Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics

Area of Science:

• Microbiology
• Genetics
• Cell Biology

Background:

• Natural genetic transformation is a key bacterial horizontal gene transfer mechanism.
• Competence, a transient state for DNA uptake and integration, is crucial for transformation.
• Streptococcus pneumoniae competence is regulated by competence-stimulating peptide (CSP).

Purpose of the Study:

• To develop and utilize a microfluidic system for real-time, single-cell monitoring of pneumococcal competence and transformation.
• To investigate the effects of CSP on bacterial growth rate and gene expression during competence development.
• To track the process of transformation and the fate of transformants in live bacterial cells.

Main Methods:

• Culturing Streptococcus pneumoniae under continuous flow in a microfluidic system.
• Employing fluorescence microscopy to monitor live cells.
• Using novel fluorescent reporters to distinguish temporally distinct competence gene expression.
• Injecting CSP and transforming DNA into microfluidic channels for real-time tracking.

Main Results:

• Established conditions for microfluidic culture of S. pneumoniae with comparable growth rates to batch cultures.
• Demonstrated that CSP perfusion reduces individual cell growth rates, consistent with natural competence.
• Successfully visualized and tracked bacterial transformation events at the single-cell level in near real-time.

Conclusions:

• The developed microfluidic approach enables detailed investigation of pneumococcal competence onset and transformation dynamics.
• This technology provides a powerful tool for studying horizontal gene transfer mechanisms in bacteria at the single-cell level.
• Real-time tracking of transformation offers new avenues for understanding bacterial adaptation and evolution.