Home LiteratureArticle Details
PMID: 14711647 Published · ppublish English Comparative Study Evaluation Study Journal Article Research Support, Non-U.S. Gov't

New molecular screening tools for analysis of free-living diazotrophs in soil.

Applied and environmental microbiology ·Vol. 70 ·No. 1 ·2004-01-00 ·Pages 240-7

Bürgmann H, Widmer F, Von Sigler W, Zeyer J

Abstract

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.

MeSH Terms
Bacteria/classification,genetics,isolation & purification Culture Media DNA Primers DNA, Bacterial/isolation & purification Molecular Sequence Data Nitrogen Fixation Oxidoreductases/genetics Polymerase Chain Reaction/methods Polymorphism, Restriction Fragment Length Sequence Analysis, DNA Soil/analysis Soil Microbiology
Chemicals
Culture Media DNA Primers DNA, Bacterial Soil Oxidoreductases nitrogenase reductase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Bürgmann Helmut
Soil Biology, Institute of Terrestrial Ecology, Swiss Federal Institute of Technology Zurich, 8952 Schlieren, Switzerland. helmut.buergmann@cnv.ethz.ch
Widmer Franco
Von Sigler William
Zeyer Josef
References (23)
23 references, click to expand
  1. Genetic diversity of nifH gene sequences in paenibacillus azotofixans strains and soil samples analyzed by denaturing gradient gel electrophoresis of PCR-amplified gene fragments.
    Appl Environ Microbiol. 1998 Aug;64(8):2770-9 PMID: 9687429
  2. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity.
    J Bacteriol. 1998 Sep;180(18):4765-74 PMID: 9733676
  3. Nitrogenase gene diversity and microbial community structure: a cross-system comparison.
    Environ Microbiol. 2003 Jul;5(7):539-54 PMID: 12823187
  4. nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass.
    Environ Microbiol. 2002 Aug;4(8):477-81 PMID: 12153588
  5. Spatial and Temporal Assessment of Diazotroph Assemblage Composition in Vegetated Salt Marsh Sediments Using Denaturing Gradient Gel Electrophoresis Analysis.
    Microb Ecol. 1999 Aug;38(2):157-167 PMID: 10441708
  6. Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora.
    Appl Environ Microbiol. 2000 Sep;66(9):3814-22 PMID: 10966395
  7. Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii.
    Appl Environ Microbiol. 1989 Oct;55(10):2522-6 PMID: 2513774
  8. Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil.
    Appl Environ Microbiol. 2002 Aug;68(8):3818-29 PMID: 12147477
  9. Phylogenetic identification and in situ detection of individual microbial cells without cultivation.
    Microbiol Rev. 1995 Mar;59(1):143-69 PMID: 7535888
  10. The ENZYME data bank.
    Nucleic Acids Res. 1993 Jul 1;21(13):3155-6 PMID: 8332535
  11. New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of Nitrogenase (nifH) genes.
    Appl Environ Microbiol. 1998 Sep;64(9):3444-50 PMID: 9726895
  12. Basic local alignment search tool.
    J Mol Biol. 1990 Oct 5;215(3):403-10 PMID: 2231712
  13. A strategy for optimizing quality and quantity of DNA extracted from soil.
    J Microbiol Methods. 2001 May;45(1):7-20 PMID: 11295193
  14. Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.
    Appl Environ Microbiol. 2000 Jul;66(7):3119-24 PMID: 10877818
  15. Comparison of nifH gene pools in soils and soil microenvironments with contrasting properties.
    Appl Environ Microbiol. 2001 May;67(5):2255-62 PMID: 11319109
  16. MEGA2: molecular evolutionary genetics analysis software.
    Bioinformatics. 2001 Dec;17(12):1244-5 PMID: 11751241
  17. Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range.
    Appl Environ Microbiol. 1999 Feb;65(2):374-80 PMID: 9925556
  18. Phylogeny of nitrogenase sequences in Frankia and other nitrogen-fixing microorganisms.
    J Mol Evol. 1989 Nov;29(5):436-47 PMID: 2515293
  19. Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences.
    J Bacteriol. 1995 Mar;177(5):1414-7 PMID: 7868622
  20. Temporal and Spatial Distribution of the nifH Gene of N(2) Fixing Bacteria in Forests and Clearcuts in Western Oregon.
    Microb Ecol. 2000 Jan;39(1):12-21 PMID: 10790513
  21. Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil.
    Res Microbiol. 2001 Jan-Feb;152(1):95-103 PMID: 11281330
  22. Interactions among plant species and microorganisms in salt marsh sediments.
    Appl Environ Microbiol. 2002 Mar;68(3):1157-64 PMID: 11872463
  23. mRNA extraction and reverse transcription-PCR protocol for detection of nifH gene expression by Azotobacter vinelandii in soil.
    Appl Environ Microbiol. 2003 Apr;69(4):1928-35 PMID: 12676666
Article Info
Journal
Applied and environmental microbiology
Abbr.
Appl Environ Microbiol
ISSN
0099-2240
DOI
10.1128/AEM.70.1.240-247.2004
Email
helmut.buergmann@cnv.ethz.ch
Published
2004-01-00
Pages
240-7
Language
English
Region
United States
NLM ID
7605801
PMCID
PMC321232
Subset
IM
Databases
GENBANK
AY196363, AY196364, AY196365, AY196366, AY196367, AY196368, AY196369, AY196370, AY196371, AY196372, AY196373, AY196374, AY196375, AY196376, AY196377, AY196378, AY196379, AY196380, AY196381, AY196382, AY196383, AY196384, AY196385, AY196386, AY196387, AY196388, AY196389, AY196390, AY196391, AY196392, AY196393, AY196394, AY196395, AY196396, AY196397, AY196398, AY196399, AY196400, AY196401, AY196402, AY196403, AY196404, AY196405, AY196406, AY196407, AY196408, AY196409, AY196410, AY196411, AY196412, AY196413, AY196414, AY196415, AY196416, AY196417, AY196418, AY196419, AY196420, AY196421, AY196422, AY196423, AY196424, AY196425, AY196426, AY196427, AY196428, AY196429, AY196430, AY196431, AY196432, AY196433, AY196434, AY196435, AY196436, AY196437, AY196438, AY196439, AY196440, AY196441, AY196442, AY196443, AY196444, AY196445, AY196446, AY196447, AY196448, AY196449, AY196450, AY196451, AY196452, AY196453, AY196454, AY196455, AY196456, AY196457, AY196458, AY196459, AY196460, AY196461, AY196462, AY196463, AY196464, AY196465
External Links
PubMed source DOI: 10.1128/AEM.70.1.240-247.2004 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2