Deciphering the Antitoxin-Regulated Bacterial Stress Response via Single-Cell Analysis

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacterial toxin-antitoxin (TA) systems confer tolerance to drugs and environmental stresses.
• Quantitative measurement of low-abundance TA proteins in single bacteria is challenging.
• TA systems are vital for prokaryotic survival and adaptation.

Purpose of the Study:

• To develop a sensitive method for detecting basal-level antitoxin expression in single bacteria.
• To investigate the heterogeneity of antitoxin MqsA levels in *Escherichia coli* under various conditions.
• To analyze the dynamic response of TA systems to environmental stress.

Main Methods:

• Development of a nano-flow cytometry (nFCM) method using a tetracysteine-tagged TA system (MqsR/MqsA) labeled with FlAsH.
• Monitoring basal-level expression and degradation of antitoxin MqsA in single *E. coli*.
• Simultaneous analysis of bacterial growth rate and MqsA production under environmental stress.

Main Results:

• Identified two distinct populations of *E. coli* with high or low MqsA levels under native conditions.
• Observed differential responses of these populations to bile acid stress, heat shock, and amino acid starvation.
• Demonstrated resumed MqsA production after amino acid stress and a 60-minute lag between MqsA production and growth response to stress.

Conclusions:

• Stochastic elevation of MqsA facilitates bacterial survival.
• Distinct bacterial subpopulations with varying MqsA levels enhance adaptation to fluctuating environments.
• The developed nFCM method provides deeper insights into TA system heterogeneity and function.