title: Visualization and direct counting of individual denitrifying bacterial cells in soil by nirK-targeted direct in situ PCR.
abstract: The abundance of denitrifying bacteria in soil has been determined primarily by the conventional most probable number (MPN) method. We have developed a single-cell identification technique that is culture-independent, direct in situ PCR, to enumerate denitrifying bacteria in soils. The specificity of this method was evaluated with six species of denitrifying bacteria using nirK as the target gene; Escherichia coli was used as a negative control. Almost all (97.3%-100%) of the nirK-type denitrifying bacteria (Agromonas oligotrophica, Alcaligenes faecalis, Achromobacter denitrificans, Bradyrhizobium japonicum, and Pseudomonas chlororaphis) were detected by direct in situ PCR, whereas no E. coli cells and only a few cells (2.4%) of nirS-type denitrifying bacteria (Pseudomonas aeruginosa) were detected. Numbers of denitrifying bacteria in upland and paddy soil samples quantified by this method were 3.3 × 10(8) to 2.6 × 10(9) cells g(-1) dry soil. These values are approximately 1,000 to 300,000 times higher than those estimated by the MPN method. These results suggest that direct in situ PCR is a better tool for quantifying denitrifying bacteria in soil than the conventional MPN method.
authors.affiliation: Division of Soil Science, Faculty of Agriculture, Saga University, Saga 840–8502, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Ryuda
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Hashimoto
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ueno
authors.forename: Koichi
authors.initials: K
authors.lastname: Inoue
authors.forename: Takashi
authors.initials: T
authors.lastname: Someya
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000235:genetics* / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology
chemical_list: D001426:Bacterial Proteins; D009573:Nitrites; D009572:Nitrite Reductases