A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation...

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Bacterial conjugation is a key mechanism for horizontal gene transfer, driving the spread of antibiotic resistance and virulence factors.
• Accurate measurement of conjugation frequency is crucial for understanding plasmid dynamics and the epidemiology of antimicrobial resistance.

Purpose of the Study:

• To develop a streamlined, fluorescence-based method for quantifying conjugation frequency of low-copy-number plasmids.
• To enable precise monitoring of plasmid transfer dynamics using flow cytometry.

Main Methods:

• Homologous recombineering was used to insert a blue fluorescent protein gene into a conjugative plasmid.
• Recipient strains were labeled with a stable, non-conjugative plasmid encoding a red fluorescent protein and a toxin-antitoxin system.
• Flow cytometry was employed to distinguish donor, recipient, and transconjugant populations based on fluorescence.

Main Results:

• The developed method allows for precise measurement of plasmid transfer frequency during filter mating.
• The use of fluorescent protein-labeled plasmids enabled clear discrimination of bacterial populations.
• The system facilitated accurate monitoring of conjugation frequencies over time in an antibiotic-free environment.

Conclusions:

• This fluorescence labeling approach provides a robust and efficient tool for studying bacterial conjugation.
• The method enhances our ability to investigate the transfer dynamics of conjugative plasmids and their role in disseminating resistance traits.