Fluorescence in situ hybridization for intracellular localization of nifH mRNA.
Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.
Pilhofer
Martin
M
Technische Universität München, Lehrstuhl für Mikrobiologie, Am Hochanger 4, 85350 Freising, Germany. martin-pilhofer@web.de
Pavlekovic
Marko
M
Lee
Natuschka M
NM
Ludwig
Wolfgang
W
Schleifer
Karl-Heinz
KH
D016948:Azotobacter vinelandii / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D015373:Bacterial Typing Techniques; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D041121:Klebsiella oxytoca / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D009586:Nitrogen Fixation; D015345:Oligonucleotide Probes; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D010088:Oxidoreductases; C029130:nitrogenase reductase