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

• DOI: 10.1007/s00248-021-01809-5
• Publication Date: 2021-08-18T05:11:32Z
• Authors: Rachel L. Harris; Maggie C. Y. Lau ...; Esta van Heerden; Errol Cason; Jan-G Vermeulen; Anjali Taneja; Thomas L. Kieft; Christina J. DeCoste; Gary S. Laevsky; Tullis C. Onstott
• Keywords: 0301 basic medicine; 2. Zero hunger; Microbiota; Archaea; 3. Good health; 03 medical and health sciences; DNA, Archaeal; RNA, Ribosomal, 16S; Methods; Escherichia coli; RNA, Messenger; Oxidoreductases; Methane; In Situ Hybridization, Fluorescence; Phylogeny

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance biomass present challenges for discovery, identification, visualization and selection. We report development fluorescent in situ hybridization transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable archaea bacteria. The FISH-TAMB method differs from existing FISH methods by absence fixatives surfactants buffers, fast time as short 15 min at target cells' growth temperature, omission washing steps. Polyarginine cell-penetrating peptides employed deliver (MBs) across prokaryotic cell walls membranes, fluorescently labeling cells when MBs hybridize mRNA sequences. Here, detailed preparation application presented. To demonstrate FISH-TAMB's ability label intracellular targets, differentiate transcriptional states, detect active rare taxa, keep viability, experiments were performed targeted messenger RNA (mRNA) methyl-coenzyme M reductase A (mcrA) expressed (1) Escherichia coli containing plasmid with partial mcrA gene methanogen Methanosarcina barkeri (E. mcrA+); (2) M. barkeri; (3) anaerobic methanotrophic (ANME) enrichment deep continental borehole. Although was initially envisioned any functional interest without requirement prior knowledge 16S ribosomal (rRNA)-based taxonomy, has potential multiplexing going beyond thus versatile addition ecologist's toolkit, potentially widespread field environmental microbiology.