Fluorescence in situ hybridization for intracellular localization of nifH mRNA. 2009

Martin Pilhofer, and Marko Pavlekovic, and Natuschka M Lee, and Wolfgang Ludwig, and Karl-Heinz Schleifer
Technische Universität München, Lehrstuhl für Mikrobiologie, Am Hochanger 4, 85350 Freising, Germany. martin-pilhofer@web.de

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.

UI MeSH Term Description Entries
D009586 Nitrogen Fixation The process in certain BACTERIA; FUNGI; and CYANOBACTERIA converting free atmospheric NITROGEN to biologically usable forms of nitrogen, such as AMMONIA; NITRATES; and amino compounds. Diazotrophy,Diazotrophic Activity,Dinitrogen Fixation,N2 Fixation,Activities, Diazotrophic,Activity, Diazotrophic,Diazotrophic Activities,Fixation, Dinitrogen,Fixation, N2,Fixation, Nitrogen
D010088 Oxidoreductases The class of all enzymes catalyzing oxidoreduction reactions. The substrate that is oxidized is regarded as a hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The recommended name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be used. Oxidase is only used in cases where O2 is the acceptor. (Enzyme Nomenclature, 1992, p9) Dehydrogenases,Oxidases,Oxidoreductase,Reductases,Dehydrogenase,Oxidase,Reductase
D012333 RNA, Messenger RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm. Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D015345 Oligonucleotide Probes Synthetic or natural oligonucleotides used in hybridization studies in order to identify and study specific nucleic acid fragments, e.g., DNA segments near or within a specific gene locus or gene. The probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the probe include the radioisotope labels 32P and 125I and the chemical label biotin. Oligodeoxyribonucleotide Probes,Oligonucleotide Probe,Oligoribonucleotide Probes,Probe, Oligonucleotide,Probes, Oligodeoxyribonucleotide,Probes, Oligonucleotide,Probes, Oligoribonucleotide
D015373 Bacterial Typing Techniques Procedures for identifying types and strains of bacteria. The most frequently employed typing systems are BACTERIOPHAGE TYPING and SEROTYPING as well as bacteriocin typing and biotyping. Bacteriocin Typing,Biotyping, Bacterial,Typing, Bacterial,Bacterial Biotyping,Bacterial Typing,Bacterial Typing Technic,Bacterial Typing Technics,Bacterial Typing Technique,Technic, Bacterial Typing,Technics, Bacterial Typing,Technique, Bacterial Typing,Techniques, Bacterial Typing,Typing Technic, Bacterial,Typing Technics, Bacterial,Typing Technique, Bacterial,Typing Techniques, Bacterial,Typing, Bacteriocin
D016948 Azotobacter vinelandii A species of gram-negative, aerobic bacteria first isolated from soil in Vineland, New Jersey. Ammonium and nitrate are used as nitrogen sources by this bacterium. It is distinguished from other members of its genus by the ability to use rhamnose as a carbon source. (From Bergey's Manual of Determinative Bacteriology, 9th ed) Azotobacter miscellum
D017404 In Situ Hybridization, Fluorescence A type of IN SITU HYBRIDIZATION in which target sequences are stained with fluorescent dye so their location and size can be determined using fluorescence microscopy. This staining is sufficiently distinct that the hybridization signal can be seen both in metaphase spreads and in interphase nuclei. FISH Technique,Fluorescent in Situ Hybridization,Hybridization in Situ, Fluorescence,FISH Technic,Hybridization in Situ, Fluorescent,In Situ Hybridization, Fluorescent,FISH Technics,FISH Techniques,Technic, FISH,Technics, FISH,Technique, FISH,Techniques, FISH
D041121 Klebsiella oxytoca A species of gram-negative bacteria causing URINARY TRACT INFECTIONS and SEPTICEMIA. Bacillus oxytocus perniciosus

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