title: Higher diversity and abundance of denitrifying microorganisms in environments than considered previously.
abstract: Denitrification is an important process in the global nitrogen cycle. The genes encoding NirK and NirS (nirK and nirS), which catalyze the reduction of nitrite to nitric oxide, have been used as marker genes to study the ecological behavior of denitrifiers in environments. However, conventional polymerase chain reaction (PCR) primers can only detect a limited range of the phylogenetically diverse nirK and nirS. Thus, we developed new PCR primers covering the diverse nirK and nirS. Clone library and qPCR analysis using the primers showed that nirK and nirS in terrestrial environments are more phylogenetically diverse and 2-6 times more abundant than those revealed with the conventional primers. RNA- and culture-based analyses using a cropland soil also suggested that microorganisms with previously unconsidered nirK or nirS are responsible for denitrification in the soil. PCR techniques still have a greater capacity for the deep analysis of target genes than PCR-independent methods including metagenome analysis, although efforts are needed to minimize the PCR biases. The methodology and the insights obtained here should allow us to achieve a more precise understanding of the ecological behavior of denitrifiers and facilitate more precise estimate of denitrification in environments.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Isobe
authors.affiliation: 1] Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan [2] Department of Bioresource Science, College of Agriculture, Ibaraki University, Ibaraki, Japan.
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Nishizawa
authors.affiliation: Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Harbin, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Niigata Agricultural Research Institute, Niigata,, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Shiratori
authors.affiliation: Department of Forest Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Nobuhito
authors.initials: N
authors.lastname: Ohte
authors.affiliation: Institute of Agriculture, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Koba
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Shigeto
authors.initials: S
authors.lastname: Otsuka
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Senoo
mesh_terms: D039903:Actinobacteria / Q000235:genetics; D001419:Bacteria / Q000235:genetics* / Q000378:metabolism; D041963:Bacteroidetes / Q000235:genetics; D041862:Chloroflexi / Q000235:genetics; D017931:DNA Primers; D058440:Denitrification; D016013:Likelihood Functions; D054892:Metagenome; D009572:Nitrite Reductases / Q000235:genetics*; D009573:Nitrites / Q000378:metabolism; D010802:Phylogeny; D020560:Proteobacteria / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012987:Soil / Q000737:chemistry; D012988:Soil Microbiology; D013144:Spirochaetales / Q000235:genetics
chemical_list: D017931:DNA Primers; D009573:Nitrites; D012987:Soil; D009572:Nitrite Reductases