@article{31979670e37248cebc2530f335bd8515,
title = "FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities",
abstract = "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{\textquoteright} 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{\textquoteright}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{\textquoteright}s toolkit, with potentially widespread application in the field of environmental microbiology.",
keywords = "ANMEs, Cell-penetrating peptides, FISH, Methanogens, Molecular beacons, mRNA",
author = "Harris, {Rachel L.} and Vetter, {Maggie C.Y.Lau} and {van Heerden}, Esta and Errol Cason and Vermeulen, {Jan G.} and Anjali Taneja and Kieft, {Thomas L.} and DeCoste, {Christina J.} and Laevsky, {Gary S.} and Onstott, {Tullis C.}",
note = "Funding Information: We are indebted to Sibanye Gold, Ltd. and the staff at the Beatrix Gold Mine for their hospitality and granting us continued access to the BE326 BH2 borehole. We would like to thank Mike Pullin, Gilbert Tetteh, Sarah Hendrickson, and Olukayode Kuloyo for their field assistance. Special thanks are extended to Rick Colwell, Hillary Morrison, and the Marine Biological Laboratory for supporting sequencing efforts through the Deep Carbon Observatory{\textquoteright}s Census of Deep Life. M.C.Y.L. and R.L.H. would like to thank Ewa Zarnowska and Tao Liang of Bruker Scientific Instruments (Florescence Microscopy) for imaging FISH-TAMB labeled E. coli at their facility in Middleton, WI. R.L.H. is thankful to Douglas H. Bartlett and his lab for hosting her visiting research supported through the Deep Carbon Observatory Deep Life Cultivation Internship (Alfred P. Sloan Foundation). Funding Information: This project was supported by funding from National Science Foundation grants DGE-1148900 to R.L.H. and DEB-1441717, EAR-1528492, DEB-1442059, and DEB-1441646 grants awarded to T.C.O. R.L.H. was also supported by the Geosciences Graduate Student Research Fund (Princeton University). Metagenomic sequencing was supported by Phase 14 of the Census of Deep Life (Deep Carbon Observatory, Alfred P. Sloan Foundation). Pioneer work was supported by funding from the Department of Geosciences, Princeton University to A.T. The Princeton University Flow Cytometry Resource Facility is supported, in part, with funding from NCI-CCSG P30CA072720-5921. Funding Information: We are indebted to Sibanye Gold, Ltd. and the staff at the Beatrix Gold Mine for their hospitality and granting us continued access to the BE326 BH2 borehole. We would like to thank Mike Pullin, Gilbert Tetteh, Sarah Hendrickson, and Olukayode Kuloyo for their field assistance. Special thanks are extended to Rick Colwell, Hillary Morrison, and the Marine Biological Laboratory for supporting sequencing efforts through the Deep Carbon Observatory{\textquoteright}s Census of Deep Life. M.C.Y.L. and R.L.H. would like to thank Ewa Zarnowska and Tao Liang of Bruker Scientific Instruments (Florescence Microscopy) for imaging FISH-TAMB labeled E. coli at their facility in Middleton, WI. R.L.H. is thankful to Douglas H. Bartlett and his lab for hosting her visiting research supported through the Deep Carbon Observatory Deep Life Cultivation Internship (Alfred P. Sloan Foundation). Publisher Copyright: {\textcopyright} 2021, The Author(s).",
year = "2022",
month = jul,
doi = "10.1007/s00248-021-01809-5",
language = "English (US)",
volume = "84",
pages = "182--197",
journal = "Microbial Ecology",
issn = "0095-3628",
publisher = "Springer New York",
number = "1",
}