FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities

• Rachel L. Harris ^{[1] [5]} ; Maggie C. Y. Lau Vetter ^{[6] [5]} ; Esta van Heerden ^{[7] [8]} ; Errol Cason ^{[2] [2]} ; Jan-G Vermeulen ^{[2] [2]} ; Anjali Taneja ^{[3] [5]} ; Thomas L. Kieft ^[4] ; Christina J. DeCoste ^[5] ; Gary S. Laevsky ^[5] ; Tullis C. Onstott ^[5]
  1. [1] Harvard University

     Harvard University

     City of Cambridge, Estados Unidos

     
  2. [2] University of the Free State

     University of the Free State

     Mangaung, Sudáfrica

     
  3. [3] Georgetown University

     Georgetown University

     Estados Unidos

     
  4. [4] New Mexico Institute of Mining and Technology

     New Mexico Institute of Mining and Technology

     Estados Unidos

     
  5. [5] Princeton University

     Princeton University

     Estados Unidos

     
  6. [6] Laboratory of Extraterrestrial Ocean Systems, Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, 572000, Hainan, China
  7. [7] Centre for Water Sciences and Management, North West University, Potchefstroom, South Africa
  8. [8] iWater Pty Ltd, 5 Walter Sisulu Rd, Park West, Bloemfontein, 9301, South Africa

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• Localización: Microbial ecology, ISSN-e 1432-184X, ISSN 0095-3628, Vol. 84, Nº. 1, 2022, págs. 182-197
• Idioma: inglés
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  • 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.