title: Primers for amplification of nitrous oxide reductase genes associated with Firmicutes and Bacteroidetes in organic-compound-rich soils.
abstract: The nosZ gene encodes nitrous oxide reductase, a key enzyme in the nitrous oxide reduction that occurs during complete denitrification. Many conventional approaches have used Proteobacteria-based primers to detect nosZ in environmental samples. However, these primers often fail to detect nosZ in non-Proteobacteria strains, including Firmicutes (Gram-positive) and Bacteroidetes. In this study, newly designed nosZ primers successfully amplified this gene from five Geobacillus species (Firmicutes). The primers were used to construct nosZ clone libraries from DNA extracted from sludge and domestic animal feedlot soils, all with high organic carbon contents. After DNA sequencing, phylogenetic analysis identified many new nosZ sequences with high levels of homology to nosZ from Bacteroidetes, probably because of the high sequence similarity of nosZ from Firmicutes and Bacteroidetes, and a predominance of Bacteroidetes in feedlot environments. Three sets of new quantitative real-time PCR (qPCR) primers based on our clone library sequences were designed and tested for their specificities. Our data showed that only Bacteroidetes-related nosZ sequences were amplified, whereas conventional Proteobacteria-based primers amplified only Proteobacteria-related nosZ. Quantitative analysis of nosZ with the new qPCR primers recovered ~10(4) copies per 100 ng DNA. Thus, it appears that amplification with conventional primers is insufficient for developing an understanding of the diversity and abundance of nosZ genes in the environment.
authors.affiliation: Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea.
authors.forename: Jaejoon
authors.initials: J
authors.lastname: Jung
authors.affiliation: Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea.
authors.forename: Sungjong
authors.initials: S
authors.lastname: Choi
authors.affiliation: Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea.
authors.forename: Hoon
authors.initials: H
authors.lastname: Jung
authors.affiliation: Department of Land, Air and Water Resources, University of California, Davis, CA 95616, USA.
authors.forename: Kate M
authors.initials: KM
authors.lastname: Scow
authors.affiliation: Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea.
authors.forename: Woojun
authors.initials: W
authors.lastname: Park
mesh_terms: D058491:Bacterial Load / Q000379:methods*; D041963:Bacteroidetes / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002244:Carbon / Q000032:analysis; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000032:analysis; D010088:Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012987:Soil / Q000737:chemistry; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D009930:Organic Chemicals; D012987:Soil; D002244:Carbon; D010088:Oxidoreductases; C022763:nitrous oxide reductase