Single-cell analysis of Bacillus subtilis biofilms using fluorescence microscopy and flow cytometry

Area of Science:

• Microbiology
• Bacterial Physiology
• Molecular Biology

Background:

• Biofilm formation is a common bacterial behavior, involving cells encased in an extracellular matrix.
• Within biofilms, distinct, spatially organized subpopulations of specialized cells often emerge.
• In Bacillus subtilis, matrix producer subpopulations are crucial for biofilm development.

Purpose of the Study:

• To detail a technical approach for visualizing and quantifying specialized cell subpopulations in Bacillus subtilis biofilms.
• To investigate the role of surfactin in the differentiation of matrix producers.
• To provide a framework for studying phenotypic heterogeneity in microbial communities.

Main Methods:

• Utilizing fluorescent reporters for genes involved in matrix and surfactin production.
• Inserting reporter genes into the Bacillus subtilis chromosome for targeted expression.
• Monitoring subpopulations via fluorescence microscopy and flow cytometry.

Main Results:

• Demonstrated visualization and quantification of matrix and surfactin producer subpopulations in B. subtilis biofilms.
• Identified surfactin as an extracellular signal influencing matrix producer differentiation.
• Showcased the coexistence of distinct specialized cell subpopulations within the biofilm.

Conclusions:

• The differentiation of specialized cell subpopulations is a key aspect of bacterial biofilm formation.
• Fluorescent reporter systems offer a powerful tool to study cellular heterogeneity in microbial communities.
• This methodology can be adapted to diverse bacterial models for investigating gene regulation and phenotypic diversity.