mRNA extraction and reverse transcription-PCR protocol for detection of nifH gene expression by Azotobacter vinelandii in soil.
The study of free-living nitrogen-fixing organisms in bulk soil is hampered by the great diversity of soil microbial communities and the difficulty of relating nitrogen fixation activities to individual members of the diazotroph populations. We developed a molecular method that allows analysis of nifH mRNA expression in soil in parallel with determinations of nitrogen-fixing activity and bacterial growth. In this study, Azotobacter vinelandii growing in sterile soil and liquid culture served as a model system for nifH expression, in which sucrose served as the carbon source and provided nitrogen-limited conditions, while amendments of NH(4)NO(3) were used to suppress nitrogen fixation. Soil RNA extraction was performed with a new optimized direct extraction protocol that yielded nondegraded total RNA. The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR. The level of nifH gene expression was estimated by PCR amplification of reverse-transcribed nifH mRNA fragments with A. vinelandii-specific nifH primers. This new approach revealed that nifH gene expression was positively correlated with bulk nitrogen fixation activity in soil (r(2) = 0.72) and in liquid culture (r(2) = 0.84) and therefore is a powerful tool for studying specific regulation of gene expression directly in the soil environment.
Bürgmann
Helmut
H
Soil Biology, Institute of Terrestrial Ecology, Swiss Federal Institute of Technology (ETH-Zürich), CH-8952 Schlieren, Switzerland. buergmann@ito.umnw.ethz.ch
Widmer
Franco
F
Sigler
William V
WV
Zeyer
Josef
J
D016948:Azotobacter vinelandii / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D009586:Nitrogen Fixation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012988:Soil Microbiology
D003470:Culture Media; D000644:Quaternary Ammonium Compounds; D012329:RNA, Bacterial; D012333:RNA, Messenger; D010088:Oxidoreductases; C029130:nitrogenase reductase