Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Fluorogenic protein tagging systems are less developed in prokaryotes compared to eukaryotes.
• Existing methods often lack the versatility for prolonged live-cell imaging in bacteria.

Purpose of the Study:

• To develop novel, noncovalent fluorogenic protein tags for bacterial systems.
• To enable long-term fluorescence microscopy of living bacterial cells under aerobic and anaerobic conditions.

Main Methods:

• Introduction of transcription factor-based tags, LmrR and RamR, termed Chemogenetic Tags with Probe Exchange (CTPEs).
• Extensive characterization of 30 reversibly binding organic fluorophores with the CTPEs.
• Evaluation of fluorophore binding specificity and photophysical properties.

Main Results:

• CTPEs impart fluorogenicity and extended fluorescence lifetime to bound organic fluorophores.
• Aromatic planar fluorophores exhibit high specificity for CTPE hydrophobic pockets.
• Successful long-term fluorescence microscopy of living bacterial cells was achieved.

Conclusions:

• CTPEs offer a versatile tool for studying bacterial protein localization, dynamics, and interactions.
• The system supports prolonged live-cell imaging, even in oxygen-free environments.
• This technology advances bacterial cell biology research through enhanced imaging capabilities.