Primers for amplification of nitrous oxide reductase genes associated with firmicutes and bacteroidetes in organic-compound-rich soils

Jaejoon Jung, Sungjong Choi, Hoon Jung, Kate M. Scow, Woojun Park

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 ~104 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.

Original languageEnglish
Pages (from-to)307-315
Number of pages9
JournalMicrobiology (United Kingdom)
Volume159
Issue number2
DOIs
Publication statusPublished - 2013 Feb 1

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Bacteroidetes
Soil
Proteobacteria
Genes
Geobacillus
Clone Cells
Denitrification
Domestic Animals
Nitrous Oxide
Sewage
Gene Library
DNA Sequence Analysis
Real-Time Polymerase Chain Reaction
Carbon
Firmicutes
nitrous oxide reductase
DNA
Enzymes

ASJC Scopus subject areas

  • Microbiology

Cite this

Primers for amplification of nitrous oxide reductase genes associated with firmicutes and bacteroidetes in organic-compound-rich soils. / Jung, Jaejoon; Choi, Sungjong; Jung, Hoon; Scow, Kate M.; Park, Woojun.

In: Microbiology (United Kingdom), Vol. 159, No. 2, 01.02.2013, p. 307-315.

Research output: Contribution to journalArticle

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