Visualization and Quantification of Genetically Adapted Microbial Cells During Preculture

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Microbial growth is influenced by culture history, necessitating adaptation for optimal performance.
• Precultures are used to acclimate microbial cells to new media, reducing lag phase duration.
• Understanding adaptation mechanisms is crucial for microbial applications and research.

Purpose of the Study:

• To investigate the adaptation process of *Escherichia coli* BL21(DE3) during sequential transfers to succinate minimal medium.
• To identify genetic changes responsible for accelerated growth and improved fitness in a new environment.
• To visualize and quantify the emergence of genetically adapted microbial populations in real-time.

Main Methods:

• Utilized *Escherichia coli* BL21(DE3) in succinate minimal medium as a model system.
• Performed sequential transfers between minimal media to observe growth rate changes.
• Employed genome and transcript analyses to identify genetic mutations.
• Used fluorescence microscopy and flow cytometry to track and quantify adapted mutant cells.

Main Results:

• Sequential transfers to minimal medium accelerated *E. coli* growth rate and increased large colony formation.
• A C-to-T point mutation in the *kgtP* promoter was identified, enhancing exogenous succinate uptake.
• Fluorescence microscopy visualized the increase of adapted cells expressing KgtP-fused green fluorescent proteins.
• Flow cytometry showed a significant increase in mutant cell proportion from 1.75% to 70.79% over three transfers.

Conclusions:

• Genetic adaptation, specifically a promoter mutation in *kgtP*, drives accelerated growth in *E. coli* during environmental shifts.
• The study demonstrates microbial population heterogeneity and its role in adapting to new conditions.
• This research offers insights into the genetic mechanisms underlying microbial adaptability and fitness enhancement.