Schlüsselwörter
(Englisch)
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Nitrogen fixation; soil; molecular biology; population; quantification
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Forschungsprogramme
(Englisch)
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COST-Action 831 - Biotechnology of soil: monitoring, conservation and remediation
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Kurzbeschreibung
(Englisch)
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See abstract
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Weitere Hinweise und Angaben
(Englisch)
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Full name of research-institution/enterprise: ETH Zürich Institut für terrestrische Ökologie ITO Fachbereich Bodenbiologie
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Partner und Internationale Organisationen
(Englisch)
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A, B, DK, FIN, F, D, H, I, NL, N, SI, E, S, CH, GB Swiss Federal Research Station for Agroecology and Agriculture (FAL Reckenholz), Zürich
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Abstract
(Englisch)
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The project 'Activity and diversity of nitrogen-fixing microorganisms. Novel tools to characterize populations in soil' developed and improved tools to study nitrogen-fixing microorganisms in soil, with an emphasis on the free-living (asymbiotic) population. A marker gene of nitrogen-fixing microorganisms (nifH) was used as a molecular marker to study the diversity, and via the detection of mRNA, also the activity of these organisms in soil. This approach allows to link structural information about a microbial community directly to the macroscopically observable activity of nitrogen fixation. This approach provides an important tool to bridge the structure-activity gap that represents a major challenge to current microbial ecology. A number of techniques were developed to advance this technology: To estimate the diversity of this functional gene and its expression, nucleic acids (DNA and RNA) have to be extracted from soil - a critical step on which all downstream analyses depend. Optimised protocols for robust extraction of DNA and RNA from soil were developed. The new protocols allow high yield extraction of DNA and isolation of intact and highly pure RNA, including mRNA. Furthermore, the protocol is capable of providing an estimate on the maximum extractable DNA content of a soil, an important parameter for establishing a quantitative basis for downstream analysis. Tools for the detection of diazotroph organisms have been improved by designing, optimising and testing group specific nifH-PCR protocols. We developed several PCR protocols capable of detecting different groups of proteobacterial nifH sequences in soil using direct amplification with newly developed primers of low degeneracy. This approach has several advantages compared to previous universal amplification protocols using nested PCR. It is e.g. less prone to amplification bias, the new protocols amplified sequences that were not detected by the universal amplification protocol, and differences of genetic fingerprints between soil populations could oftentimes be observed more clearly with the focused amplification protocols. Furthermore all of these protocols amplify the same gene fragment, so that they are mutually compatible for downstream analysis with e.g. fingerprinting methods. The developed methodology was used to study the nifH expression in Azotobacter vinelandii growing in sterile soil. Intact mRNA was extracted, reverse transcribed and amplified using specific nifH PCR protocol for Azotobacter. The results were compared with data on cell densities, nitrogen fixation activity, and other parameters. The regulatory effect of mineral nitrogen on the expression of nifH could be demonstrated for the first time in a soil matrix. The method was sensitive enough to allow relative quantification of the cellular level of nifH transcription in relation to the observed fixation activity. The method was also shown to be suitable for the detection of complex mixtures of nifH mRNA from natural soil samples. Our work has contributed reliable tools to study the effect of environmental conditions on the pattern and intensity of nifH gene transcription in natural soil samples. It is now possible to obtain more detailed information on regulatory mechanisms of diazotrophs in their natural soil environment as opposed to liquid culture, and to study interactions and community response to various stimuli in a much more detailed way. The methodology should furthermore be adaptable to other functions and genes.
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Datenbankreferenzen
(Englisch)
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Swiss Database: COST-DB of the State Secretariat for Education and Research Hallwylstrasse 4 CH-3003 Berne, Switzerland Tel. +41 31 322 74 82 Swiss Project-Number: C99.0036
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