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

Area of Science:

• Microbiology
• Molecular Ecology
• Environmental Science

Background:

• Keystone species and ecological engineers are crucial for ecosystem health but challenging to identify due to low abundance.
• Existing methods for microbial identification often face limitations in speed, specificity, or sample preservation.

Purpose of the Study:

• To develop and present a novel, fixation-free protocol for detecting and visualizing specific microbial taxa.
• To enable the identification of active and rare prokaryotes based on their gene expression.

Main Methods:

• Developed fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol.
• Utilized polyarginine cell-penetrating peptides to deliver molecular beacons (MBs) into prokaryotic cells.
• Targeted messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) in various microbial samples, including Escherichia coli, Methanosarcina barkeri, and an ANME enrichment.

Main Results:

• FISH-TAMB successfully labeled intracellular mRNA targets in prokaryotes without fixation.
• The method demonstrated rapid hybridization (as short as 15 minutes) and preserved cell viability.
• FISH-TAMB differentiated transcriptional states and detected active, rare taxa, including methanogens.

Conclusions:

• FISH-TAMB is a versatile and efficient tool for molecular ecologists, enabling the detection of specific microbial taxa based on functional gene expression.
• The protocol overcomes limitations of traditional methods, offering a valuable addition for environmental microbiology research.
• FISH-TAMB has potential for multiplexing and applications beyond mRNA detection, expanding its utility in microbial studies.