Labeling of prokaryotic mRNA in live cells using fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB)
| Autor: | Tullis C. Onstott, Anjali Taneja, Errol D. Cason, Esta van Heerden, Rachel L. Harris, Gary Laevsky, Maggie C. Y. Lau, Jan-G Vermeulen, Christina DeCoste, Thomas L. Kieft |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0303 health sciences
Messenger RNA medicine.diagnostic_test 030306 microbiology ved/biology ved/biology.organism_classification_rank.species In situ hybridization Cell sorting Biology Molecular biology Cell biology Flow cytometry Cell wall 03 medical and health sciences Molecular beacon medicine Methanosarcina barkeri Intracellular 030304 developmental biology |
| DOI: | 10.1101/178368 |
| Popis: | High-throughput sequencing and cellular imaging have expanded our knowledge of microbial diversity and expression of cellular activity. However, it remains challenging to characterize low-abundance, slow-growing microorganisms that play key roles in biogeochemical cycling. With the goal of isolating transcriptionally active cells of these microorganisms from environmental samples, we developed fluorescentin situhybridization of transcript-annealing molecular beacons (FISH-TAMB) to label living prokaryotic cells. FISH-TAMB utilizes polyarginine cell-penetrating peptides to deliver molecular beacons across cell walls and membranes. Target cells are fluorescently labeled via hybridization between molecular beacons and messenger RNA of targeted functional genes. FISH-TAMB’s target specificity and deliverance into both bacterial and archaeal cells were demonstrated by labeling intracellular methyl-coenzyme M reductase A (mcrA) transcripts expressed byEscherichia colimcrA+,Methanosarcina barkeri,and a methanogenic enrichment of deep continental fracture fluid. Growth curve analysis supported sustained cellular viability following FISH-TAMB treatment. Flow cytometry and confocal microscopy detected labeled single cells and single cells in aggregates with unlabeled cells. As FISH-TAMB is amenable to target any functional gene of interest, when coupled with cell sorting, imaging, and sequencing techniques, FISH-TAMB will enable characterization of key uncharacterized rare biosphere microorganisms and of the syntrophically activated metabolic pathways between physically associated microorganisms. |
| Databáze: | OpenAIRE |
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