A Microplate Reader-Based System for Visualizing Transcriptional Activity During in vivo Microbial Interactions in...

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Understanding bacterial responses to fungal interactions is crucial for microbial ecology and disease research.
• Real-time monitoring of gene expression during such interactions is challenging with current methods.

Purpose of the Study:

• To develop and validate a novel microplate reader-based system for real-time visualization of bacterial gene expression dynamics.
• To investigate the temporal regulation of bacterial antifungal secondary metabolite gene clusters in response to fungal hyphae.

Main Methods:

• A reporter bacterial strain expressing mCherry fluorescent protein under the control of the nunF promoter was engineered.
• Time-lapse, multi-channel imaging was performed using a microplate reader, integrating microbial growth measurements.
• Fluorescent signals from the reporter and metabolites were correlated with bacterial growth phases and physical contact with fungi.

Main Results:

• Bacterial nunF-regulated gene transcription was activated during the deceleration growth phase.
• Gene transcription initiation coincided with physical contact between bacterial cells and fungal hyphae.
• The system successfully visualized real-time gene expression dynamics and metabolite production.

Conclusions:

• The developed microplate reader system offers a versatile tool for live imaging of microbial interactions.
• This technique facilitates the precise timing of destructive sampling for complementary analyses like transcriptomics.
• It provides insights into the molecular mechanisms governing bacterial-fungal communication and defense responses.