyclales dominated in the rice paddy soil (Saito et al, 2008). A possible reason was that plant communities or the rhizosphere and soil factors could affect the structure and activity of the denitrifying community (Kowalchuk et al, 2002;Philippot et al, 2002). Based on the nosZ clone library analysis, the overwhelming majority of clones (cluster IeIII, and cluster VeVII) were related to Rhizobiales (Brucellaceae, Rhizobiaceae and Bradyrhizobiaceae) and Burkholderiales (Alcaligenaceae, Burkholderiaceae and Oxalobacteraceae) and only few clones in cluster IV were related to Rhodospirillales (Rhodospirillaceae), and the NosZ-based denitrifiers composition from our studied soil was similar to that from other farmland soils (Enwall et al, 2005;Horn et al, 2006).…”

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

“…Only few non-target sequences occurred and were excluded from further analyses; 95±7% of sequences generated from amplicons obtained with a certain structural gene-specific (that is, narG, nirK, nirS or nosZ) primer set were related to publicly available target genes of that primer set. Such amplification specificity is above or in the range of values obtained for other structural gene marker analyses (for example, average of 87±11% for narG, dsrAB, [FeFe]-hydrogenase and dioxygenase genes; Philippot et al, 2002;Loy et al, 2004;Iwai et al, 2010;Schmidt et al, 2010).…”

“…narG, nirK, nirS and nosZ were amplified using the primer pairs narG1960f (TAYGTSGGSCARGAR-AA)/narG2650r (TTYTCRTACCABGTBGC; Philippot et al, 2002), F1aCu (ATCATGGTSCTGCCGCG)/ R3Cu (GCCTCGATCAGRTTGTGGTT; Throbäck et al, 2004), cd3aF (GTSAACGTSAAGGARACSG G)/R3cd (GASTTCGGRTGSGTCTTGA; Throbäck et al, 2004) and nosZF (CGCTGTTCITCGACAGYC AG)/nosZR (ATGTGCAKIGCRTGGCAGAA; Rich et al, 2003), respectively. Each primer was preceeded by a 6-bp-long barcode (AGCGTC for unturbated and ATATAC for cryoturbated soil samples) to separate sequences after pyrosequencing.…”

“…Organisms hosting both types of nitrite reductase are unknown to date (Heylen et al, 2006). The genes coding for the above-named oxidoreductases are commonly used as structural gene markers for the analysis of nitrate reducer and denitrifier communities (Braker et al, 2000;Philippot et al, 2002;Prieme et al, 2002;Rich et al, 2003;Horn et al, 2006;Enwall et al, 2010;Jones and Hallin, 2010;Palmer et al, 2010;Bru et al, 2011). The main products of denitrification that are released into the atmosphere are N 2 or N 2 O. Denitrifiers might lack nitrate reductases and/or N 2 O reductases, and occupy diverse ecological niches (Tiedje, 1988;Zumft, 1997;Shapleigh, 2006).…”