FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities
| Autor: | Esta van Heerden, Rachel L. Harris, Jan G. Vermeulen, Thomas L. Kieft, Maggie C. Y. Lau Vetter, Tullis C. Onstott, Anjali Taneja, Gary Laevsky, Christina DeCoste, Errol D. Cason |
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| Rok vydání: | 2020 |
| Předmět: |
ved/biology.organism_classification_rank.species
Soil Science Biology medicine.disease_cause Plasmid Molecular beacon RNA Ribosomal 16S medicine Escherichia coli RNA Messenger Ecology Evolution Behavior and Systematics In Situ Hybridization Fluorescence Phylogeny Messenger RNA Ecology ved/biology Microbiota Ribosomal RNA biology.organism_classification Archaea DNA Archaeal Biochemistry Methanosarcina barkeri Oxidoreductases Methane Bacteria |
| Zdroj: | Microbial ecology. 84(1) |
| ISSN: | 1432-184X |
| Popis: | Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells’ growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology. |
| Databáze: | OpenAIRE |
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